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Prevalence, Diversity, and Load of Borrelia species in Ticks That Have Fed on Humans in Regions of Sweden and Åland Islands, Finland with Different Lyme Borreliosis Incidences

  • Peter Wilhelmsson ,

    peter.wilhelmsson@liu.se

    Affiliation Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

  • Pontus Lindblom,

    Affiliation Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

  • Linda Fryland,

    Affiliation Division of Clinical Immunology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

  • Jan Ernerudh,

    Affiliation Division of Clinical Immunology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

  • Pia Forsberg,

    Affiliations Division of Infectious Diseases, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden, Clinic of Infectious Diseases, County Council of Östergötland, Linköping, Sweden

  • Per-Eric Lindgren

    Affiliations Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden, Medical Services, Department of Microbiology, Ryhov County Hospital, Jönköping, Sweden

Prevalence, Diversity, and Load of Borrelia species in Ticks That Have Fed on Humans in Regions of Sweden and Åland Islands, Finland with Different Lyme Borreliosis Incidences

  • Peter Wilhelmsson, 
  • Pontus Lindblom, 
  • Linda Fryland, 
  • Jan Ernerudh, 
  • Pia Forsberg, 
  • Per-Eric Lindgren
PLOS
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Abstract

The incidence of Lyme borreliosis (LB) in a region may reflect the prevalence of Borrelia in the tick population. Our aim was to investigate if regions with different LB incidences can be distinguished by studying the prevalence and diversity of Borrelia species in their respective tick populations. The Borrelia load in a feeding tick increases with the duration of feeding, which may facilitate a transmission of Borrelia Spirochetes from tick to host. Therefore, we also wanted to investigate how the Borrelia load in ticks that have fed on humans varies with the duration of tick feeding. During 2008 and 2009, ticks that had bitten humans were collected from four regions of Sweden and Finland, regions with expected differences in LB incidence. The duration of tick feeding was estimated and Borrelia were detected and quantified by a quantitative PCR assay followed by species determination. Out of the 2,154 Ixodes ricinus ticks analyzed, 26% were infected with Borrelia and seven species were identified. B. spielmanii was detected for the first time in the regions. The tick populations collected from the four regions exhibited only minor differences in both prevalence and diversity of Borrelia species, indicating that these variables alone cannot explain the regions’ different LB incidences. The number of Borrelia cells in the infected ticks ranged from fewer than ten to more than a million. We also found a lower number of Borrelia cells in adult female ticks that had fed for more than 36 hours, compared to the number of Borrelia cells found in adult female ticks that had fed for less than 36 hours.

Introduction

Ticks transmit a wide range of infections to humans, including Lyme borreliosis (LB) and relapsing fever borreliosis (RF). LB is a multi-systemic inflammatory disease caused by spirochetes belonging to the Borrelia burgdorferi sensu lato (sl) complex, which comprises at least 18 species [1]. In Europe, 8 species of this complex have been reported: B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), B. valaisiana, B. lusitaniae, B. spielmanii, B. bavariensis and B. bissettii. Among them, B. afzelii, B. garinii, and B. burgdorferi ss are the most frequently reported in human clinical specimens. Borrelia burgdorferi sl are predominantly transmitted by Ixodes ticks. Borrelia miyamotoi, a member of the relapsing fever group has been detected in Ixodes ricinus ticks [2] and has recently been associated with disease in humans [3,4], characterized by recurring episodes of fever. Ixodes ricinus, the predominant Ixodes species in Western Europe, has three active life stages and may feed on more than 200 animal species, including mammals, birds, and reptiles [5]. Different groups of animals are more or less suitable as hosts to certain life stages of the I. ricinus [6,7], and more or less suitable as reservoirs to certain Borrelia species [8,9]. Each life stage of the tick may therefore harbor a certain diversity of Borrelia species.

