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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Ticks Tick Borne Dis. 2010 Dec 1;1(4):151–158. doi: 10.1016/j.ttbdis.2010.09.002

Multilocus sequence analysis of Borrelia bissettii strains from North America reveals a new Borrelia species, Borrelia kurtenbachii

Gabriele Margos a,*, Andrias Hojgaard b, Robert S Lane c, Muriel Cornet d, Volker Fingerle e, Nataliia Rudenko f, Nicholas Ogden g, David M Aanensen h, Durland Fish i, Joseph Piesman b
PMCID: PMC3000690  NIHMSID: NIHMS245956  PMID: 21157575

Abstract

Using multilocus sequence analyses (MLSA), we investigated the phylogenetic relationship of spirochaete strains from North America previously assigned to the genospecies Borrelia bissettii. We amplified internal fragments of 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) located on the main linear chromosome by polymerase chain reaction. Phylogenetic analysis of concatenated sequences of the 8 loci showed that the B. bissettii clade consisted of 4 closely related clusters which included strains from California (including the type strain DN127-Cl9-2/p7) and Colorado that were isolated from Ixodes pacificus, I. spinipalpis, or infected reservoir hosts. Several strains isolated from I. scapularis clustered distantly from B. bissettii. Genetic distance analyses confirmed that these strains are more distant to B. bissettii than they are to B. carolinensis, a recently described Borrelia species, which suggests that they constitute a new Borrelia genospecies. We propose that it be named Borrelia kurtenbachii sp. nov. in honour of the late Klaus Kurtenbach. The data suggest that ecological differences between B. bissettii and the new Borrelia genospecies reflect different transmission cycles. In view of these findings, the distinct vertebrate host-tick vector associations and the distributions of B. bissettii and B. kurtenbachii require further investigation.

Keywords: Borrelia bissettii, Borrelia kurtenbachii sp. nov, Ixodes, Multilocus sequence analysis, Molecular ecology

Introduction

Lyme borreliosis (LB) is caused by several species of bacteria belonging to the LB group of spirochaetes, also referred to as Borrelia burgdorferi sensu lato (s.l.) species complex. These bacteria are maintained in nature by numerous vertebrate hosts and are transmitted by ticks in the Ixodes persulcatus species complex (reviewed by Kurtenbach et al., 2006). Today, LB is the most frequently reported vector-borne disease in the temperate zones of the Northern Hemisphere. In the United States, human cases exceeding 20,000 per year currently are being reported with 90% of the cases occurring in 2 regional foci, the Northeast and Upper Midwest. Human cases are far less frequently reported in the western and southern regions of the country (Bacon et al., 2008). In North America, B. burgdorferi sensu stricto (hereinafter referred to as B. burgdorferi) is the sole species associated with human disease (Mathiesen et al., 1997; Wormser et al., 2008). The reasons for the skewed spatial distribution of LB cases in the United States are not entirely clear but may be due to ecological factors, such as the abundance of, and spirochaete infection prevalences in, tick vectors, the presence or absence of disseminating, disease-causing B. burgdorferi genotypes, differences in tick host-seeking behaviour (in particular the immature stages), or possibly other factors such as land-use changes and regional differences in human behaviour (Burgdorfer et al., 1985; Oliver, 1996; Lane and Quistad, 1998; Wright et al., 2000; Slowik and Lane, 2001; Lane et al., 2004; Diuk-Wasser et al., 2006; Eisen et al., 2006; Killilea et al., 2008; Gatewood et al., 2009; Hamer et al., 2010; Humphrey et al., 2010).

The aetiological agent of LB was identified in 1981 and named Borrelia burgdorferi after its discoverer, Willy Burgdorfer (Burgdorfer et al., 1982; Johnson et al., 1984). Over the following 2 decades, many isolates derived from human patients, ticks, or reservoir host animals were analysed biochemically and genetically, and a high diversity within B. burgdorferi was found which led to the delineation of new genospecies in Europe, Asia, and North America (Baranton et al., 1992; Kawabata et al., 1993; Maupin et al., 1994; Postic et al., 1994; Marconi et al., 1995; Fukunaga et al., 1996; Le Fleche et al., 1997; Mathiesen et al., 1997; Wang et al., 1997, 1999; Postic et al., 1998; Masuzawa et al., 1999, 2001; Lin et al., 2001). The LB group of spirochaetes now forms a species complex consisting of 17 named genospecies (Kurtenbach et al., 2010) reflecting the complex ecology of these bacteria (Kurtenbach et al., 2006). Of the named species, 6 occur in North America including B. andersonii, B. bissettii, B. californiensis, B. carolinensis, B. americana, and B. burgdorferi. The true species richness doubtless is higher as several strains phylogenetically distinct from all other species have not yet been named (Postic et al., 1998, 2007).

B. bissettii enzootic transmission cycles in the United States involving tick species such as I. spinipalpis, I. minor, I. affinis, and various vertebrate host species were found in Colorado, California, and some Southern states (Bissett and Hill, 1987; Brown and Lane, 1992; Maupin et al., 1994; Lane and Quistad, 1998; Schneider et al., 2000; Oliver et al., 2003). In 1990, Anderson and coworkers described a non-pathogenic variant of B. burgdorferi, strain 25015. This strain was isolated from 4 blood-engorged I. scapularis larvae (then termed I. dammini) that had been removed from 3 Peromyscus leucopus mice collected in Dutchess County, New York, in 1987 (Anderson et al., 1988, 1990). In 1998, the species B. bissettii was delineated using restriction patterns of the rrf-rrl intergenic spacer as well as sequences of the intergenic spacer and 16S rDNA. Samples included mainly strains isolated from I. pacificus or I. neotomae (now I. spinipalpis) in California (Postic et al., 1998). Strain 25015 was included in the genospecies B. bissettii, although differences in rrf-rrl restriction patterns between strain 25015 and DN127 were observed (Mathiesen et al., 1997; Postic et al., 1998; Lin et al., 2003). In 1996, 1997, and 1998, Borrelia isolates were obtained from the white-footed mouse (Peromyscus leucopus), the meadow vole (Microtus pennsylvanicus), and the meadow jumping mouse (Zapus hudsonius) in forested, suburban areas of Chicago, Illinois (Picken and Picken, 2000). These isolates were compared to B31, DN127, and 25015 by RFLP analysis. They were designated B. bissettii because most of them exhibited a restriction pattern similar to that of strain 25015, and 2 strains exhibited a macrorestriction pattern like that of the type strain DN127 (Picken and Picken, 2000).

