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1.  Isolation of Lyme Disease Borrelia from Puffins (Fratercula arctica) and Seabird Ticks (Ixodes uriae) on the Faeroe Islands 
Journal of Clinical Microbiology  1999;37(4):890-896.
This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans.
PMCID: PMC84640  PMID: 10074497
2.  Genetic Heterogeneity of Borrelia burgdorferi Sensu Lato in the Southern United States Based on Restriction Fragment Length Polymorphism and Sequence Analysis 
Journal of Clinical Microbiology  2001;39(7):2500-2507.
Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.
doi:10.1128/JCM.39.7.2500-2507.2001
PMCID: PMC88176  PMID: 11427560
3.  Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order. 
Journal of Bacteriology  1995;177(10):2769-2780.
We have constructed physical and genetic maps of the chromosomes of 21 Lyme disease agent spirochetes from geographically diverse locations. All have linear chromosomes whose lengths range from 935 to 955 kbp, and all contain multiple linear plasmids in the 16- to 175-kbp size range. The locations of 11 gene clusters on the chromosomes of these different isolates are indistinguishable at the resolution achieved in this study, indicating that the members of this related group of species have highly conserved chromosomal gene orders. However, chromosomal restriction endonuclease cleavage site maps are unique for nearly all isolates. The 22 chromosomal maps currently available define eight classes of Lyme disease agents. Four of these correspond to the previously proposed species Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, and Borrelia japonica. In addition, the North American isolates 21038, DN127 c19-2, 25015, and CA55 typify four additional chromosomal types that are as phylogenetically distinct as the species listed above. These findings support the idea that comparison of restriction maps is currently the most robust and definitive method for determining overall chromosomal relationships among closely related bacteria. In the course of this work, we located on the chromosome the previously unmapped outer surface protein-encoding LA7 gene and genes homologous to the Escherichia coli priA, plsC, parE, and parC genes, and we have substantially refined the locations of the recA, fla, p22A, and flgE genes.
PMCID: PMC176948  PMID: 7751287
4.  Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons 
Journal of Clinical Microbiology  2004;42(4):1444-1449.
We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.
doi:10.1128/JCM.42.4.1444-1449.2004
PMCID: PMC387532  PMID: 15070987
5.  Borrelia burgdorferi Sensu Stricto Is Clonal in Patients with Early Lyme Borreliosis ▿  
Applied and Environmental Microbiology  2008;74(16):5008-5014.
Lyme borreliosis, the most commonly reported vector-borne disease in North America, is caused by the spirochete Borrelia burgdorferi. Given the extensive genetic polymorphism of B. burgdorferi, elucidation of the population genetic structure of the bacterium in clinical samples may be relevant for understanding disease pathogenesis and may have applicability for the development of diagnostic tests and vaccine preparations. In this investigation, the genetic polymorphism of the 16S-23S rRNA (rrs-rrlA) intergenic spacer and ospC was investigated at the sequence level in 127 clinical isolates obtained from patients with early Lyme borreliosis evaluated in suburban New York City. Sixteen distinct rrs-rrlA and 16 distinct ospC alleles were identified, representing virtually all of the genotypes previously found in questing Ixodes scapularis nymphs in this region. In addition, a new ospC group was identified in a single patient. The strong linkage observed between the chromosome-located rrs-rrlA and plasmid-borne ospC genes suggests a clonal structure of B. burgdorferi in these isolates, despite evidence of recombination at ospC.
doi:10.1128/AEM.00479-08
PMCID: PMC2519259  PMID: 18539816
6.  Borrelia carolinensis sp. nov., a New (14th) Member of the Borrelia burgdorferi Sensu Lato Complex from the Southeastern Region of the United States ▿  
Journal of Clinical Microbiology  2008;47(1):134-141.
