# Exploring Diversity among NorwegianBorrelia Strains Originating from Ixodes ricinus Ticks

Authors: Ann-Kristin Tveten
Journal: International Journal of Microbiology
 (2014)

---

## Abstract

Characterisation ofBorrelia strains from Ixodes ricinus ticks is important in the epidemiological surveillance of vector-borne pathogens. Multilocus sequences analysis (MLSA) is a molecular genotyping tool with high discriminatory power that has been applied in evolutionary studies and for the characterisation of Borrelia genospecies. MLSA was used to study genetic variations in Borrelia strains isolated from I. ricinus ticks collected from the woodlands in Skodje. The results demonstrate that the 50 Borrelia strains were separated into 36 sequence types (STs) that were not previously represented in the MLST database. A distance matrix neighbour-joining tree (bootstrapped 500 iterations) showed four deeply branched clusters, and each deeply branched cluster represented one Borrelia genospecies. The mean pairwise genetic differences confirm the genospecies clustering. The combination of alleles separates the Borrelia strains from northwest Norway from the strains in the MLST database, thus identifying new STs. Although a highly divergent B. afzelii population could be expected, the heterogeneity among the B. garinii strains is more unusual. The present study indicates that the circulation of strains between migrating birds and stationary birds in this coastal region may play a role in the evolution of B. garinii strains.


---

## Body

## 1. Introduction

Ticks are considered important carriers of pathogenic microorganisms in the northern hemisphere [1], andIxodes ricinus ticks are associated with a diverse microbiota [2].Borrelia spirochetes are one of the pathogens transmitted to humans byI. ricinus ticks [3], andBorrelia burgdorferi sensu lato (s.l.) represents a bacterial species complex that comprises severalBorrelia genospecies associated with Lyme borreliosis (LB) [4].Borrelia burgdorferi sensu stricto (s.s.),Borrelia afzelii, andBorrelia garinii are causative agents of human LB in Norway [5], and the prevalence of LB is monitored by the Norwegian Surveillance System for Communicable Diseases (MSIS). A fourthBorrelia genospecies,Borrelia valaisiana, was discovered in Norwegian ticks in 2010 [5]. These four genospecies have been detected inI. ricinus ticks in northwest Norway [6].During the recent years, several genotyping methods have been established for analysis ofBorrelia to characterise strains and determine the phylogenetic relationships between strains [7–11]. Multilocus sequence typing (MLST) is a molecular typing tool that is used to characterise pathogenic microorganisms [12], and MLST can be used as a tool for the epidemiological surveillance and tracking of infectious diseases [13]. MLST, as defined by Urwin and Maiden, is a genotyping tool based on the sequences of housekeeping genes that evolve slowly. The sequences used for MLST are approximately 400–500 bp in size and are located throughout the genome to avoid bias [13]. Multilocus sequence analysis (MLSA) applies MLST to characterise closely related species using a distance-based procedure for phylogenetic characterisation, and it includes pairwise genetic similarities. MLST/MLSA have been applied in population studies and for analysis ofBorrelia species in geographic areas and for evolutionary studies and characterisation of newBorrelia species. While some MLST/MLSA schemes combine housekeeping genes with hypervariable regions and noncoding loci [9–11], the MLST scheme published in 2008 by Margos et al. is based on housekeeping genes that fulfill the strict criteria defined by Urwin and Maiden [7, 13]. TheBorrelia MLST scheme is based on amplification, sequencing, and bioinformatic analysis of internal fragments of eight housekeeping genes (clpA, clpX, nifS, pepX, pyrG, rplB, recG, anduvrA) [7].TheBorrelia MLST scheme is available through the MLST network (http://www.mlst.net/). The MLST network enables easy access to sequences from all over the world [14]. Recently, studies of phylogenetic relationship ofBorrelia genospecies using MLSA have resulted in the definition of two newBorreliaspecies,Borrelia bavariensis sp. [15] andBorrelia kurtenbachii [8]. These new findings demonstrate the high discriminatory power that MLST/MLSA schemes provide. Rudenko et al. utilised MLST to describe recombination at thenifS locus amongB. burgdorferi s.s. andBorrelia americana strains. Their findings indicated that, to a degree, strain diversity is influenced by the host [16]. The aim of this study is to utilise MLSA to characteriseBorrelia strains isolated fromI. ricinus ticks collected from the fauna in northwest Norway. Multilocus sequence analysis of Norwegian strains could provide knowledge about the strain diversity amongBorreliastrains found in this region and describe their evolution compared to European strains.