DNA-DNA hybridization and sequence analyses of the 16S rRNA locus were used as standard procedures to delineate bacterial species for many years, but were not without drawbacks (Gevers et al., 2005; Stackebrandt and Ebers, 2006). The invention of multilocus sequence typing (MLST) during the late 1990s (Maiden et al., 1998; Enright and Spratt, 1999; Spratt, 1999; Feil and Enright, 2004) led to the development of multilocus sequence analysis (MLSA), a tool that has been used since to resolve inter-species relationships of bacteria and to delineate bacterial species (Gevers et al., 2005; Bishop et al., 2009). Several different multiple-loci schemes have been used for the LB group of spirochaetes (Richter et al., 2006; Postic et al., 2007; Margos et al., 2008; Rudenko et al., 2009a, 2009b). One of them employs housekeeping genes (Margos et al., 2008) and resembles typical MLSA schemes reported for other microorganisms (see http://www.mlst.net).

Here, we used this MLSA system (Margos et al., 2008, 2009) to investigate the phylogenetic relationships among B. bissettii strains isolated from reservoir hosts or ticks removed from them in different regions of North America. The data confirm that B. bissettii forms a heterogeneous clade (Postic et al., 1998). We also demonstrate that strain 25015 and several other strains collected in Illinois and Nova Scotia that were assigned previously to B. bissettii represent a phylogenetically distinct group, and we propose to name this group Borrelia kurtenbachii sp. nov.

Materials and methods

Tick collection and isolation of LB spirochaetes

The samples used in this study are listed in Table 1. They mainly represent cultured isolates principally obtained from ticks that were collected from rodents trapped in California, Colorado, Illinois, and New York. Isolation of LB spirochaetes followed standard procedures (Maupin et al., 1994). One sample, NS07-121, was not a cultured isolate but DNA-extracted from a nymphal I. scapularis collected in Nova Scotia from a human in 2007.

Table 1.

Borrelia isolates evaluated during this study.

ST Strain ID Country of origin Region Species Biological origin Year of collection Collected by
156 CA128 USA Hopland Field Station, Mendocino County, CA B. bissettii Ixodes neotomae (now known as I. spinipalpis) ex Neotoma fuscipes 1991 R.S. Lane
282 CA370 USA Alameda County, CA B. bissettii N. fuscipes ear biopsy 1992 R.S. Lane
283 CA371 USA Alameda County, CA B. bissettii N. fuscipes ear biopsy 1992 R.S. Lane
270 CA389 USA Tilden Regional Park, Berkeley, CA B. bissettii I. pacificus 1993 R.S. Lane
272 DN127-Cl9-2/p7 USA Del Norte County, CA B. bissettii I. pacificus 1985 J.R. Clover
gom93-267a USA Larimer County, CO B. bissettii I. spinipalpis ex Neotoma mexicana 1993 Gary O. Maupin
273 gom93-268 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-270a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-271a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-272a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
273 gom93-274 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
273 gom93-275 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-277a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
273 gom93-278 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-280a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
160 gom93-283 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
273 gom93-284 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
274 gom93-286 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
273 gom93-287 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-289a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
271 gom93-296 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
273 gom93-297 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-298a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
158 gom93-299 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
gom93-300a USA Larimer County, CO B. bissettii I. spinipalpis ex Peromyscus difficilis 1993 Gary O. Maupin
gom93-304a USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
275 gom93-305 USA Larimer County, CO B. bissettii I. spinipalpis ex P. difficilis 1993 Gary O. Maupin
276 gom93-310 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
277 gom93-501 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
277 gom93-543 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
277 gom93-544 USA Larimer County, CO B. bissettii I. spinipalpis ex N. mexicana 1993 Gary O. Maupin
278 IL 96-255 USA Poplar Creek, Cook County, IL B. kurtenbachiib Microtus pennsylvanicus 1996 R. Picken
279 IL 97-236u USA Poplar Creek, Cook County, IL B. kurtenbachiib M. pennsylvanicus 1996 R. Picken
280 25015 USA New York State B. kurtenbachiib I. scapularis 1987 J.F. Anderson
281 NS07-121 Canada Nova Scotia B. kurtenbachiib I. scapularis 2007 N. Ogden
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a

These strains showed mixed infections and could not be included into phylogenetic and multilocus sequence analysis.

b

These strains form a distinct group that is genetically sufficiently distant from B. bissettii to represent a new species. We propose the name Borrelia kurtenbachii spec. nov. in honour of Klaus Kurtenbach.