Approximately 118 Borrelia isolates were cultured from a variety of rodents, birds, and ticks collected in the southern United States. In addition to a highly diverse group of Borrelia bissettii strains and a homogenous group of Borrelia burgdorferi sensu stricto strains, a group of 16 isolates with unusual characteristics was found. The isolates were cultured from ear biopsy samples of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina. A multilocus sequence analysis of the rrf-rrl intergenic spacer, 16S rRNA, fla, ospA, and p66 genes were used to clarify the taxonomic status of the new group of B. burgdorferi sensu lato isolates. Thirteen species of the B. burgdorferi sensu lato complex were used as controls. Unique restriction fragment length polymorphism patterns of the rrf-rrl intergenic spacer region and fla gene were recognized. Unique signature nucleotides were also found in the 16S rRNA gene. A phylogenetic analysis shows that the 16 new isolates cluster together but separately from the other species in the B. burgdorferi sensu lato complex. Our data strongly support the recognition of the 16 isolates as a new B. burgdorferi sensu lato species. We propose to name this genospecies “Borrelia carolinensis” with respect to the place of its currently known geographic location.
doi:10.1128/JCM.01183-08
PMCID: PMC2620843  PMID: 19020062
7.  Specificities and Sensitivities of Four Monoclonal Antibodies for Typing of Borrelia burgdorferi Sensu Lato Isolates 
Borrelia burgdorferi, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana. In the United States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East Coast were differentiated. In Asia, B. japonica has been identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of rrf (5S)-rrl (23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55 B. burgdorferi ss isolates but showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45 B. afzelii isolates. Monoclonal antibody D6 was 100% specific for B. garinii but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new Borrelia isolates.
doi:10.1128/CDLI.8.2.376-384.2001
PMCID: PMC96066  PMID: 11238225
8.  Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains. 
Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.
PMCID: PMC168014  PMID: 8779571
9.  Mixed infection of different Borrelia species among Apodemus speciosus mice in Hokkaido, Japan. 
Journal of Clinical Microbiology  1995;33(2):490-492.
The wood mouse (Apodemus speciosus) serves as a wildlife reservoir for Lyme disease spirochetes in Hokkaido, Japan. To isolate Borrelia species, we captured 34 wood mice in an area where Borrelia species are endemic during October 1993. The earlobes (right and left), heart, spleen, and urinary bladder from each mouse were used as culture sources. As a result of culture 73 isolates from 21 mice were classified by rRNA gene restriction fragment length polymorphism analysis. Ribotype groups III (Borrelia afzelii) and IV (unknown species) were detected among those isolates. Thirty-one (77.5%) of 40 earlobe isolates were classified as group IV. In contrast, 6 (40.0%) of 15 heart isolates, 5 (50.0%) of 10 spleen isolates, and 7 (87.5%) of 8 urinary bladder isolates were B. afzelii. Seven mice showed mixed infection with B. afzelii and group IV. The data indicate that different Borrelia species can coexist in a reservoir host that is suitable for them.
PMCID: PMC227974  PMID: 7536218
10.  Distribution of Borrelia burgdorferi Sensu Lato in China▿† 
Journal of Clinical Microbiology  2011;49(2):647-650.
We genotyped 102 Borrelia burgdorferi sensu lato strains isolated from ticks, animals, and patients in 11 provinces in China by PCR–restriction fragment length polymorphism (PCR-RFLP) amplification of 5S (rrf)-23S (rrl) rRNA gene spacer amplicons and multilocus sequence analysis (MLSA). The results showed that Borrelia garinii was the main genotype in China (65/102) and that it was distributed mainly in northern China. Borrelia afzelii was the second most frequently found species (22/102), and it was distributed in both northern and southern China. All Borrelia valaisiana strains were isolated from Guizhou Province. Additionally, one B. burgdorferi strain was isolated from Hunan Province. Our results show the diversity and wide distribution of B. burgdorferi sensu lato in China.
doi:10.1128/JCM.00725-10
PMCID: PMC3043522  PMID: 21106783
11.  Isolation and Characterization of Borrelia burgdorferi Sensu Lato Strains in an Area of Italy Where Lyme Borreliosis Is Endemic 
Journal of Clinical Microbiology  2001;39(6):2254-2260.
Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to “San Martino” Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.
doi:10.1128/JCM.39.6.2254-2260.2001
PMCID: PMC88120  PMID: 11376066
12.  Prevalence of Lyme disease spirochetes in Ixodes persulcatus and wild rodents in far eastern Russia. 
Applied and Environmental Microbiology  1996;62(10):3887-3889.
Borrelia spirochetes were isolated from the adult ixodid tick (Ixodes persulcatus) in three areas of far eastern Russia, namely, Khabarovsk, Vladivostok, and Yuzhno-Sakhalinsk. Borrelia infective rates of ticks in those areas were 24.5, 41.4, and 25.1%, respectively (total rate was 26.6%). Spirochetes were also isolated from the tissues of small mammals captured at Khabarovsk (infective rate was 20.8%). Samples were classified by rRNA gene restriction fragment length polymorphism (RFLP) analysis. The isolated spirochetes from ticks were classified mainly RFLP ribotype group IV (B. garinii), followed by groups II (B. garinii), III (B. afzelii), and V (B. garinii), showing that B. garinii is a dominant species among them. Both B. garinii and B. afzelii were also found in rodents, and multiple infections with those two species were observed in some rodents. B. burgdorferi sensu stricto (group I) was not isolated from either ticks or rodents.
PMCID: PMC168201  PMID: 8837448
13.  Molecular Typing of Borrelia burgdorferi Sensu Lato: Taxonomic, Epidemiological, and Clinical Implications 
Clinical Microbiology Reviews  1999;12(4):633-653.
Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the LB-related spirochetes and their potential association with distinct clinical syndromes. These methods include serotyping, multilocus enzyme electrophoresis, DNA-DNA reassociation analysis, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis, plasmid fingerprinting, randomly amplified polymorphic DNA fingerprinting analysis, species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and sequence analysis of 16S rRNA and other conserved genes. On the basis of DNA-DNA reassociation analysis, 10 different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, and Borrelia bissettii sp. nov. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well known to be responsible for causing human disease. Different Borrelia species have been associated with distinct clinical manifestations of LB. In addition, Borrelia species are differentially distributed worldwide and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomic status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especiallly correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail.
PMCID: PMC88929  PMID: 10515907
14.  Transhemispheric exchange of Lyme disease spirochetes by seabirds. 
Journal of Clinical Microbiology  1995;33(12):3270-3274.
Lyme disease is a zoonosis transmitted by ticks and caused by the spirochete Borrelia burgdorferi sensu lato. Epidemiological and ecological investigations to date have focused on the terrestrial forms of Lyme disease. Here we show a significant role for seabirds in a global transmission cycle by demonstrating the presence of Lyme disease Borrelia spirochetes in Ixodes uriae ticks from several seabird colonies in both the Southern and Northern Hemispheres. Borrelia DNA was isolated from I. uriae ticks and from cultured spirochetes. Sequence analysis of a conserved region of the flagellin (fla) gene revealed that the DNA obtained was from B. garinii regardless of the geographical origin of the sample. Identical fla gene fragments in ticks obtained from different hemispheres indicate a transhemispheric exchange of Lyme disease spirochetes. A marine ecological niche and a marine epidemiological route for Lyme disease borreliae are proposed.
PMCID: PMC228686  PMID: 8586715
15.  Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis. 
Journal of Clinical Microbiology  1996;34(5):1306-1309.
Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.
PMCID: PMC229006  PMID: 8727927
16.  Genetic Characteristics of Borrelia coriaceae Isolates from the Soft Tick Ornithodoros coriaceus (Acari: Argasidae) 
Journal of Clinical Microbiology  2000;38(7):2678-2682.