## 2. Materials and Methods

### 2.1. Samples

DNA from questingI. ricinus ticks collected in 2012 (531 nymphs/71 adults) and 2013 (539 nymphs/64 adults) (Figure 1) was isolated, and the DNA samples were analysed by qPCR to detect the presence ofBorrelia genospecies as previously described [6]. A total of 86 samples (79 nymphs/7 adults) were positive forBorrelia in 2012, and 89 samples (83 nymphs/8 adults) were positive forBorrelia in 2013. Multilocus sequence analysis was performed using the MLST scheme developed by Margos et al. A total of 50 samples were amplified across all eight housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, anduvrA) [7] and were submitted for sequencing. The 50 strains used in this study are listed in Table 1.Table 1
Allele and sequence types (STs).
Source
Species
clpA
clpX
nifS
pepX
pyrG
recG
rplB
uvrA
ST
I. ricinus
B. afzelii
109
24
23
85
22
29
23
29
481
I. ricinus
B. afzelii
36
24
96
31
119
29
23
28
483
I. ricinus
B. afzelii
36
24
96
31
22
30
23
28
484
I. ricinus
B. garinii
46
30
33
42
29
88
79
36
485
I. ricinus
B. garinii
47
30
33
42
111
88
79
36
486
I. ricinus
B. garinii
47
30
33
42
111
88
79
36
486
I. ricinus
B. garinii
46
30
33
38
32
76
32
36
487
I. ricinus
B. garinii
46
30
29
38
32
76
32
36
488
I. ricinus
B. afzelii
37
24
23
35
96
30
99
142
489
I. ricinus
B. afzelii
51
82
23
86
85
27
28
29
490
I. ricinus
B. afzelii
51
82
23
86
85
27
29
29
491
I. ricinus
B. afzelii
172
24
23
85
85
27
29
29
492
I. ricinus
B. afzelii
37
135
24
87
22
29
23
28
493
I. ricinus
B. garinii
45
30
32
96
29
38
30
36
494
I. ricinus
B. burgdorferis.s.
14
8
14
10
10
137
9
129
495
I. ricinus
B. afzelii
36
24
23
35
26
33
23
28
496
I. ricinus
B. afzelii
36
24
23
32
22
33
23
28
497
I. ricinus
B. garinii
46
30
29
96
32
76
79
33
498
I. ricinus
B. garinii
95
74
34
96
32
88
79
85
499
I. ricinus
B. afzelii
36
82
23
32
119
27
23
28
500
I. ricinus
B. afzelii
36
82
23
87
22
27
23
28
501
I. ricinus
B. afzelii
109
24
23
35
22
98
23
29
502
I. ricinus
B. afzelii
109
24
23
85
22
98
23
29
503
I. ricinus
B. afzelii
36
24
24
87
22
29
29
30
504
I. ricinus
B. afzelii
36
24
23
31
22
29
29
30
505
I. ricinus
B. afzelii
36
24
23
31
20
29
29
30
506
I. ricinus
B. afzelii
36
24
23
32
20
29
29
28
507
I. ricinus
B. afzelii
36
24
96
32
92
33
29
28
508
I. ricinus
B. afzelii
37
135
24
87
22
29
23
28
493
I. ricinus
B. afzelii
37
24
23
35
96
30
99
142
489
I. ricinus
B. afzelii
36
24
96
31
119
29
23
28
483
I. ricinus
B. afzelii
37
135
24
87
22
29
23
28
509
I. ricinus
B. afzelii
109
24
23
85
22
29
23
29
481
I. ricinus
B. afzelii
36
24
23
31
22
29
29
30
505
I. ricinus
B. garinii
46
74
29
38
35
40
32
33
510
I. ricinus
B. garinii
46
74
29
38
35
40
32
33
510
I. ricinus
B. garinii
45
30
32
96
29
38
30
36
494
I. ricinus
B. garinii
95
74
34
96
34
76
79
85
511
I. ricinus
B. garinii
45
30
32
96
29
38
30
36
494
I. ricinus
B. afzelii
36
24
23
35
85
29
28
29
512
I. ricinus
B. afzelii
51
24
23
86
85
30
99
142
513
I. ricinus
B. afzelii
36
24
23
35
85
29
28
29
512
I. ricinus
B. afzelii
51
82
23
86
119
29
28
29
514
I. ricinus
B. garinii
95
74
34
96
32
76
79
85
515
I. ricinus
B. garinii
46
30
29
96
32
76
32
33
516
I. ricinus
B. garinii
46
30
29
96
32
76
32
33
516
I. ricinus
B. valaisiana
96
76
36
98
84
44
78
86
517
I. ricinus
B. valaisiana
50
76
36
98
38
44
78
86
518
I. ricinus
B. valaisiana
96
76
36
98
84
44
78
86
517
I. ricinus
B. valaisiana
96
76
36
98
84
44
78
86
517Figure 1
Map of the collection site. The map indicates the location site in northwest Norway and the tracking route used. The altitude of each collection site and the distance between the sites are indicated in the bottom part of the map.


