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Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii, Borrelia carolinensis, and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.
Spirochetes of the Borrelia burgdorferi sensu lato complex parasitize vertebrates and are transmitted by hard-bodied ticks (Ixodidae) throughout the temperate zones of the northern hemisphere (48). Four species of Ixodes, Ixodes scapularis, Ixodes pacificus, Ixodes ricinus, and Ixodes persulcatus, account for the majority of Ixodes species-vectored human disease. Certain Ixodes ticks are host specific, whereas others are not. Those with nonspecific feeding habits, (e.g., I. scapularis, I. pacificus, I. ricinus, and I. persulcatus) not only feed on species that are competent reservoirs for multiple tick-borne pathogens but also readily bite humans. The list of potential reservoir hosts is great and variable and includes species in classes Mammalia, Aves, and Reptilia (9, 15). The worldwide distribution of B. burgdorferi sensu lato may be caused by long-distance dispersal of infected birds that serve as hosts for ticks (11, 46, 53).
The blacklegged tick, I. scapularis, is the main vector of B. burgdorferi sensu lato for humans in the eastern half of the United States, both in the northeastern and southern parts, while I. pacificus is the main vector in the far-western part of the United States. Other species of Ixodes known to be naturally infected and to transmit B. burgdorferi among wildlife include Ixodes spinipalpis (formerly Ixodes neotomae) (38), Ixodes jellisoni (25), and Ixodes angustus (47) in the western United States and Ixodes dentatus, Ixodes affinis, and Ixodes minor in the eastern part of the country (39). The non-human-biting tick I. spinipalpis serves as a maintenance vector, and I. pacificus serves as the “bridge” vector for humans. In some areas of the southeastern United States, I. minor (which usually does not bite humans) appears to be more important as a maintenance vector in the enzootic cycle of B. burgdorferi sensu lato than the “bridge” vector I. scapularis, which feeds on nonhuman species and humans. The role of I. minor and I. affinis and several species of birds in the enzootic cycle of B. burgdorferi in the southern United States is currently under investigation (J. H. Oliver, Jr., unpublished data).
Data generated during the last decade demand reevaluation of the previously held concepts about Lyme borreliosis in the United States, particularly in the western and southern United States. Recent results confirm the presence of well-established populations of B. burgdorferi sensu stricto, Borrelia andersonii, Borrelia bissettii, and the recently described Borrelia carolinensis in the southern part of the country (29-32, 39-45, 58). Now we present data that support delineation of another species from the B. burgdorferi sensu lato complex, Borrelia americana sp. nov. B. americana includes American strains isolated in California from 1989 to 1991 that were designated as genomospecies 1 in 2007 and South Carolina strains isolated from 1994 to 1995 and identified as Borrelia sp. nov. in 2007. The enzootiology of Lyme disease in California differs fundamentally from that reported in the northeastern United States but is quite similar to the enzootiology of Lyme borreliosis in the southeastern region of the United States. The explanation for the presence of identical strains of the same species, B. americana sp. nov., on opposite sides of the United States (California and South Carolina) is an enigma worthy of further investigation.
Seven Borrelia strains were isolated from nymphs of the hard tick I. minor collected from birds in November 1994 (four strains) and May 1995 (three strains) on the Wedge Plantation, Charleston County, South Carolina. Five of seven nymphs (strains SCW-30e, SCW-30g, SCW-41, SCW-42b, and SCW-42c) were collected from Carolina wrens (Thryothorus ludovicianus), and two nymphs (strains SCW-32 and SCW-33) were collected from Eastern towhees (Pipilo erythrophthalmus). Borrelia isolates from I. minor were cultured in Barbour-Stoenner-Kelly-H medium supplemented with 6% rabbit serum, 0.15% agarose (SeaKem; FMC BioProducts, Rockland, ME), antibiotics (rifampin, phosphomycin), and fungicide (amphotericin B). The cultures were incubated in 5% CO2 at 33°C. When the cultures reached a cell density of 2 × 106 spirochetes/ml, they were stored at −80°C.