Molecular methods and MLST analyses

Borrelia DNA was extracted from cultured isolates or frozen stocks using DNA STAT-60 (Tel-Test, Friendswood, TX, USA). Briefly, cells were lysed in 1 ml of DNA isolation reagent and 0.2 ml chloroform. After centrifugation (12,000 × g for 15 min at 4°C), the upper layer was isolated and added to an equal volume of isopropanol, followed by incubation at room temperature for 5 min. The sample was centrifuged at 12,000 × g for 10 min at 4°C, followed by a wash in 75% ethanol. After drying, the sample was resuspended in 50 μl H2O. For strain NS07-121, no isolation was attempted, but tick and Borrelia DNA was purified as described earlier (Ogden et al., 2010).

Amplification of the 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) was conducted by nested PCR using primers and conditions principally as described previously (Margos et al., 2009; Ogden et al., 2010; see also http://borrelia.mlst.net). Template DNA was diluted to 10 pg/μl, and 2 μl were used in a total reaction volume of 15 μl for the first amplification step except for clpA for which 4 μl of template DNA were used instead. The nested step of amplification was slightly modified: HotStarTaq Mastermix (Qiagen, Germany) was used and 1 pmol/μl of primer in a final reaction volume of 30 μl. Three μl from the first amplification step were used as template DNA. A touchdown PCR starting at 58°C with a decrease of 1°C at each cycle until 50°C was used for the second amplification step, followed by 34 cycles of amplification at an annealing temperature of 50°C. All PCR products were sequenced in both forward and reverse directions. Quality control of DNA traces was conducted manually in DNASTAR (Lasergene plc, USA). Sequences with more than one base peak at the same position in forward and reverse sequence were considered mixed isolates and removed from further analysis.

Sequences of individual housekeeping genes were compared to each other and to sequences in the MLST database. Sequences were assigned allelic numbers, and novel sequences received consecutive numbers. New consecutive sequence type numbers were assigned to allelic profiles with novel combinations or containing novel alleles (Table 2) (Margos et al., 2008). Sequences were aligned and concatenated in Mega 4.0 (Kumar et al., 2004), which also was used to determine genetic distances and to generate a neighbour-joining tree using the Maximum Composite Likelihood model. All sequences are available on the MLST website, http://borrelia.mlst.net, hosted at Imperial College London, U.K.

Table 2.

Sequence types and allelic profiles of B. bissettii and B. kurtenbachii isolates.

Strain ST clpA clpX nifS pep pyrG recG rplB uvrA
DN127T, Cl9-2/p7 272 117 69 68 102 99 99 83 89
CA128 156 91 69 68 81 77 73 69 73
CA389 270 118 70 83 103 100 100 84 90
CA370 282 119 83 88 81 101 101 89 73
CA371 283 120 69 68 104 99 74 83 91
Gom93-268 273 121 84 83 103 102 102 84 92
Gom93-274 273 121 84 83 103 102 102 84 92
Gom93-275 273 121 84 83 103 102 102 84 92
Gom93-278 273 121 84 83 103 102 102 84 92
Gom93-283 160 92 70 69 82 78 74 70 74
Gom93-284 273 121 84 83 103 102 102 84 92
Gom93-286 274 92 70 69 105 78 103 84 93
Gom93-287 273 121 84 83 103 102 102 84 92
Gom93-296 271 118 70 83 103 100 100 84 94
Gom93-297 273 121 84 83 103 102 102 84 92
Gom93-299 158 93 71 70 83 79 74 71 75
Gom93-305 275 122 70 84 106 103 104 84 94
Gom93-310 276 92 70 69 105 78 74 84 93
Gom93-501 277 123 70 85 103 100 100 84 95
Gom93-543 277 123 70 85 103 100 100 84 95
gom93-544 277 123 70 85 103 100 100 84 95
IL 96-255 278 124 85 86 107 104 105 85 96
IL 97-236u 279 125 86 87 108 105 106 86 97
25015 280 126 87 87 109 106 107 87 97
NS07-121 281 127 88 87 107 105 108 88 98
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Results

A total of 33 strains designated previously as B. bissettii (Postic et al., 1998; Picken and Picken, 2000) was analysed by MLST. Of these isolates, 30% (n=10) contained mixed infections probably reflecting isolation from hosts infected with multiple strains. This included the type strain DN127, but further analysis of a cloned isolate (cl9-2/p7; 2/18/93) yielded unambiguous results. The remaining 23 samples fell into 2 clades. One clade consisted of 4 closely related clusters from California (i.e., CA128, CA389, CA370, CA371, and DN127-Cl9-2/p7) and Colorado (all strains isolated in 1993, designated here gom93-267 to gom93-544). The second clade was genetically distant and included strain 25015 (Fig. 1, Table 3). The pair-wise genetic similarities of strains in the first clade were >98.5% (Table 3), which is above the genetic distance used to designate distinct genospecies in the MLST scheme used here (i.e. 98.3%) (Margos et al., 2009). Some isolates of different geographic origin (i.e. Colorado – isolate gom93-299, California – DN127-Cl9-2/p7) have a high degree of similarity, and therefore occurred in the same cluster (Fig. 1). The number of alleles for the B. bissettii strains found in individual genes varied from 4 to 9 (Table 4). The G+C contents of the gene fragments ranged from 26.6 to 36.5% and are close to the average G+C contents of the B. burgdorferi main chromosome (Fraser et al., 1997). The nucleotide diversity per site was particularly high for the pyrG locus (Table 4) due to high diversity of this gene in DN127-Cl9-2/p7.

Fig. 1.

Fig. 1

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Phylogenetic analysis of Lyme borreliosis (LB) genospecies. The tree was generated using concatenated sequences of 8 chromosomally located housekeeping gene fragments. For the phylogenetic reconstruction, the maximum composite likelihood model available in MEGA 4 was used with 1000 bootstrap replicates. Scale bar indicates 1% divergence.

Table 3.