### 2.2. Amplification and Sequencing

The amplification of each housekeeping gene was carried out in a 15µL reaction volume containing 7.5 µL Mastermix 2.0x SYBR GREEN (Applied Biosystems), 0.75 µL of each primer (10 µM), 3.00 µL ddH2O, and 3.0 µL template DNA. Amplification of the eight housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, anduvrA) was performed with primers and nested touchdown PCR conditions forclpA, nifS, pepX, pyrG, recG, anduvrA as previously described [7, 8]. Amplification ofclpX andrplB was performed with primers described by Margos et al. combined with a nested touchdown PCR composed of 1 cycle of denaturation (10 min, 95°C), followed by 9 cycles of denaturation (30 sec, 95°C), touchdown annealing (30 sec, 58°C–50°C), and extension (1 min, 72°C) and 30 cycles of denaturation (30 sec, 95°C), annealing (30 sec, 50°C), and extension (1 min, 72°C) with a final cycle of extension (10 min, 72°C). Gel electrophoresis was used to confirm the size and approximate concentration of the PCR products for each amplified gene. Gel electrophoresis was performed with a 2% Tris-acetate EDTA buffered (TBE) gel followed by staining with GelRed (Affymetrix, Santa Clara, USA).The PCR products were purified in a two-step process by ExoSAP-IT (Affymetrix, Santa Clara, USA) according to the manufacturers’ protocol. The sequencing reaction was carried out in a 10µL reaction volume containing 1 µL Big Dye Terminator 3.1 (Applied Biosystems, Carlsbad, USA), 1 µL 5x Sequencing Buffer (Applied Biosystems, Carlsbad, USA), 0.32 µL primer (10 µM), 4.68 µL ddH2O, and 3 µL template DNA. Amplification was performed in a 2720 thermal cycler (Applied Biosystems, Carlsbad, USA) with 1 cycle of denaturation (6 min, 96°C), followed by 25 cycles of denaturation (10 sec, 96°C), annealing (5 sec, 50°C), and extension (4 min, 60°C). Finally, 10 µL ddH2O was added to each well upon completion of the sequencing reaction, and the samples were stored at –20°C until sequencing. Sequencing was performed by Eurofins MWG Operon (Germany).


### 2.3. Alignment and Multilocus Sequence Analysis

The sequences for each housekeeping gene were aligned by the MUSCLE algorithm and concatenated in MEGA 5.1 [17]. The sequences were analysed with BLAST to confirm the correctBorrelia genospecies [18]. Each locus and the concatenated sequence of all eight loci were analysed. The MEGA 5.1 software was used to draw a distance matrix neighbour-joining tree (bootstrapped 500 iterations) with the concatenated DNA sequences. Sequences fromB. duttonii were added from the BLAST database [18] to root the neighbour-joining tree.B. duttonii is aBorrelia species that causes relapsing fever [19]. Sequences from four reference species from the MLST database were included in the analysis,B. burgdorferi s.s. (B31),B. afzelii (VS461),B. valaisiana (70865B), andB. garinii (20047). To compare allele sequences and sequences types (STs) and to identify identical sequences, the nonredundant database (NRDB) written by Warren Gish at Washington University was used (http://pubmlst.org/analysis/). All known alleles and STs were retrieved from theBorrelia MLST database for comparison (http://borrelia.mlst.net/). STs that were not found in the MLST database were submitted to the MLST database for proper numbering. Statistical analysis was performed with MEGA 5.1. The mean pairwise genetic difference (within and between groups) was calculated using the Kimura 2-parameter model. Average nonsynonymous substitutions and synonymous substitutions (dN/dS) were calculated using the modified Nei-Gojobori method (Jukes-Cantor). The number of polymorphic sites (PS) and the nucleotide diversity (π) were calculated using Tajima’s test of neutrality. The index of association, both standard and classic, was calculated using START2 [20].To compare the relationship between NorwegianBorrelia strains and EuropeanBorrelia strains a goeBURST minimum spanning tree was generated using PHYLOViZ 1.0 software [21].