Two Borrelia isolates (CA434 and CA435) cultured from the soft tick Ornithodoros coriaceus were analyzed by contour-clamped homogeneous electric field gel electrophoresis of unrestricted and ApaI-restricted DNA, standard electrophoresis of BamHI- and HindIII-restricted DNA, Southern hybridization, restriction fragment length polymorphism and sequencing of the 16S rRNA gene, and amplification of the 5S-23S intergenic spacer region. These isolates were compared with Borrelia coriaceae type strain Co53, B. burgdorferi sensu stricto strain CA4, and the relapsing-fever spirochete B. parkeri (undesignated). The 16S rRNA region of CA434 and CA435 differed from that of B. coriaceae type strain Co53 by the presence of 1 base (C) at position 367 (GenBank accession no. U42286). The linear plasmid profile of CA434 was similar to that of Co53, and the ApaI, BamHI, and HindIII restriction fingerprints of the total cellular DNA of CA434 and Co53 were similar. In contrast, CA435 differed somewhat from CA434 and Co53, which demonstrates that B. coriaceae is genetically diverse. Southern hybridization showed that the DNAs of CA434 and CA435 hybridized strongly with the digoxigenin-labeled DNA of Co53. Low homology was found between the DNA of Co53 and that of B. parkeri. The 16S rRNA sequence of B. parkeri was identical to previously published results for B. parkeri strain M3001 (GenBank accession number U42296). CA434 and CA435 represent only the second and third isolates of B. coriaceae obtained from any source since its initial isolation from an O. coriaceus tick in 1985. All three B. coriaceae isolates were derived from adult ticks collected from the same locality in northwestern California. Difficulties encountered in detecting B. coriaceae in, and isolating this spirochete from, the tissues of O. coriaceus are discussed. The lack of concordance between different detection or isolation methods suggests that reliance upon a single technique may grossly underestimate the true prevalence of spirochetal infection in wild-caught O. coriaceus ticks.
PMCID: PMC86996  PMID: 10878063
17.  Associations of passerine birds, rabbits, and ticks with Borrelia miyamotoi and Borrelia andersonii in Michigan, U.S.A. 
Parasites & Vectors  2012;5:231.
Background
Wild birds contribute to maintenance and dissemination of vectors and microbes, including those that impact human, domestic animal, and wildlife health. Here we elucidate roles of wild passerine birds, eastern cottontail rabbits (Sylvilagus floridanus), and Ixodes dentatus ticks in enzootic cycles of two spirochetes, Borrelia miyamotoi and B. andersonii in a region of Michigan where the zoonotic pathogen B. burgdorferi co-circulates.
Methods
Over a four-year period, wild birds (n = 19,631) and rabbits (n = 20) were inspected for tick presence and ear tissue was obtained from rabbits. Samples were tested for Borrelia spirochetes using nested PCR of the 16S-23S rRNA intergenic spacer region (IGS) and bidirectional DNA sequencing. Natural xenodiagnosis was used to implicate wildlife reservoirs.
Results
Ixodes dentatus, a tick that specializes on birds and rabbits and rarely bites humans, was the most common tick found, comprising 86.5% of the 12,432 ticks collected in the study. The relapsing fever group spirochete B. miyamotoi was documented for the first time in ticks removed from wild birds (0.7% minimum infection prevalence; MIP, in I. dentatus), and included two IGS strains. The majority of B. miyamotoi-positive ticks were removed from Northern Cardinals (Cardinalis cardinalis). Borrelia andersonii infected ticks removed from birds (1.6% MIP), ticks removed from rabbits (5.3% MIP), and rabbit ear biopsies (5%) comprised twelve novel IGS strains. Six species of wild birds were implicated as reservoirs for B. andersonii. Frequency of I. dentatus larval and nymphal co-feeding on birds was ten times greater than expected by chance. The relatively well-studied ecology of I. scapularis and the Lyme disease pathogen provides a context for understanding how the phenology of bird ticks may impact B. miyamotoi and B. andersonii prevalence and host associations.