Genetic distances of LB spirochaetes. The lower left panel shows genetic distances as calculated in MEGA 4.0 using Kimura-2 parameter Model. The upper right panel shows percent genetic similarity, calculated by the fomula: 100 − (genetic distance × 100). B. bissettii strains are highlighted in yellow, B. kurtenbachii strains are highlighted in blue, percent genetic distance between B. bissettii and B. kurtenbachii are highlighted in purple.

DN127 CA128 CA389 CA370 CA371 gom93-268 gom93-274 gom93-275 gom93-278 gom93-283 gom93-284 gom93-286 gom93-287 gom93-296 gom93-297 gom93-299 gom93-305 gom93-310 gom93-501 gom93-543 gom93-544 IL 96-255 IL 97_236u 25015 NS07-121 SCJ1 CA443 QLM4P1 B31 VS461 20047 VS116 B. andersoni A14S PBi CA2 CA8 PoTiBL37 HK501 HO14 Ya501
DN127,cl9-2/p7- B. bissettii 98.97 98.54 99.5 99.81 98.71 98.71 98.71 98.71 98.58 98.71 98.58 98.71 98.52 98.71 99.05 98.45 98.6 98.34 98.34 98.34 96.5 96.55 96.37 96.53 97.32 95.47 90.56 94.13 91.81 92.03 91.33 93.36 90.97 91.91 94.54 94.04 91.82 91.72 91.71 91.53
CA128 0.0103 98.86 99.1 99.12 98.9 98.9 98.9 98.9 98.73 98.9 98.73 98.9 98.88 98.9 99.03 98.77 98.75 98.71 98.71 98.71 96.84 96.88 96.7 96.86 97.69 95.42 90.56 94.1 94.191.56 91.98 91.23 93.36 90.75 91.89 94.24 93.99 91.7 91.72 91.53 91.41
CA389 0.0146 0.0114 98.63 98.6 99.41 99.41 99.41 99.41 99.16 99.41 99.16 99.41 99.98 99.41 98.86 99.54 99.18 99.81 99.81 99.81 96.61 96.65 96.55 96.63 97.87 95.26 90.23 93.91 91.45 91.72 90.98 93.14 90.84 91.57 93.75 93.78 91.48 91.59 91.28 91.41
CA370 0.005 0.009 0.0137 99.6 98.84 98.84 98.84 98.84 98.67 98.84 98.67 98.84 98.65 98.84 99.1 98.63 98.69 98.48 98.48 98.48 96.6 96.64 96.46 96.62 97.46 95.51 90.74 94.2 91.92 92.2 91.48 93.31 91.02 92.06 94.56 94.08 91.8 91.82 91.8 91.73
CA371 0.0019 0.0088 0.014 0.0040 98.72 98.72 98.72 98.72 98.65 98.72 98.65 98.72 98.58 98.72 99.12 98.56 98.67 98.47 98.41 98.41 96.62 96.66 96.48 96.64 97.44 95.54 90.76 94.2 91.94 92.1 91.4 93.43 91.04 91.96 94.6 94.18 91.9 91.82 91.82 91.63
gom93-268 0.0129 0.011 0.0059 0.0116 0.0123 100 100 100 99.37 100 99.61 100 99.43 100 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-274 0.0129 0.011 0.0059 0.0116 0.0123 0 100 100 99.37 100 99.41 100 99.43 100 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-275 0.0129 0.011 0.0059 0.0116 0.0123 0 0 100 99.37 100 99.41 100 99.43 100 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-278 0.0129 0.011 0.0059 0.0116 0.0123 0 0 0 99.37 100 99.41 100 99.43 100 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-283 0.0142 0.0127 0.0084 0.0133 0.0136 0.0063 0.0063 0.0063 0.0063 99.37 99.83 99.37 99.18 99.37 98.86 99.24 99.85 99.01 99.01 99.01 96.66 96.7 96.59 96.72 97.69 95.47 90.33 93.89 91.43 91.84 91.03 93.1 90.79 91.7 93.91 93.87 91.63 91.57 91.38 91.39
gom93-284 0.0129 0.011 0.0059 0.0116 0.0123 0 0 0 0 0.0063 99.41 100 99.43 100 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-286 0.0142 0.0127 0.0084 0.0133 0.0136 0.0059 0.0059 0.0059 0.0059 0.0017 0.0059 99.41 99.18 99.41 98.86 99.24 99.85 99.01 99.01 99.01 96.66 96.7 96.59 96.72 97.69 95.47 90.33 93.89 91.43 91.84 91.03 93.1 90.79 91.7 93.91 93.87 91.63 91.57 91.38 91.39
gom93-287 0.0129 0.011 0.0059 0.0116 0.0123 0 0 0 0 0.0063 0 0.0059 99.43 100 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-296 0.0148 0.0112 0.0002 0.0136 0.0142 0.0057 0.0057 0.0057 0.0057 0.0082 0.0057 0.0082 0.0057 99.43 98.88 99.56 99.2 99.83 99.83 99.83 96.63 96.68 96.57 96.66 97.89 95.28 90.26 93.94 91.47 91.74 91.01 93.17 90.87 91.6 93.77 93.8 91.51 91.62 91.31 91.44
gom93-297 0.0129 0.011 0.0059 0.0116 0.0123 0 0 0 0 0.0063 0 0.0059 0 0.0057 98.9 99.28 99.39 99.26 99.26 99.26 96.72 96.74 96.64 96.72 97.82 95.44 90.43 94.01 91.52 91.81 91.13 93.29 90.89 91.72 94.01 93.92 91.63 91.69 91.43 91.39