## 2.1. Samples

DNA from questingI. ricinus ticks collected in 2012 (531 nymphs/71 adults) and 2013 (539 nymphs/64 adults) (Figure 1) was isolated, and the DNA samples were analysed by qPCR to detect the presence ofBorrelia genospecies as previously described [6]. A total of 86 samples (79 nymphs/7 adults) were positive forBorrelia in 2012, and 89 samples (83 nymphs/8 adults) were positive forBorrelia in 2013. Multilocus sequence analysis was performed using the MLST scheme developed by Margos et al. A total of 50 samples were amplified across all eight housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, anduvrA) [7] and were submitted for sequencing. The 50 strains used in this study are listed in Table 1.Table 1
Allele and sequence types (STs).
Source
Species
clpA
clpX
nifS
pepX
pyrG
recG
rplB
uvrA
ST
I. ricinus
B. afzelii
109
24
23
85
22
29
23
29
481
I. ricinus
B. afzelii
36
24
96
31
119
29
23
28
483
I. ricinus
B. afzelii
36
24
96
31
22
30
23
28
484
I. ricinus
B. garinii
46
30
33
42
29
88
79
36
485
I. ricinus
B. garinii
47
30
33
42
111
88
79
36
486
I. ricinus
B. garinii
47
30
33
42
111
88
79
36
486
I. ricinus
B. garinii
46
30
33
38
32
76
32
36
487
I. ricinus
B. garinii
46
30
29
38
32
76
32
36
488
I. ricinus
B. afzelii
37
24
23
35
96
30
99
142
489
I. ricinus
B. afzelii
51
82
23
86
85
27
28
29
490
I. ricinus
B. afzelii
51
82
23
86
85
27
29
29
491
I. ricinus
B. afzelii
172
24
23
85
85
27
29
29
492
I. ricinus
B. afzelii
37
135
24
87
22
29
23
28
493
I. ricinus
B. garinii
45
30
32
96
29
38
30
36
494
I. ricinus
B. burgdorferis.s.
14
8
14
10
10
137
9
129
495
I. ricinus
B. afzelii
36
24
23
35
26
33
23
28
496
I. ricinus
B. afzelii
36
24
23
32
22
33
23
28
497
I. ricinus
B. garinii
46
30
29
96
32
76
79
33
498
I. ricinus
B. garinii
95
74
34
96
32
88
79
85
499
I. ricinus
B. afzelii
36
82
23
32
119
27
23
28
500
I. ricinus
B. afzelii
36
82
23
87
22
27
23
28
501
I. ricinus
B. afzelii
109
24
23
35
22
98
23
29
502
I. ricinus
B. afzelii
109
24
23
85
22
98
23
29
503
I. ricinus
B. afzelii
36
24
24
87
22
29
29
30
504
I. ricinus
B. afzelii
36
24
23
31
22
29
29
30
505
I. ricinus
B. afzelii
36
24
23
31
20
29
29
30
506
I. ricinus
B. afzelii
36
24
23
32
20
29
29
28
507
I. ricinus
B. afzelii
36
24
96
32
92
33
29
28
508
I. ricinus
B. afzelii
37
135
24
87
22
29
23
28
493
I. ricinus
B. afzelii
37
24
23
35
96
30
99
142
489
I. ricinus
B. afzelii
36
24
96
31
119
29
23
28
483
I. ricinus
B. afzelii
37
135
24
87
22
29
23
28
509
I. ricinus
B. afzelii
109
24
23
85
22
29
23
29
481
I. ricinus
B. afzelii
36
24
23
31
22
29
29
30
505
I. ricinus
B. garinii
46
74
29
38
35
40
32
33
510
I. ricinus
B. garinii
46
74
29
38
35
40
32
33
510
I. ricinus
B. garinii
45
30
32
96
29
38
30
36
494
I. ricinus
B. garinii
95
74
34
96
34
76
79
85
511
I. ricinus
B. garinii
45
30
32
96
29
38
30
36
494
I. ricinus
B. afzelii
36
24
23
35
85
29
28
29
512
I. ricinus
B. afzelii
51
24
23
86
85
30
99
142
513
I. ricinus
B. afzelii
36
24
23
35
85
29
28
29
512
I. ricinus
B. afzelii
51
82
23
86
119
29
28
29
514
I. ricinus
B. garinii
95
74
34
96
32
76
79
85
515
I. ricinus
B. garinii
46
30
29
96
32
76
32
33
516
I. ricinus
B. garinii
46
30
29
96
32
76
32
33
516
I. ricinus
B. valaisiana
96
76
36
98
84
44
78
86
517
I. ricinus
B. valaisiana
50
76
36
98
38
44
78
86
518
I. ricinus
B. valaisiana
96
76
36
98
84
44
78
86
517
I. ricinus
B. valaisiana
96
76
36
98
84
44
78
86
517Figure 1
Map of the collection site. The map indicates the location site in northwest Norway and the tracking route used. The altitude of each collection site and the distance between the sites are indicated in the bottom part of the map.