Seven Borrelia strains analyzed in this paper were from a group of 118 Borrelia isolates cultured from a variety of rodents, birds, and ticks collected in the southern United States from 1993 until 1999. DNA purification, PCR amplification, sequencing, primers used for analysis of borrelia sequences, analyses of borrelia sequences, and phylogenetic analysis were conducted according to our previously described scheme (58). Multilocus sequence analysis of the rrf-rrl intergenic spacer, 16S rRNA genes, the gene encoding flagellin, p66, and ospA were used to clarify the taxonomic status of this group of B. burgdorferi sensu lato isolates. Briefly, total borrelia DNA was purified using the DNeasy blood and tissue kit (Qiagen) strictly according to the manufacturer's recommendations. The MasterTaq kit (Eppendorf, Germany) was used for amplification of selected genes using the following previously described gene-specific primers: 5S(rrf)F-5′-CTGCGAGTTCGCGGGAGA-3′ and 23S(rrl)R-5′-TCCTAGGCATTCACCATA-3′ for amplification of the rrf-rrl intergenic spacer region (IGS) (51), 16SMF-5′-AGAGTTTGATCCTGGCTTAG-3′ and 16SMR-5′-CCTCCCTTACGGGTTAGAA-3′ for amplification of 16S rRNA (17), Fla out F-5′-AARGAATTGGCAGTTCAATC-3′ and Fla out R-5′-GCATTTTCWATTTTAGCAAGTGATG-3′ for amplification of the gene encoding flagellin (10), outer1-5′-CGAAGATACTAAATCTGT-3′ and outer2-5′-GCTGCTTTTGAGATGTGTCC-3′ for amplification of p66 (10), and N1-5′-GAGCTTAAAGGAACTTCTGATAA-3′ and C1-5′-GTATTGTTGTACTGTAATTGT-3′ for amplification of ospA (18). PCR products of the expected size were cut off the gel, purified, and submitted in 96-well skirted PCR plates for direct sequencing to the University of Washington High-Throughput Genomics Unit (Seattle, WA). Sequencing was conducted in both directions, using the same specific primers that were used for amplification of each gene. All sequences were analyzed with DNAStar software (DNAStar, United Kingdom). Database searches were conducted with the BLAST programs of the NCBI (Bethesda, MD). Restriction fragment length polymorphism (RFLP) analysis of borrelia sequences was done in silico (4). The rrf-rrl IGS was digested with MseI and DraI restriction endonucleases; the gene encoding flagellin (14) was digested with HapII, HhaI, HincII, CelII, and DdeI. All obtained RFLP patterns were compared with the previously published patterns (13, 51, 64). Available sequences from the same loci (rrf-rrl, 16S rRNA, fla, p66, ospA) of all 14 control species of the B. burgdorferi sensu lato complex and genomospecies 1 and 2 were used as controls in phylogenic analysis. The alignments were done with ClustalX (version 1.81) (62). Identical sequences were excluded from analyses. Alignment was edited manually using BioEdit 7.0.9 (19), and unaligned and ambiguously aligned positions were deleted from the data set. Phylogenetic reconstruction for the molecular data was inferred by using a maximum-parsimony heuristic search performed in PAUP* 4.0, beta version 10. The best model for DNA evolution was calculated by ModelTest (49). A maximum-likelihood phylogenetic tree was inferred using a GTR+Γ+I (with gamma distribution in four categories) model in PhyML 3.0 (16). Branch support was calculated by bootstrap analysis (500 replicates for molecular data set).
Sequences determined in this study have been deposited into GenBank and given the indicated accession numbers as follows: EU076517 to EU076523 for the rrf-rrl IGS, EU081282 to EU081288 for the 16S rRNA gene sequences, EU081289 to EU081295 for the fla gene sequences, EU076524 to EU076530 for the p66 gene sequences, and EU081296 for the ospA gene sequences of isolate SCW-33.
All seven cultivated isolates reported here proved to be free from bacterial or yeast contamination. Repeated double-directional sequencing of PCR products from five selected loci gave identical results for every discussed B. americana isolate, confirming in this way the presence of one Borrelia species in each culture. The type strain SCW-41T and strain SCW-42c were deposited into the American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSMZ, Germany).