Two meta-analysis studies demonstrate that countries with high LB incidences (e.g. Austria, Czech Republic, Slovenia, Switzerland, and Sweden) also have a high prevalence of Borrelia in “their” field-collected tick populations, while countries with low LB incidences (e.g. France, Great Britain, Italy, and Poland) have a low prevalence of Borrelia in “their” field-collected tick populations [10,11]. Even if ticks collected from field are considered to be host-seeking it is not evident that they would attach to and bite a human that passes by. Besides, the methods used to collect ticks in field are influenced by weather conditions both before and during sampling [12]. Further, the different life stages of I. ricinus have different seasonal host-seeking patterns that are quite variable and not yet fully understood [13,14]. From a clinical point of view it would be more relevant to study the prevalence and diversity of Borrelia in ticks that have actually bitten humans, and only few such European studies exist [2,1517]. It would also be interesting to evaluate if such prevalence reflects the incidences of LB in the same area as the tick bite occurred. To investigate this, a direct comparison of the Borrelia content in ticks from different regions with dissimilar incidences of LB is required.

The duration of tick feeding influences the risk to get infected with Borrelia after a tick bite. Crippa et al. (2008) observed that B. burgdorferi ss was only transferred to mice when Ixodes nymphs fed for more than 48 hours [18]. Further, one out of seven and four out of eight mice exposed to B. afzelii infected ticks for 24 hours and 48 hours, respectively, became infected; thereafter the transmission risk increased. The duration of tick feeding also influences the Borrelia load in a feeding tick. Piesman et al. (2001) observed a sixfold increase of B. burgdorferi ss cells when Ixodes nymphs had fed on mice for 48 hours [19]. All together, this means that a longer duration of tick feeding leads to a higher load of Borrelia in the tick that could facilitate a transmission of Borrelia spirochetes from tick to host; the higher load the higher infection probability. To our knowledge, the Borrelia load in ticks that have bitten humans has never been related to the duration of tick feeding. Such investigation will improve our understanding of when a feeding tick may be able to transmit Borrelia spirochetes to humans.

An ongoing prospective Nordic epidemiological investigation, denoted the Tick-Borne Diseases (TBD) STING-study, was initiated in 2007 [2,20]. The overall aim of the TBD STING-study is to investigate the prevalence of tick-borne pathogens in ticks that have fed on humans and to evaluate if parameters such as pathogen load, species and type, the life stage of tick, and duration of tick feeding influence the risk of pathogen transmission and the development of symptomatic infections. The present study focuses on the Borrelia content in those ticks with the aim to investigate if regions with different incidences of LB can be distinguished by studying the prevalence and diversity of Borrelia species in their respective tick populations. We expect that regions with high incidence of LB also have a high prevalence of Borrelia infected ticks, and vice versa, which has previously been demonstrated in other European regions [10,11]. We also wanted to investigate if there are any tick stage-related differences regarding prevalence and diversity of Borrelia species. Finally, we wanted to examine how the Borrelia load in ticks that have bitten humans is influenced by the duration of tick feeding. To investigate this, ticks that had bitten humans in two regions with different incidences of LB (located in the Finnish Åland Islands and Southernmost Sweden) and two regions with unknown incidences of LB (Northern Sweden and South Central Sweden) were collected. The duration of tick feeding was estimated by microscopic methods and Borrelia were detected and quantified by real-time PCR followed by species determination. The prevalence and diversity of Borrelia species found in the tick populations collected from the different regions were compared. We also compared the prevalence and diversity of Borrelia species in ticks at different life stages. In addition, the Borrelia load in ticks that had fed on humans was related to the duration of tick feeding.

Materials and Methods

Ethics Statement

Ethical permission for the TBD STING-study was granted by the Ethics Committee of the Medical Faculty, Linköping University (M132-06), and by the local Ethics Committee of the Åland Health Care, 2008-05-23.