## 2.2. Amplification and Sequencing

The amplification of each housekeeping gene was carried out in a 15µL reaction volume containing 7.5 µL Mastermix 2.0x SYBR GREEN (Applied Biosystems), 0.75 µL of each primer (10 µM), 3.00 µL ddH2O, and 3.0 µL template DNA. Amplification of the eight housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, anduvrA) was performed with primers and nested touchdown PCR conditions forclpA, nifS, pepX, pyrG, recG, anduvrA as previously described [7, 8]. Amplification ofclpX andrplB was performed with primers described by Margos et al. combined with a nested touchdown PCR composed of 1 cycle of denaturation (10 min, 95°C), followed by 9 cycles of denaturation (30 sec, 95°C), touchdown annealing (30 sec, 58°C–50°C), and extension (1 min, 72°C) and 30 cycles of denaturation (30 sec, 95°C), annealing (30 sec, 50°C), and extension (1 min, 72°C) with a final cycle of extension (10 min, 72°C). Gel electrophoresis was used to confirm the size and approximate concentration of the PCR products for each amplified gene. Gel electrophoresis was performed with a 2% Tris-acetate EDTA buffered (TBE) gel followed by staining with GelRed (Affymetrix, Santa Clara, USA).The PCR products were purified in a two-step process by ExoSAP-IT (Affymetrix, Santa Clara, USA) according to the manufacturers’ protocol. The sequencing reaction was carried out in a 10µL reaction volume containing 1 µL Big Dye Terminator 3.1 (Applied Biosystems, Carlsbad, USA), 1 µL 5x Sequencing Buffer (Applied Biosystems, Carlsbad, USA), 0.32 µL primer (10 µM), 4.68 µL ddH2O, and 3 µL template DNA. Amplification was performed in a 2720 thermal cycler (Applied Biosystems, Carlsbad, USA) with 1 cycle of denaturation (6 min, 96°C), followed by 25 cycles of denaturation (10 sec, 96°C), annealing (5 sec, 50°C), and extension (4 min, 60°C). Finally, 10 µL ddH2O was added to each well upon completion of the sequencing reaction, and the samples were stored at –20°C until sequencing. Sequencing was performed by Eurofins MWG Operon (Germany).


## 2.3. Alignment and Multilocus Sequence Analysis

The sequences for each housekeeping gene were aligned by the MUSCLE algorithm and concatenated in MEGA 5.1 [17]. The sequences were analysed with BLAST to confirm the correctBorrelia genospecies [18]. Each locus and the concatenated sequence of all eight loci were analysed. The MEGA 5.1 software was used to draw a distance matrix neighbour-joining tree (bootstrapped 500 iterations) with the concatenated DNA sequences. Sequences fromB. duttonii were added from the BLAST database [18] to root the neighbour-joining tree.B. duttonii is aBorrelia species that causes relapsing fever [19]. Sequences from four reference species from the MLST database were included in the analysis,B. burgdorferi s.s. (B31),B. afzelii (VS461),B. valaisiana (70865B), andB. garinii (20047). To compare allele sequences and sequences types (STs) and to identify identical sequences, the nonredundant database (NRDB) written by Warren Gish at Washington University was used (http://pubmlst.org/analysis/). All known alleles and STs were retrieved from theBorrelia MLST database for comparison (http://borrelia.mlst.net/). STs that were not found in the MLST database were submitted to the MLST database for proper numbering. Statistical analysis was performed with MEGA 5.1. The mean pairwise genetic difference (within and between groups) was calculated using the Kimura 2-parameter model. Average nonsynonymous substitutions and synonymous substitutions (dN/dS) were calculated using the modified Nei-Gojobori method (Jukes-Cantor). The number of polymorphic sites (PS) and the nucleotide diversity (π) were calculated using Tajima’s test of neutrality. The index of association, both standard and classic, was calculated using START2 [20].To compare the relationship between NorwegianBorrelia strains and EuropeanBorrelia strains a goeBURST minimum spanning tree was generated using PHYLOViZ 1.0 software [21].