gom93-299 0.0095 0.0097 0.0114 0.0091 0.0089 0.011 0.011 0.011 0.011 0.0114 0.011 0.0114 0.011 0.0112 0.011 98.77 98.88 98.71 98.71 98.71 96.48 96.52 96.35 96.5 97.39 95.4 90.33 94.1 91.52 91.86 91.11 93.21 90.77 91.69 94.44 93.94 91.6 91.52 91.28 91.29
gom93-305 0.0155 0.0123 0.0046 0.0138 0.0144 0.0072 0.0072 0.0072 0.0072 0.0076 0.0072 0.0076 0.0072 0.0044 0.0072 0.0123 99.26 99.52 99.52 99.52 96.75 96.79 96.68 96.77 97.95 95.37 90.41 94.01 91.57 91.65 91.13 93.19 90.99 91.58 93.87 93.85 91.56 91.6 91.46 91.54
gom93-310 0.014 0.0125 0.0082 0.0131 0.0133 0.0061 0.0061 0.0061 0.0061 0.0015 0.0061 0.0002 0.0061 0.008 0.0061 0.0112 0.0074 99.03 99.03 99.03 96.68 96.72 96.61 96.7 97.72 95.44 90.3 93.91 91.4 91.77 91.06 93.07 90.77 91.72 93.89 93.85 91.61 91.59 91.4 91.41
gom93-501 0.0166 0.0129 0.0019 0.0153 0.0159 0.0074 0.0074 0.0074 0.0074 0.0099 0.0074 0.0099 0.0074 0.0017 0.0074 0.0129 0.0048 0.0097 100 100 96.5 96.54 96.43 96.52 97.85 95.33 90.31 93.89 91.4 91.65 91.11 93.14 90.91 91.53 93.75 93.8 91.39 91.57 91.28 91.36
gom93-543 0.0166 0.0129 0.0019 0.0153 0.0159 0.0074 0.0074 0.0074 0.0074 0.0099 0.0074 0.0099 0.0074 0.0017 0.0074 0.0129 0.0048 0.0097 0 100 96.5 96.54 96.43 96.52 97.85 95.33 90.31 93.89 91.4 91.65 91.11 93.14 90.91 91.53 93.75 93.8 91.39 91.57 91.28 91.36
gom93-544 0.0166 0.0129 0.0019 0.0153 0.0159 0.0074 0.0074 0.0074 0.0074 0.0099 0.0074 0.0099 0.0074 0.0017 0.0074 0.0129 0.0048 0.0097 0 0 96.5 96.54 96.43 96.52 97.85 95.33 90.31 93.89 91.4 91.65 91.11 93.14 90.91 91.53 93.75 93.8 91.39 91.57 91.28 91.36
IL 96-255 - B. kurtenbachii 0.035 0.0316 0.0339 0.0341 0.0339 0.0328 0.0328 0.0328 0.0328 0.0334 0.0328 0.0334 0.0328 0.0337 0.0328 0.0352 0.0325 0.0332 0.035 0.035 0.035 99.66 99.39 99.52 96.95 95.46 90.39 94.01 91.74 91.99 91.21 93.4 90.87 91.99 93.94 93.66 91.56 91.89 91.87 91.51
IL 97-236u 0.0345 0.0312 0.0335 0.0337 0.0334 0.0326 0.0326 0.0326 0.0326 0.033 0.0326 0.033 0.0326 0.0332 0.0326 0.0348 0.0321 0.0328 0.0346 0.0346 0.0346 0.0034 99.56 99.56 97.01 95.46 90.36 94.12 91.74 91.91 91.14 93.43 90.82 91.84 93.98 93.66 91.56 91.77 91.89 91.39
25015 0.0363 0.033 0.0345 0.0354 0.0352 0.0336 0.0336 0.0336 0.0336 0.0341 0.0336 0.0341 0.0336 0.0343 0.0336 0.0365 0.0332 0.0339 0.0357 0.0357 0.0357 0.0061 0.0044 99.41 96.9 95.28 90.24 93.92 91.68 91.77 91.02 93.31 90.8 91.73 93.85 93.5 91.59 91.7 91.83 91.35
NS07-121 0.0347 0.0314 0.0337 0.0339 0.0336 0.0328 0.0328 0.0328 0.0328 0.0328 0.0328 0.0332 0.0328 0.0334 0.0328 0.035 0.0323 0.033 0.0348 0.0348 0.0348 0.0048 0.0044 0.0059 96.97 95.37 90.32 93.98 91.7 91.89 91.04 93.24 90.7 91.75 93.82 93.59 91.61 91.72 91.77 91.27
SCJ1 - B. carolinensis 0.0268 0.0231 0.0213 0.0255 0.0257 0.0218 0.0218 0.0218 0.0218 0.0231 0.0218 0.0231 0.0218 0.0211 0.0218 0.0261 0.0205 0.0228 0.0215 0.0215 0.0215 0.0305 0.0299 0.031 0.0303 95.38 90.37 93.97 91.41 91.43 91.05 93.22 90.82 91.53 94.08 93.92 91.62 91.55 91.8 91.42
CA443 - B. californensis 0.0453 0.0458 0.0474 0.0449 0.0447 0.0456 0.0456 0.0456 0.0456 0.0453 0.0456 0.0458 0.0456 0.0472 0.0456 0.046 0.0463 0.0456 0.0467 0.0467 0.0467 0.0454 0.0454 0.0472 0.0463 0.0462 91.3 94.47 92.35 92.39 91.92 93.9 91.55 92.37 94.89 94.45 92.07 92.01 92.08 91.63
QLM4P1 - B. yangtze 0.0944 0.0944 0.0977 0.0927 0.0934 0.0957 0.0957 0.0957 0.0957 0.0967 0.0957 0.0972 0.0957 0.0974 0.0957 0.0967 0.0959 0.097 0.0969 0.0969 0.0969 0.0961 0.0964 0.0976 0.0968 0.0963 0.087 91.43 91.83 92.05 96.45 90.31 90.94 92.22 91.3 90.8 92.18 94.18 91.71 91.39