Selection of regions and collection of ticks

Four regions: the Finnish Åland Islands and three separate regions of the Swedish mainland were selected, based on known or expected differences in LB incidences (Figure 1). The Åland Islands are a hyper-endemic region with an annual LB incidence in the range of 710-890/100,000 inhabitants [21], and a seroprevalence of 19.7% [22]. The region of Southernmost Sweden has an annual LB incidence in the range of 69-464/100,000 [23,24]. The LB incidence in South Central and Northern Sweden are unknown. In South Central Sweden, however, we previously reported a Borrelia seroprevalence of 11% among tick-bitten humans in the county of Östergötland [20], a lower seroprevalence than in the general population on the Åland Islands.

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Figure 1. Map, showing the four regions where the 34 primary health care centers (PHCs) are located.

Southernmost Sweden (10 PHCs), South Central Sweden (20 PHCs), Northern Sweden (3 PHCs), and Åland Islands (1 PHCs). SE: Sweden, FI: Finland.

http://dx.doi.org/10.1371/journal.pone.0081433.g001

Individuals aged 18 years or older were recruited into the TBD STING-study if they had recently been bitten by a tick. The study subjects were asked to bring their tick(s) to one of the 34 participating primary health care centers (PHCs) located in the four studied regions (Figure 1). At the initial visit at the PHCs, the bitten persons signed a written consent to participate, donated tick(s), blood sample, and completed a health questionnaire. Another blood sample was drawn and a second health questionnaire was filled in at a three-month follow-up visit to the PHC. Ticks and blood samples were transported to Linköping University within three days, where they were frozen at -70°C. Ticks were collected from May 2008 to November 2009.

Identification and measurement of ticks

Ticks were photographed and measured dorsally and ventrally, using a USB-microscope (Dino-Lite Long AM4013TL, AnMo Electronics Corp., Taiwan) to determine species, life stage, sex of adults, and to estimate feeding duration for adult female ticks and nymphs using coxal and scutal indices as described elsewhere [25].

Tick homogenization, total nucleic acid extraction and reverse-transcription of nucleic acid

Forty-six ticks, one positive control (5 µl of Borrelia burgdorferi sensu stricto B31 ATCC 35210 [108 cells/ml]), and one negative control, H2O, in separate 2 ml safe-lock microcentrifuge tubes were processed simultaneously. For each individual tick sample, 450 µl RLT buffer (Qiagen, Hilden, Germany) supplemented with 1% β-Mercaptoethanol (Sigma-Aldrich Sweden, Stockholm, Sweden) was added to each tube together with one 5-mm stainless steel bead (Qiagen). Ticks were homogenized for 2 min at 25 Hz at RT, using a TissueLyser (Qiagen). After a centrifugation step (3 min at 20,000 × g), 400 µl of the supernatants were collected in separate 2 ml micro tubes followed by automated total nucleic acid (NA) extraction.

Total NA was extracted with a BioRobot M48 Workstation (Qiagen), using the MagAttract® RNA Tissue Mini M48 Kit (Qiagen), according to the manufacturer's instructions with the exception of not adding DNase to the supplied RDD buffer, thus obtaining total NA including DNA. This will allow targeting non-coding regions (DNA) of the Borrelia genome as PCR-template for species determination. NA was eluted in a final volume of 50 µl RNAse free water. Twenty µl NA extract was used for reverse-transcribed NA (RTNA) synthesis, the remaining NA extract was stored at -70°C.

RTNA synthesis was performed using illustra™ Ready-to-Go RT-PCR Beads kit (GE Healthcare, Amersham Place, UK) according to manufacturer's protocol, and 10 µl (0.25µg/µl) random hexamer primers (pd(N)6) were incubated with 20 µl NA for 5 min at 97°C. Beads were dissolved in 20 µl RNAse free water and transferred to the solution containing NA and primers, followed by incubation for 30 min at 42°C, and subsequently for 5 min at 95°C, resulting in a final volume of 50 µl RTNA. Pipetting was performed using a CAS-1200 pipetting robot (Corbett Robotics Inc., San Francisco, CA) and incubation using a PTC-100 thermal cycler (M. J. Research, Inc., Waltham, MA).