## 3. Results

A total of 50 strains were sequenced across all eight loci for the present study. A distance matrix neighbour-joining tree (bootstrapped 500 iterations) (Figure2) demonstrated that the 50 strains were separated into 36 sequence types (STs) (Table 1) that were not previously found in the MLST database. The neighbour-joining tree formed four deeply branched clusters, and, between the four clusters, there were no shared alleles. All strains cluster together with their respective reference species and contain combinations of alleles that are new entries into the MLST database. The mean pairwise genetic difference confirms the genospecies clustering (Figure 3) based on the genospecies threshold (0.017) as defined by Margos et al. [15]. Table 2 summarises the characteristics of each of the eight housekeeping genes included in the multilocus sequencing analysis ofBorrelia strains.Table 2
Characteristics of each housekeeping gene.
Species
Gene
Size (bp)
Avg. G+C content %
PS (%)a
π
b
dN/dSc
B. burgdorferis.s.
clpA
579
52.2
—d
—
—
B. afzelii
0,69
0,001759
0,001810
B. garinii
1,73
0,005484
0,005205
B. valaisiana
0,69
0,003454
0,003768
B. burgdorferis.s.
clpX
624
56.8
—
—
—
B. afzelii
0,48
0,001255
0,00122
B. garinii
6,08
0,02191
0,001835
B. valaisiana
—e
—
—
B. burgdorferis.s.
nifS
564
55.8
—
—
—
B. afzelii
0,36
0,00087
0,00090
B. garinii
1,06
0,0054
0,00434
B. valaisiana
—
—
—
B. burgdorferis.s.
pepX
570
55.6
—
—
—
B. afzelii
0,88
0,00328
0,00223
B. garinii
1,23
0,00484
0,00501
B. valaisiana
—
—
—
B. burgdorferis.s.
pyrG
603
51.0
—
—
—
B. afzelii
0,83
0,00240
0,00211
B. garinii
1,49
0,00805
0,00609
B. valaisiana
1,32
0,0073
0,00637
B. burgdorferis.s.
recG
651
53.0
—
—
—
B. afzelii
0,62
0,00209
0,00157
B. garinii
1,54
0,00545
0,00463
B. valaisiana
—
—
—
B. burgdorferis.s.
rplB
624
56.9
—
—
—
B. afzelii
0,61
0,00261
0,00143
B. garinii
1,28
0,00669
0,00386
B. valaisiana
—
—
—
B. burgdorferis.s.
uvrA
570
54.4
—
—
—
B. afzelii
0,88
0,00234
0,00223
B. garinii
1,05
0,00335
0,003172
B. valaisiana
—
—
—
aPercent polymorphic sites (PS (%)).
bNucleotide diversity (π).
cAverage nonsynonymous substitutions versus synonymous substitutions (dN/dS).
dIt was not possible to perform statistics with only one isolate in this group.
eIt was not possible to perform statistics with only one allele variant.Figure 2
Description of clades based on the neighbour-joining tree (bootstrapped 500 iterations) calculated based on concatenated DNA sequences showing the relationship betweenB. burgdorferi s.l. strains.Figure 3
Mean pairwise difference within and betweenBorrelia genospecies. The line indicates species threshold (0.017) as defined by Margos et al. [15].The comparative goeBURST analysis (level 8 grouping) displays the relationship between the Norwegian strains and European strains (Figure4). The goeBURST analysis shows that the NorwegianBorrelia population is the most closely related to the Latvian and French populations. The dataset contains 175 unique STs and information for approximately 246 strains. A goeBURST analysis was performed at the SLV (single locus variant) level by dividing the dataset into 32 groups with 2 or more STs and 64 singletons. The largest clonal complex 0 (CC0) has 13 unique STs and represents 33 strains. CC0 represents strains identified asB. valaisiana and contains 6 SLVs. All of the SLVs share thepepX, pyrG, rplB, anduvrA alleles. In CC1 (9 STs, 22 strains), the predicted ancestral genotype is ST 86, with 8 SLVs representing 10 strains; thus this is the highest ranking ancestral genotype. All SLVs have the sameclpA, clpX, andrplB alleles. A goeBURST analysis conducted at the DLV (double locus variant) level divided the dataset into 54 CCs consisting of 26 groups with 2 or more STs and 28 singletons. A goeBURST analysis conducted at the TLV (three loci variant) level divided the dataset into 33 CCs consisting of 21 groups with 2 or more STs and 11 singletons. A level 4 grouping was required forB. valaisiana to be represented by one CC in the goeBURST analysis, and a level 5 grouping was required forB. afzelii to be represented by one CC. A level 7 grouping was required forB. garinii andB. burgdorferi s.s. to be represented by one CC.Figure 4
goeBURST. The goeBURST is coloured based on the geographic origin of each ST. Multiple colours indicated multiple origins.The Norwegian STs are characterised as both SLVs and DLVs. The Norwegian strains are spread throughout the goeBURST tree and are clustered together with their respective genospecies from other European countries. Four branches are formed from the primary founder. These four branches contain STs related to the four genospecies,B. garinii, B. afzelii, B. valaisiana, andB. burgdorferi s.s. The highest ranking ancestral genotype, ST 86, is aB. garinii strain from Latvia.