The exact sizes of the 5S-23S IGS of the tested isolates were determined by direct sequencing of the purified amplicons. Analysis of B. americana sequences showed the existence of two types of IGS with the size of 254 nt (SCW-30e, SCW-30g, SCW-32, and SCW-41) and 253 nt (SCW-33, SCW-42b, and SCW-42c). RFLP patterns of the B. americana IGS differ from any known for B. burgdorferi sensu lato species but are similar to those of California strains CA-8B-89 and CA-29-91, known as genomospecies 1, and consist of six fragments after digestion by MseI (107, 52/51, 38, 28, 16, 13) and four fragments after restriction with DraI (144, 81/80, 16, 13).
The similarity matrix revealed great conservation in the case of sequences of the gene encoding flagellin. Strains SCW-32, SCW-33, SCW-41, SCW-42b, and SCW-42c were 100% identical on a protein level. Strains SCW-30e and SCW-30g were 100% identical to each other and differ from the rest of the B. americana strains by having isoleucine instead of methionine at amino acid position 160. The DdeI RFLP pattern of the B. americana fla gene is unique and has not been described previously (13). Restriction endonucleases HapII, HhaI, HincII, and CelII had no sites on the fla amplicon of B. americana.
In B. americana strains, unique signature nucleotides were detected at position 168 (G168) in 16S rRNA sequences of isolates from both subgroups (A and B) and at position 219 (A219) in isolates from subgroup B (SCW-42b, SCW-42c, and SCW-33). The same unique signature nucleotides at G168 and A219 were detected in 16S rRNA amplicons of borrelia genomospecies 1 isolates only. B. burgdorferi B31 was used as a “baseline” of the alignment, and the signature nucleotide positions were numbered according to the full rrs sequence of this species.
Analysis of the adjusted region of the p66 gene confirmed definite separation of B. americana isolates into two subgroups: strains SCW-30e, SCW-30g, SCW-32, and SCW-41 comprised subgroup A, and SCW-33, SCW-42b, and SCW-42c comprised subgroup B, as was already shown by the RFLP pattern of IGS.
Analysis of B. americana ospA amplicons adjusted in size to available sequences showed the ospA fragment having highest similarity to genomospecies 1 (96.8%). The next-highest similarity of 91.4% was confirmed for both genomospecies 2 and Borrelia spielmanii strain A14S.
The distance matrix (1,827 to 1,835 nt) was generated from the adjusted-in-size and concatenated sequences of the partial fla gene (269 nt), the partial 16S rRNA gene (1,311 nt), and the IGS (247 to 255 nt) of B. americana strains, 14 control species from B. burgdorferi sensu lato complex, and genomospecies 1 and 2. Analysis of the concatenated sequences showed that B. americana strains clustered together with genomospecies 1 and separately from any other species (Fig. (Fig.1).1). The addition of a 269-nt amplicon of the p66 gene and a 93-nt amplicon of the ospA sequence to the concatenated sequences confirmed the separation of all spirochete species into two groups. One group includes species from Eurasia (Borrelia afzelii, Borrelia garinii, Borrelia japonica, Borrelia lusitaniae, B. spielmanii, Borrelia turdi, and Borrelia valaisiana), whereas the other group includes species typically found in the United States (B. andersonii, B. bissettii, Borrelia californiensis, B. carolinensis, genomospecies 1 and 2, and B. americana that formed the separate cluster with genomospecies 1 in the group of American species [data not shown]). B. burgdorferi B31 was linked to both groups. The results of analyses of five different genomic loci in B. americana strains as well as phylogenetic analysis confirmed once again that isolates of B. americana were closely associated with genomospecies 1 and constitute a new taxon in the B. burgdorferi sensu lato complex.
In conclusion, using the results of molecular and phylogenetic analyses, we delineate a 15th named genomospecies in the B. burgdorferi sensu lato complex, Borrelia americana. It is comprised of the strains isolated in California, formerly named genomospecies 1, and Borrelia sp. nov. strains isolated in South Carolina.