Conclusions
Given the current invasion of I. scapularis, a human biting species that serves as a bridge vector for Borrelia spirochetes, human exposure to B. miyamotoi and B. andersonii in this region may increase. The presence of these spirochetes underscores the ecological complexity within which Borrelia organisms are maintained and the need for diagnostic tests to differentiate among these organisms.
doi:10.1186/1756-3305-5-231
PMCID: PMC3497883  PMID: 23057837
Ticks; Borrelia miyamotoi; Borrelia andersonii; Ixodes; Wild birds; Eastern cottontail rabbit; Relapsing fever; Lyme disease
18.  Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from The Netherlands. 
Journal of Clinical Microbiology  1995;33(1):119-125.
Sixty-three Borrelia burgdorferi isolates recovered from Ixodes ricinus ticks collected in 17 locations in The Netherlands and three Dutch human skin isolates were characterized by rRNA gene restriction fragment length polymorphism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting). All three human isolates belonged to B. burgdorferi group VS461. Of the tick isolates, 29 (46%) were B. burgdorferi sensu stricto, 2 (3%) were group VS461, 19 (30%) were Borrelia garinii, and 13 (21%) were different from any previously described genomic species. On the basis of the criteria described, 12 isolates formed a distinct genomic group, designated M19. rRNA gene restriction patterns of the group M19 isolates resembled but were not identical to the B. garinii patterns. Hybridization of digested DNA with a flagellin probe confirmed the separation of group M19 from the B. garinii isolates. One isolate, M63, was different from all the others. In conclusion, the occurrence of B. burgdorferi sensu stricto, B. garinii, and B. burgdorferi group VS461 in ticks from The Netherlands corresponds with the occurrence of these genomic species among tick isolates from other European countries. However, our findings suggest that B. burgdorferi sensu lato probably contains more than three genomic species.
PMCID: PMC227892  PMID: 7699027
19.  Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States 
The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
doi:10.1128/AEM.69.8.4561-4565.2003
PMCID: PMC169074  PMID: 12902243
20.  Lyme Disease Borrelia Species in Northeastern China Resemble Those Isolated from Far Eastern Russia and Japan 
Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.
PMCID: PMC106449  PMID: 9647853
21.  Borrelia burgdorferi Genotype Predicts the Capacity for Hematogenous Dissemination during Early Lyme Disease 
The Journal of infectious diseases  2008;198(9):1358-1364.
Background
Lyme disease, the most common tickborne disease in the United States, is caused exclusively by Borrelia burgdorferi sensu stricto in North America. The present study evaluated the genotypes of >400 clinical isolates of B. burgdorferi recovered from patients from suburban New York City with early Lyme disease associated with erythema migrans; it is the largest number of borrelial strains from North America ever to be investigated.
Methods
Genotyping was performed by restriction fragment–length polymorphism polymerase chain reaction analysis of the 16S–23S ribosomal RNA spacer and reverse line blot analysis of the outer surface protein C gene (ospC). For some isolates, DNA sequence analysis was also performed.
Results
The findings showed that the 16S–23S ribosomal spacer and ospC are in strong linkage disequilibrium. Most B. burgdorferi genotypes characterized by either typing method were capable of infecting and disseminating in patients. However, a distinct subset of just 4 of the 16 ospC genotypes identified were responsible for >80% of cases of early disseminated Lyme disease.
Conclusions
This study identified the B. burgdorferi genotypes that pose the greatest risk of causing hematogenous dissemination in humans. This information should be considered in the future development of diagnostic assays and vaccine preparations.