## 4. Discussion

Northwest Norway is characterised by geographical features such as islands, several deep fjords, and high mountains. The landscape shows signs of overgrowth and little maintenance of the vegetation, thus providing suitable growth conditions for diverse wildlife. More than 4000 different Animalia species have been observed in this region [22]. The region has mild winters and humid summers, creating good conditions forIxodes ricinus ticks.Borrelia spirochetes are maintained in nature by the circulation between the vector and the host, and the wide range of available hosts may influence the diversity among strains ofBorrelia [23].Borrelia spirochetes have an obligate parasitic lifestyle, and their circulation depends on an interaction between reservoir hosts and vectors [24]. Birds are reservoir hosts forB. garinii whileB. afzelii are usually associated with rodents [15, 25]. Evolution and a wide range of determinants that affect reservoir hosts and vectors are likely to influence the diversity among strains differently [23, 24, 26]. Reservoir hosts and vectors are submitted to a number of factors such as climate, nutrition, and pollution that have different impacts depending on the geographic locations [23, 24]. In addition to the influence that reservoir hosts provide, the complex microbial communities associated withI. ricinus ticks also subjectBorrelia spirochetes to microbial competition and natural selection. These microbial communities are composed of bacteria, endosymbionts, viruses, and protozoa from the tick’s natural habitat and hosts, and, therefore, they vary between different geographical locations [2, 27, 28].Multilocus sequence analysis of theBorrelia strains found inI. ricinus ticks from this region shows signs of high diversity. The majority of the strains areB. afzelii andB. garinii, the most prevalent genospecies within this region [6]. FiftyBorrelia strains were separated into 36 STs, and the genetic relationship between the strains was visualised in a neighbour-joining tree. The first deeply branched cluster contains strains identified asB. afzelii, and 29 strains were separated into 22 STs. These 29B. afzelii strains form a highly structured population consisting of STs that have not been previously described in the MLST database. In 2011, Vollmer et al. demonstrated that there is limited movement ofB. afzelii strains between geographic locations and that only twoB. afzelii STs have been identified in more than one geographic location [22, 29].B. afzelii strains are described as heterogenic, and a comparative goeBURST analysis of strains from Scotland and strains from England showed that there is a limited movement of strains between the north and south in the United Kingdom [24, 29]. The dispersal ofBorrelia spirochetes is linked to the movement of their hosts.B. afzelii is associated with small rodent hosts with a limited movement pattern, thus creating a limited dispersal ofB. afzelii strains [30]. All strains included in the present study were collected from four sites within one geographically defined area (Figure 1). Some of theB. afzelii strains were identified in more than one sample, but they were always within the collection site.The second deeply branched cluster contains fourB. valaisiana strains divided into 2 STs that represent new entries into the MLST database. The third deeply branched cluster contains 16B. garinii strains divided into 11 STs that are new entries into the MLST database. BothB. valaisiana andB. garinii are bird-associated genospecies, and previous studies have found that these twoBorrelia genospecies have sequence types that have been identified in different geographic locations throughout Europe [29]. Vollmer et al. reported thatB. valaisiana strains are not completely homogenised within Europe whileB. garinii shows signs of being a mixed population across central Europe [29]. Migrating birds play an important role in the dispersal ofB. garinii strains between geographic locations. Kjelland et al. studied the prevalence ofBorrelia infection in 31 species of migrating birds and 6 species of resident birds in southern Norway.Borrelia infection was found in all bird species [31]. Studies also suggest thatB. garinii strains have the ability to circulate both in terrestrial cycles and in seabirds with their associated tickIxodes uriae [32–34]. A study by Comstedt et al. from 2011 determined thatB. garinii was found in Norwegian populations of seabirds andI. uriae [35]. Sequencing of the intergenic spacer (IGS) fromB. garinii strains has indicated that the seabird-associatedB. garinii strains are more diverse than the terrestrialB. garinii strains [36]. All strains included in the present study are collected from a geographic location with a high number of observed bird species. In Møre and Romsdal county, 290 