There are 14 Borrelia burgdorferi sensu lato species recognized today worldwide, including 9 strictly associated with Eurasia (B. afzelii, B. garinii, B. japonica, B. lusitaniae, B. spielmanii, Borrelia sinica, Borrelia tanukii, B. turdi, and B. valaisiana), 3 associated only with the United States (B. andersonii, B. californiensis, and B. carolinensis) (2, 8, 12, 21, 22, 28, 33, 36, 50, 52, 55, 58, 63), and 2 present in both the Old and New World (B. burgdorferi sensu stricto and B. bissettii) (20, 35, 54, 56, 57, 61). The last decade of the 20th century brought a significant increase in the number of identified Borrelia strains in the United States (1, 5, 34, 37, 51, 66). During 1992 to 1993, isolation of four unassigned American Borrelia sp. strains in California from I. pacificus and I. spinipalpis were reported (51, 60, 65). In 2007, the MLSA analysis of these strains confirmed their clustering into two groups distant from any other known species. They were proposed as genomospecies 1 and genomospecies 2 but were not named because of insufficient information and a low number of isolates (50). Publication of the results mentioned above caused some confusion when the authors shortened the full designations of the isolates they received from the laboratory where strains were originated. Both isolates comprising genomospecies 1 group were originally isolated from I. pacificus by Tom Schwan (National Institutes of Health [NIH], Rocky Mountain Laboratories), and their original designations were CA-8B-89 and CA-29-91. Isolate CA-28-91 representing genomospecies 2 was isolated at the same laboratory. The second representative of genomospecies 2, the CA-2 strain, isolated from I. spinipalpis and first mentioned in 1989, originated in the Robert Lane laboratory (University of California, Berkeley), as was documented by our literature search (23, 27). Genomospecies 1 strains CA-8B-89 and CA-29-91 were 98.9% related to one another (50), which corresponds to the sequence similarity between B. americana subgroups A and B in most of the loci analyzed (see Results). The genetic distance among B. americana and genomospecies 1 strains ranged from 0.008 to 0.011 in 5S-23S IGS, corresponding to 98.8% to 99.2% sequence similarity, and 0.001 to 0.003 in the 16S rRNA locus, corresponding to 99.7% to 99.9% sequence similarity. No additional sequences in databanks clustered with genomospecies 1 isolates until now. A recent GenBank search showed that 16S rRNA genes of the other isolates, SCCH-5 (EF570071), SCW-30f (EF570069), and SCW-42a (EF570073), collected in South Carolina previously (T. Lin, L. Gao, and J. H. Oliver, Jr., unpublished data) share the unique signature nucleotides G168 and A219 at positions 168 and 219 as determined for B. americana isolates and are 100% identical to them. Assignment of isolates SCCH-5, SCW-30f, and SCW-42a to B. americana must be taken as preliminary as it is based on the analysis of a single 16S rRNA locus. Recently published results of RFLP analysis of 5S-23S IGS of isolate SCCH-5 (43) support the hypothesis of its belonging to the newly described species B. americana, but a definitive conclusion requires additional analysis. Data presented in this paper confirm the validity of a single new species, B. americana sp. nov, consisting of California and South Carolina isolates and suggest that populations of B. americana are well established in the southeastern region of the United States. The enzootiology of Lyme disease in California likewise is more complicated and differs fundamentally from that reported from the northeastern United States where the typical I. scapularis vector and Peromyscus leucopus reservoir cycle predominates (44). Dusky-footed wood rats and western gray squirrels, not mice, serve as the primary reservoir hosts, and I. pacificus and the non-human-biter I. spinipalpis (formerly Ixodes neotomae) maintain B. burgdorferi and B. bissettii, respectively, in overlapping but discrete enzootic cycles in California (6, 7, 24, 26, 59). Isolates of B. burgdorferi from California exhibit considerable heterogeneity, and some isolates differ strikingly from isolates recovered from this and other geographic regions (6, 7). This situation is analogous to the enzootiology of Lyme borreliosis in the southeastern United States. There I. minor, a non-human-biting tick, appears to be more important in some areas as a maintenance vector in the enzootic cycle of B. burgdorferi sensu lato than the “bridge” vector I. scapularis (39; J. H. Oliver, Jr., C. W. Banks, K. L. Clark, and A. M. James, unpublished data). In addition to the major reservoir hosts, Peromyscus gossypinus and Sigmodon hispidus, the impact of the eastern wood rat Neotoma floridana, several bird species, and the vector I. minor in the enzootic cycles in the southeastern region was confirmed (41, 42). There could be either several enzootic separate parallel cycles or overlapping cycles operating. For example, there could be separate wood rat and I. minor cycles or a bird and I. minor cycle. Alternatively, because I. minor feeds on birds, wood rats, cotton mice, cotton rats, gray squirrels, spotted skunks, and other small mammals, the two cycles might not be separate but might form a weblike overlap (39). The seven Borrelia isolates described in this paper were isolated from I. minor nymphs that were feeding on two bird species. The Carolina wren (Thryothorus ludovicianus) is a common species in the eastern part of the United States, and the Eastern towhee (Pipilo erythrophthalmus) breeds in brushy areas across eastern North America. All strains from the new genomospecies group that we named Borrelia americana sp. nov. are associated with I. pacificus and I. minor and their rodent and bird hosts. The vertebrate host(s) of B. americana in California is still unknown.
It is not known whether B. americana can infect humans. All strains isolated from humans in the United States so far belong to B. burgdorferi sensu stricto. Both I. spinipalpis and I. minor are primarily non-human-biting ticks and rarely attach to humans; this suggests that they do not transmit Borrelia americana to humans. But I. pacificus and I. scapularis bite humans and feed on the same hosts as I. spinipalpis and I. minor. Isolation of B. americana from I. pacificus confirms that this human-biting tick can acquire this species from hosts, so theoretically it might transmit B. americana to humans (65). B. americana appears to be cycling silently among wild animals.
B. burgdorferi sensu lato is endemic to many foci over large areas of the southeastern United States, but rather small numbers of human cases are reported, and presumably many Lyme infections go undiagnosed or neglected—especially if the characteristic skin rash does not occur initially. Most methods used in the laboratory confirmation of Lyme disease involve detection of serum antibodies. Inconsistent or negative serological findings might be related to the use of primarily B. burgdorferi sensu stricto as the antigen, the genetic profile of which might differ markedly from any other spirochete species that might possibly be involved in Lyme disease in some parts of the United States or possibly even to different ospC genotypes comprising this spirochete.
A description of Borrelia americana sp. nov. is as follows: amer.i.'ca.na. L. fem. adj. americana, referring to the United States of America, where the organism was first isolated. Morphology is as described previously for the genus (3). Cultural properties are as described for B. burgdorferi sensu lato (21). B. americana was differentiated from other B. burgdorferi sensu lato species by multilocus sequence analysis of five genomic loci and phylogenetic analysis. RFLP patterns of 5S-23S IGS of B. americana consist of six fragments after digestion by MseI (107, 52/51, 38, 28, 16, 13) and four fragments after digestion by DraI (144, 80/81, 16, 13) and contain unique signature nucleotides in the 16S rRNA gene (G168 and A219). B. americana was isolated from I. minor nymphs collected in South Carolina from two bird species that are hosts of Borrelia in the United States, the Carolina wren (Thryothorus ludovicianus) and the Eastern towhee (Pipilo erythrophthalmus). The type strain, SCW-41Τ, has been deposited into the ATCC and DSMZ (Germany).
We are grateful to Robert S. Lane and Tom Schwan for their comments, suggestions, and help in improving our manuscript. We thank Tomaš Chrudimský for his help and advice in phylogenetic analysis of samples.
This research was supported in part by grant R37AI-24899 from the NIH and cooperative agreement U50/CCU410282 from the Centers for Disease Control and Prevention (CDC). This work was also partially supported by the grants MSM 6007665801, LC06009 (Ministry of Education of CR), and Z60220518 (Institute of Parasitology AS CR).
The opinions expressed are the responsibility of the authors and do not necessarily represent the official views of the NIH or CDC.
Published ahead of print on 21 October 2009.