LUX real-time PCR assay used for detection and quantification of Borrelia

Two microliters of RTNA per reaction were used in a Light Upon eXtensionTM (LUX) real-time PCR assay to detect and quantify Borrelia 16S rRNA, using genus-specific primers, as previously described [2]. PCR reagents and template RTNA were added to 96-well real-time PCR plates using CAS-1200 pipetting robot (Corbett Robotics Inc.).

To quantify the number of Borrelia cells, a serial dilution of plasmid standard was used. The plasmid contained the target sequence of the LUX real-time PCR assay, spanning the nucleotides 465284 - 465556 of the B. burgdorferi ss B31 chromosome (acc. no NC_001318.1). PCR product of the target sequence was synthesized and cloned into the pPCR-Script Amp SK(+) cloning vector (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

Estimation of real-time PCR inhibition

To estimate real-time PCR inhibition, a representative subset (15%) of all the extracted tick samples (61 adult ticks, 216 nymphs, 9 larvae, and 46 ticks that could neither be determined to species nor life stage (ND) was analyzed with a SYBR green real-time PCR assay to detect the Ixodes tick mitochondrial 16S rRNA, as previously described [2]. The Ixodes tick mitochondrial 16S rRNA was detected in all samples. The range of quantification cycle (Cq) values differed according to the life stage of the tick. The Cq values for adult ticks ranged from 9 to 20 (median 13, interquartile range [IQR] 11-15); for nymphs 9 to 21 (median 13, IQR 12-16), and for larvae 12 to 22 (median 16, IQR 13-21). Cq values for adult females and nymphs did not correlate with time of tick feeding. Cq values for the ND samples ranged from 10 to 37 (median 22, IQR 16-32). ND samples showed a higher median compared to the other groups, which was probably due to poor condition of the ticks.

Possible PCR inhibition for the SYBR green real-time PCR assay was identified by measuring amplification performance using dilution series of samples to be targeted. Serial dilutions (1:1, 1:10, 1:100, and 1:1000) of RTNA processed from six fully engorged ticks (three adult ticks and three nymphs) were prepared in RNAse free water and analyzed, as described above. The amplification of undiluted and diluted samples showed parallel curves with a Cq difference of 3.4-3.5 with dilution. This was considered as PCR non-inhibitory.

To check for possible inhibition in the Borrelia 16S rRNA LUX real-time PCR assay, serial dilutions (1:1, 1:10, 1:100, and 1:1000) of RTNA from three fully engorged adult ticks containing B. valaisiana, B. burgdorferi ss, and B. spielmanii, respectively, and three fully engorged nymphal ticks containing B. afzelii, B. garinii, and B. miyamotoi, respectively, were prepared in RNAse free water and analyzed, as described earlier. Prior to the test, the Borrelia species of these samples had been determined with the use of the 5S-23S and 16S-23S intergenic spacer PCR assays, as described below. The amplification of undiluted and diluted samples for all Borrelia species showed parallel curves with a highest Cq difference of 3.4 - 3.8 with dilution. Since the Cq values corresponds to an amplification efficiency between 83% and 97%, undiluted samples were used to screen RTNA samples for the presence of Borrelia.

Comparison of LUX real-time PCR sensitivity targeting 16S rRNA (RTNA) and 16S rRNA gene (DNA)