doi:10.1086/592279
PMCID: PMC2776734  PMID: 18781866
22.  Borrelia burgdorferi Spirochetes That Harbor Only a Portion of the lp28-1 Plasmid Elicit Antibody Responses Detectable with the C6 Test for Lyme Disease▿  
Detection of antibody to C6, a peptide that reproduces the sequence of the sixth invariable region within the central domain of the VlsE protein of Borrelia burgdorferi, is used currently for the serologic diagnosis of Lyme disease in humans. B. burgdorferi isolates taken from infected humans can be categorized into specific genetic subtypes (designated RST1, -2, and -3) by restriction fragment length polymorphisms in the 16S to 23S rRNA spacer sequence. Many of these, usually categorized as RST2, retain only segments of the linear plasmid lp28-1, which encodes VlsE. The VlsE genetic region is retained, but altered expression of this molecule could affect diagnosis by the C6 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients infected with each of the three genotypes and from mice infected with three RST2 isolates were tested with the C6 ELISA. Such isolates elicited marked C6 responses in infected mice. The sensitivity of C6 antibody detection in patients infected with RST2 spirochetes was statistically indistinguishable from detection of RST1 and RST3 infections. These findings demonstrate that diagnosis by C6 ELISA remains effective for infection with all B. burgdorferi genotypes, including those with incomplete lp28-1 plasmids.
doi:10.1128/CVI.00266-06
PMCID: PMC1797709  PMID: 17108288
23.  Divergence of Borrelia burgdorferi sensu lato spirochetes could be driven by the host: diversity of Borrelia strains isolated from ticks feeding on a single bird 
Background
The controversy surrounding the potential impact of birds in spirochete transmission dynamics and their capacity to serve as a reservoir has existed for a long time. The majority of analyzed bird species are able to infect larval ticks with Borrelia. Dispersal of infected ticks due to bird migration is a key to the establishment of new foci of Lyme borreliosis. The dynamics of infection in birds supports the mixing of different species, the horizontal exchange of genetic information, and appearance of recombinant genotypes.
Methods
Four Borrelia burgdorferi sensu lato strains were cultured from Ixodes minor larvae and four strains were isolated from Ixodes minor nymphs collected from a single Carolina Wren (Thryothorus ludovicianus). A multilocus sequence analysis that included 16S rRNA, a 5S-23S intergenic spacer region, a 16S-23S internal transcribed spacer, flagellin, p66, and ospC separated 8 strains into 3 distinct groups. Additional multilocus sequence typing of 8 housekeeping genes, clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA was used to resolve the taxonomic status of bird-associated strains.
Results
Results of analysis of 14 genes confirmed that the level of divergence among strains is significantly higher than what would be expected for strains within a single species. The presence of cross-species recombination was revealed: Borrelia burgdorferi sensu stricto housekeeping gene nifS was incorporated into homologous locus of strain, previously assigned to B. americana.
Conclusions
Genetically diverse Borrelia strains are often found within the same tick or same vertebrate host, presenting a wide opportunity for genetic exchange. We report the cross-species recombination that led to incorporation of a housekeeping gene from the B. burgdorferi sensu stricto strain into a homologous locus of another bird-associated strain. Our results support the hypothesis that recombination maintains a majority of sequence polymorphism within Borrelia populations because of the re-assortment of pre-existing sequence variants. Even if our findings of broad genetic diversity among 8 strains cultured from ticks that fed on a single bird could be the exception rather than the rule, they support the theory that the diversity and evolution of LB spirochetes is driven mainly by the host.
doi:10.1186/1756-3305-7-4
PMCID: PMC3892016  PMID: 24383476
Borrelia burgdorferi sensu lato; Ixodes minor; Bird migration; Bird reservoir host; Multilocus sequence analysis; Multilocus sequence typing; Recombinant genotypes; Southeastern United States
24.  Genetic Divergence and Evolutionary Instability in ospE-Related Members of the Upstream Homology Box Gene Family in Borrelia burgdorferi Sensu Lato Complex Isolates 
Infection and Immunity  1998;66(10):4656-4668.
A series of related genes that are flanked at their 5′ ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.
PMCID: PMC108573  PMID: 9746562
25.  rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi. 
Journal of Bacteriology  1992;174(11):3757-3765.
Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.
Images
PMCID: PMC206066  PMID: 1350586

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