B31 - B. burgdorferi 0.0587 0.059 0.0609 0.0581 0.0581 0.0599 0.0599 0.0599 0.0599 0.0611 0.0599 0.0611 0.0599 0.0606 0.0599 0.059 0.0599 0.0609 0.0611 0.0611 0.0611 0.0599 0.0588 0.0608 0.0602 0.0603 0.0553 0.0857 92.18 92.21 91.9 93.97 91.44 92.12 95.43 94.79 92.12 92.07 91.6 91.54
VS461 - B. afzelii 0.0819 0.0835 0.0855 0.0809 0.0806 0.0848 0.0848 0.0848 0.0848 0.0857 0.0848 0.0863 0.0848 0.0853 0.0848 0.0848 0.0843 0.086 0.086 0.086 0.086 0.0826 0.0826 0.0832 0.083 0.0859 0.0765 0.0817 0.0782 93.31 92.47 91.9 93.32 93.55 92.61 91.52 93.04 92.61 93.22 92.51
20047 - B. garinii 0.0797 0.0802 0.0828 0.0780 0.0790 0.0819 0.0819 0.0819 0.0819 0.0816 0.0819 0.0826 0.0819 0.0826 0.0819 0.0814 0.0835 0.0823 0.0835 0.0835 0.0835 0.0801 0.0809 0.0823 0.0811 0.0857 0.0761 0.0795 0.0779 0.0669 92.94 91.71 92.22 98 92.78 91.76 92.99 93.09 93.22 92.7
VS116 - B. valaisiana 0.0867 0.0877 0.0902 0.0852 0.0860 0.0887 0.0887 0.0887 0.0887 0.0897 0.0887 0.0897 0.0887 0.0899 0.0887 0.0889 0.0887 0.0894 0.0889 0.0889 0.0889 0.0879 0.0886 0.0898 0.0896 0.0895 0.0808 0.0355 0.081 0.0753 0.0706 90.69 91.77 93.11 91.97 91.45 92.47 95.12 92.46 91.88
B. andersoni 0.0664 0.0664 0.0686 0.0669 0.0657 0.0671 0.0671 0.0671 0.0671 0.069 0.0671 0.0695 0.0671 0.0683 0.0671 0.0679 0.0681 0.0693 0.0686 0.0686 0.0686 0.066 0.0657 0.0669 0.0676 0.0678 0.061 0.0969 0.0603 0.081 0.0829 0.0931 90.49 91.52 94.2 94.02 91.41 91.27 91.37 90.51
A14S - B. spielmanii 0.0903 0.0925 0.0916 0.0898 0.0896 0.0911 0.0911 0.0911 0.0911 0.0921 0.0911 0.0926 0.0911 0.0913 0.0911 0.0923 0.0901 0.0923 0.0909 0.0909 0.0909 0.0913 0.0918 0.092 0.093 0.0918 0.0845 0.0906 0.0856 0.0668 0.0778 0.0823 0.0951 92.41 91.7 90.98 92.08 91.72 91.95 91.52
PBi - B. bavariensis 0.0809 0.0811 0.0843 0.0794 0.0804 0.0828 0.0828 0.0828 0.0828 0.083 0.0828 0.083 0.0828 0.084 0.0828 0.0831 0.0842 0.0828 0.0847 0.0847 0.0847 0.0801 0.0816 0.0827 0.0825 0.0847 0.0763 0.0778 0.0788 0.0645 0.02 0.0689 0.0848 0.0759 92.71 91.81 93.28 93.21 93.39 92.83
CA2 - genomospec 2 0.0546 0.0576 0.0625 0.0544 0.0539 0.0599 0.0599 0.0599 0.0599 0.0609 0.0599 0.0613 0.0599 0.0623 0.0599 0.0556 0.0613 0.0611 0.0625 0.0625 0.0625 0.0606 0.0602 0.0615 0.0618 0.0592 0.0511 0.087 0.0457 0.0739 0.0722 0.0803 0.058 0.083 0.0729 95.01 92.62 91.97 92.08 91.7
CA8 - B. americana 0.0596 0.0601 0.0622 0.0592 0.0587 0.0608 0.0608 0.0608 0.0608 0.0613 0.0608 0.0613 0.0608 0.062 0.0608 0.0606 0.0615 0.0615 0.062 0.062 0.062 0.0634 0.0634 0.065 0.0641 0.0608 0.0555 0.092 0.0521 0.0848 0.0824 0.0855 0.0598 0.0902 0.0819 0.0499 91.67 91.78 91.27 91.06
PoTiBL37 - B. lusitaniae 0.0818 0.083 0.0852 0.0820 0.0811 0.0837 0.0837 0.0837 0.0837 0.0837 0.0837 0.0842 0.0837 0.0849 0.0837 0.084 0.0844 0.0839 0.0861 0.0861 0.0861 0.0844 0.0844 0.0841 0.0839 0.0838 0.0793 0.0782 0.0788 0.0696 0.0701 0.0753 0.0859 0.0792 0.0672 0.0738 0.0833 92.78 92.73 92.18
HK501 - B. tanukii 0.0828 0.0828 0.0841 0.0819 0.0819 0.0831 0.0831 0.0831 0.0831 0.0843 0.0831 0.0843 0.0831 0.0838 0.0831 0.0848 0.084 0.0841 0.0843 0.0843 0.0843 0.0811 0.0823 0.083 0.0828 0.0845 0.0799 0.0582 0.0793 0.0739 0.0691 0.0488 0.0873 0.0828 0.0679 0.0803 0.0824 0.0722 92.75 92.3
HO14 - B. japonica 0.0828 0.0847 0.0872 0.0821 0.0818 0.0857 0.0857 0.0857 0.0857 0.0862 0.0857 0.0857 0.0857 0.0869 0.0857 0.0872 0.0854 0.086 0.0872 0.0872 0.0872 0.0813 0.0811 0.0817 0.0823 0.082 0.0792 0.0829 0.0804 0.0678 0.0678 0.0754 0.0863 0.0805 0.0661 0.0792 0.0873 0.0727 0.0725 92.03
Ya501 - B. turdi 0.0847 0.0859 0.0859 0.0827 0.0837 0.0861 0.0861 0.0861 0.0861 0.0861 0.0861 0.0861 0.0861 0.0856 0.0861 0.0871 0.0846 0.0859 0.0864 0.0864 0.0864 0.0849 0.0861 0.0865 0.0873 0.0858 0.0837 0.0861 0.0846 0.0749 0.073 0.0814 0.0949 0.0848 0.0717 0.083 0.0894 0.0782 0.077 0.0797
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Table 4.