bird species have been observed in 2013 by ornithologist [37]. These birds represent a variety of different reservoir hosts forB. garinii, and the nesting on the island provides a possibility of transferringB. garinii into fauna, inducing circulation between migrating and stationary hosts. The high number of migrating birds along the coastal area of northwest Norway enables introduction ofB. garinii strains from multiple different geographic origins and multiple reservoir hosts and the process of circulation between reservoir hosts has been suggested to play a role in the genetic evolution ofBorrelia genospecies [29]. SomeB. garinii strains from the present study are DLVs and TLVs of the strains from central Europe, while others have levels 4 and 5 connections to the central European STs. Within the NorwegianB. garinii strains, some are SLVs and DLVs, and some are more distantly related. This may indicate that the different strains have been introduced into the fauna by migrating birds at different times and that some strains might have been maintained by stationary hosts for some time. Calculation of the nucleotide diversity (π value) reveals that the π values were at least two times greater forB. garinii than forB. afzelii. Assuming a similar generation time betweenB. garinii andB. afzelii, the different reservoir hosts might have influenced the evolutionary rate differently between genospecies [26]. The limited geographic dispersal ofB. afzelii strains has allowed them to evolve differently between geographic locations, but the nucleotide diversity within a geographic location is relatively low.B. garinii associated with migratory birds is subjected to a higher degree of ecological determinants, and the nucleotide diversity may reflect that strains have been introduced to the fauna at different times.The fourth deep branched cluster contains oneB. burgdorferi s.s. isolate and its associated reference species. TheB. burgdorferi s.s. strain was isolated from an adult female tick. The low prevalence of this genospecies [6], in combination with certain challenges associated with amplification across all eight loci, caused this strain to be the onlyB. burgdorferi s.s. included in this study.The number of polymorphic sites (PS) and the nucleotide diversity (π) were calculated to further describe theBorrelia strains. A sequence analysis was conducted to identify point mutations found in multiple strains. These point mutations have a minor contribution to the genetic diversity between concatenated DNA sequences from northwest Norway compared to other worldwide strains. Most of the point mutations are silent or missense mutations with little or no effect on the genetic function. The ratio between nonsynonymous substitutions and synonymous substitutions (dN/dS) < 1 indicates that the number of synonymous substitutions is higher than the number of nonsynonymous substitutions. This indicates that the genes are not under selective pressure and are suitable for MLSA analysis. The index of association (IA) describes the influence of recombination in a population and was calculated in two ways: using the classical method defined by Smith et al. and using the standard method defined by Haubold and Hudson [38, 39]. The IA would be significant for a clonal population. The calculated values for this population were IAclassic=2.2054 and IAstandard=0.3151, and significant linkage disequilibrium was detected. Estimation of the index of association indicated that the population is clonal and that recombination has not played an important role in the evolution of this population.Multilocus sequencing analysis has provided knowledge about the genetic variations betweenBorrelia genospecies originating fromI. ricinus ticks from northwest Norway. The combinations of alleles separate these strains from other strains in the MLST database. This is the first multilocus sequence analysis ofBorrelia strains identified from NorwegianI. ricinus ticks, and the 50Borrelia strains from Norway represent a large contribution to the MLST database. This enabled comparative studies between Norwegian and European strains, thus creating a better understanding of the strain development ofBorrelia genospecies in different geographic locations in Europe. The high level of divergence in theB. afzelii population was expected, but the divergence within theB. garinii population differs from previous MLST/MLSA studies. Migrating birds are constantly introducing new strains to the fauna along the coastline in northwest Norway, and circulation between migrating and stationary hosts would influence the strain diversity. Some of theB. garinii strains show signs of being newly introduced to the fauna while others have most likely been maintained in stationary hosts for a longer period of time.



---

*Source: 397143-2014-08-27.xml*