Genetic diversity within B. bissettiia.

Locus Gene product/Description Fragment length analysed (bp) % G/C π value No. of alleles
clpA Clp protease subunit A 579 26.6 0.00929 8
clpX Clp protease subunit X 624 32.2 0.00349 4
nifS aminotransferase 564 28.6 0.00427 6
pepX dipeptidyl aminopeptidase 570 30.6 0.00349 7
pyrG CTP synthase 603 32.0 0.02009 7
recG DNA recombinase 651 31.0 0.00631 6
rplB 50S ribosomal protein L2 624 36.5 0.00865 5
uvrA exonuclease ABC, SU A 570 35.7 0.00882 9
concatenated 4785 31.7 0.00767
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a

Only samples identified as B. bissettii (Table 1) are included.

The genetic similarity of the strains that formed the second clade (Fig. 1) (i.e. 25015, 96-255, 97-236u, NS07-121) ranged from 98.7 to 99.9%. The genetic distance of these strains from those of B. bissettii and B. carolinensis was >3%, and it was even higher for all the other Borrelia species included (Table 3). Therefore, we conclude that the strains belonging to the second clade represent a novel genospecies. In fact, the genetic distances between those strains and the remaining B. bissettii strains was higher than it was between B. bissettii and B. carolinensis (Fig. 1, Table 2), a recently described species from South Carolina (Rudenko et al., 2009a). The second clade included 2 samples from Illinois (96-255, 97-236u), one from New York (25015), and one from Nova Scotia, (NS07-121) (Table 1).

Overall, the phylogenetic tree produced 2 major clades – one that clustered the Eurasian species, the other clustering the North American species. One genospecies, B. sinica, was not included in this study, but we consider it highly unlikely that the strains we propose as a new genospecies are similar to B. sinica. Firstly, B. sinica is only known from I. ovatus and murid rodents in southern China (Masuzawa et al. 2001). Secondly, B. bissettii strain DN127, a phylogenetic neighbour of 25015, was quite distant from B. sinica when analyzed by DNA-DNA hybridization (Masuzawa et al. 2001). Thirdly, published DraI and MseI restriction fragment length patterns of the 5S-23S rrf-rrl intergenic spacer of strain 25015 and B. sinica differ considerably (Masuzawa et al., 2001; Chu et al., 2008; Lin et al., 2001).

Discussion

In this study, we have reinvestigated the phylogenetic relatedness of spirochaete strains previously assigned to the genospecies B. bissettii (Postic et al., 1998; Picken and Picken, 2000). We found that while strains from California and Colorado clustered with the B. bissettii type strain DN127-Cl9-2/p7, other strains from Illinois and New York fell into a different cluster. A genetic distance analysis indicated that these strains are more distant to B. bissettii than they are to B. carolinensis, a newly described Borrelia species (Rudenko et al., 2009a). Thus, we propose to name this group Borrelia kurtenbachii sp. nov., in honour of the late Klaus Kurtenbach who contributed significantly to our understanding of the ecology and evolution of the LB spirochaetes (Kurtenbach et al., 2006).

All isolates from Colorado were obtained either from I. spinipalpis removed from the Mexican woodrat Neotoma mexicana or from the woodrats themselves, whereas strains from California were either isolated from I. pacificus or I. spinipalpis that had been removed from the dusky-footed woodrat Neotoma fuscipes. These strains clustered together phylogenetically, and the genetic distance analysis indicated that they are true members of the species B. bissettii. Natural transmission cycles of B. bissettii involve either I. spinipalpis or I. pacificus; in contrast, strain 25015 and the Canadian strain were both detected in the eastern North American tick vector, I. scapularis. Although it has been demonstrated experimentally that B. bissettii can be transmitted between hosts by I. scapularis (Oliver et al., 2003), our data support the notion that B. kurtenbachii strains may be maintained in enzootic transmission cycles that differ ecologically from those of B. bissettii. This needs, however, to be further explored since isolation of strain 25015 from blood-fed larvae does not comprise proof of vector competence. Both strains from Illinois were isolated from the rodent Microtus pennsylvanicus (Picken and Picken, 2000). In the southeastern United States, strains assigned to B. bissettii have been isolated from the eastern woodrat (Neotoma floridana), the cotton rat (Sigmodon hispidus), the cotton mouse (Peromyscus gossypinus), and the vectors I. affinis and I. minor (Lin et al., 2001, 2005; Oliver et al., 2003). However, the data presented here render the association of B. bissettii with I. minor uncertain because the strain isolated from this tick showed a DraI restriction pattern similar to that of strain 25015, and it also differed in its MseI restriction pattern from all other B. bissettii isolates (Lin et al., 2001). Taken together, our data suggest that the transmission cycles of B. bissettii and B. kurtenbachii differ, but the vector(s) for B. kurtenbachii needs to be determined throughout its geographic distribution, particularly in Illinois.

In Europe, B. bissettii DNA has been detected in, and was isolated from, human patients (Picken et al., 1996a, 1996b; Strle et al., 1997; Fingerle et al., 2008; Rudenko et al., 2008, 2009c), but it rarely has been found in questing I. ricinus ticks (Hulinska et al., 2007). It is, therefore, tempting to speculate that the transmission of B. bissettii is maintained by an unknown vector in an enzootic cycle with occasional spill over into the human population. Alternatively, if B. bissettii is transmitted by I. ricinus, the spirochaete distribution may be highly focal and restricted by its host associations since human cases have been recorded quite regionally. However, any association of B. bissettii with particular tick vectors and hosts in Europe is currently at best speculative and requires further investigation. This is also true for B. kurtenbachii(-related) strains that may occur in Europe (Picken et al., 1996a, 1996b). Of the tick species known to transmit B. bissettii in the United States, i.e., I. pacificus and I. spinipalpis in the Far West and Southwest and I. affinis in the Southeast (Bissett and Hill, 1987; Maupin et al., 1994; Lin et al., 2001, 2003), only I. pacificus attaches to humans with any frequency. This may partly explain why B. bissettii has not been incriminated as a human pathogen in the United States so far (Maupin et al., 1994). In addition, using a murine model for LB, it was demonstrated that B. bissettii strains in the United States differed considerably in their potential to induce arthritis in mice (Schneider et al., 2008). Early investigations into the pathogenic potential of strain 25015 revealed that this strain was non-arthritogenic in mice (Anderson et al., 1990), although subsequent studies demonstrated that it might be mildly pathogenic when passaged repeatedly through laboratory mice (Fikrig et al., 1992). In North America, strain 25015 and related strains have been detected in I. scapularis, a tick that frequently attaches to humans. However, the rarity of finding B. bissettii-like strains in I. scapularis in recent years (Hoen et al., 2009, Hamer et al., 2010) or in patients underpins the need for further studies to clarify whether I. scapularis is the primary vector for B. kurtenbachii or whether the rare findings of this Borrelia species in I. scapularis can be attributed to spill-over from another enzootic transmission cycle. Although the number of B. kurtenbachii strains evaluated during the present study is restricted, most of the strains found in the previous study in Illinois revealed a macrorestriction pattern similar to that of strain 25015 (Picken and Picken, 2000). Such strains also have been described from the southeastern United States and perhaps Wisconsin indicating that B. kurtenbachii may be more widespread in its distribution and common in certain habitats than realized so far (Picken and Picken, 2000; Lin et al., 2001, 2005; Oliver et al., 2003; Caporale et al., 2005). At the time when B. bissettii was described, strain 25015 was the only example found in I. scapularis in upstate New York (Postic et al., 1998) suggesting that these strains are more common in Illinois and the southern United States. As mentioned above, the discovery of these strains in New York and eastern Canada are unusual because such strains have not been detected in host-seeking I. scapularis during recent studies in the northeastern or upper midwestern United States (Hoen et al., 2009; Hamer et al., 2010). In Illinois, the bacteria were isolated from voles and mice in consecutive years, and a rodent-associated transmission cycle might limit the distributional range. More research is needed to clarify the relationships among strains in these locations.

In the United States, the species with the widest distribution is B. burgdorferi (s.s.), which occurs in the southern, northeastern, Midwestern, and western states (Fig. 2). B. andersonii and B. kurtenbachii seem to be restricted to the east of the Rocky Mountains, whereas B. carolinensis and B. californiensis appear to be limited to South Carolina and California, respectively. Two species, B. bissettii and B. americana, are found in the southeastern and far-western regions, with B. bissettii also being present in Colorado. The occurrence of several genospecies across the continent suggests a dynamic evolutionary history of LB spirochaetes in the United States and is in agreement with recent suggestions that B. burgdorferi has been present in the country for several thousand or even millions of years (Hoen et al., 2009). They also imply that demographic events related to glacial and interglacial periods may have shaped the current population structure. Collectively, our own and previous findings on the diversity of LB spirochaetes in the United States underscore the need for additional research to fully comprehend the ecology and evolutionary history of these spirochaetes and the impact of this spirochaetal diversity, if any, on human health.

Fig. 2.

Fig. 2

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Present day distribution of LB species in the United States based on published records.

Description of Borrelia kurtenbachii sp. nov

Borrelia kurtenbachii (kur.ten.ba’chi. L. gen in honour of the late Klaus Kurtenbach and his numerous contributions to research on the Lyme borreliosis group of spirochaetes).

The type strain 25015T was isolated from an I. scapularis larva in upstate New York (Anderson et al., 1990). The morphology matches that of previously described Borrelia species in that it displays the typical helical morphology of spirochaetal bacteria (Barbour and Hayes, 1986). Its in vitro culture properties are as described for the genus (Johnson et al., 1984). B. kurtenbachii can be distinguished from all other LB species by means of its restriction pattern of the rrf-rrl intergenic spacer (Postic et al., 1998) and its housekeeping gene sequences using multilocus sequence analysis.

Acknowledgments

This work was funded by NIH-NIAID under grant number 5R21AI065848.

Footnotes

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