We previously reported a LUX real-time PCR detection limit of fewer than 101 16S rRNA gene copies of B. burgdorferi ss (B31) when DNA was used as template [2]. Ornstein and Barbour (2006) reported a 100-fold higher numbers of copies per Borrelia cell for 16S rRNA compared to 16S rRNA gene (DNA) [26]. Their findings also indicated that rRNA copy numbers in Borrelia cells was less affected by the cell’s growth phase than DNA copy numbers, i.e. rRNA copies may be more appropriate to use to quantify Borrelia cells under different growth conditions. Here, we wanted to compare the LUX real-time PCR sensitivity targeting 16S rRNA (RTNA) and 16S rRNA gene (DNA). B. burgdorferi ss (B31) were cultivated in BSK medium and counted in phase-contrast microscope, as previously described [2]. Two 10-fold serial dilutions of B. burgdorferi ss (B31) ranging from 107 to 101 were prepared in PBS and subsequently used for either total NA extraction followed by RTNA synthesis, as described above, or DNA extraction using DNeasy blood and tissue kit (Qiagen, Hilden, Germany), as previously described [2]. This was followed by LUX real-time PCR amplification, as described above. Figure S1 shows a comparison of Cq values obtained with the 16S rRNA (analyzed template RTNA) and 16S rRNA gene (analyzed template DNA) tests. The mean Cq values were approximately 5 units (range 4.2 - 5.3) lower per dilution step with RTNA than DNA (data not shown), indicating a 10 to 100-fold higher number of 16S rRNA-molecules than 16S rRNA genes per Borrelia cell, with the assumption of 100% yield in RTNA synthesis and equal yields of total NA and DNA extraction, respectively. The mean efficiency of the LUX real-time PCR for three replicates of each dilution step with RTNA and DNA was 91% (-3.58 ± 0.02, r2 = 0.99).

Conventional PCR assays and sequencing method used for Borrelia species identification

To determine the Borrelia species in LUX real-time PCR positive samples, a nested, conventional PCR assay using primers targeting the non-coding intergenic spacer region (IGS) between 5S rRNA and 23S rRNA genes was applied, as previously described [2]. Tick samples positive for Borrelia in the LUX real-time PCR that failed to produce PCR products with primers targeting the 5S-23S IGS were analyzed with primers targeting the 16S-23S IGS [27]. This allowed identification of other Borrelia species, such as B. miyamotoi, that lack the 5S-23S IGS.

Tick samples positive in the LUX real-time PCR that failed to produce PCR products with primers targeting both 5S-23S IGS and 16S-23S IGS were regarded as untypeable.

Sequencing of all PCR products was performed by Macrogen Inc. (Seoul, South Korea). Mixed sequencing chromatograms were analyzed using the RipSeq Mixed web application (http://www.ripseq.com/login/login.aspx) (iSentio, Bergen, Norway). The RipSeq Mixed algorithm searches against the ‘5S-23S rRNA intergenic spacer’ database containing 43 sequences from six different Borrelia species. Non-mixed chromatograms were initially edited using BioEdit Software (V7.0) (Tom Hall, Ibis Therapeutics, Carlsbad, CA) followed by a standard BLAST search against the GenBank database (http://blast.ncbi.nlm.nih.gov). To confirm the genetic identity of the Borrelia species detected in ticks, 5S-23S and 16S-23S sequences were aligned with the corresponding B. afzelii, B. garinii, B. valaisiana, B. burgdorferi ss, B. miyamotoi, B. spielmanii, and B. lusitaniae, sequences available in GenBank using ClustalW2 [28]. Within each species, sequences of the intergenic spacer regions showed a sequence similarity between 94 and 100%.

5S-23S IGS and 16S-23S IGS obtained in this investigation have been deposited in GenBank with accession numbers JX909617 to JX910061 (n = 445), and JX910062 to JX910079 (n = 18), respectively.

Statistical analysis

Fisher’s exact test or Chi square test were applied to compare the prevalence of Borrelia infected ticks and number of different Borrelia species between the regions, and within the collecting season (comparison between months). Comparisons of the Borrelia load between different Borrelia species were analyzed using Kruskal-Wallis test, which, if significant, was followed by Dunn´s post test. The Spearman correlation coefficient was used to investigate any correlation between the duration of tick feeding and the Borrelia load in ticks. The test was applied on ticks in different life stages. Mann-Whitney test was used to compare the loads of Borrelia in ticks that had fed for more than 36 hours with ticks that had fed for less than 36 hours. Results were reported as median values with interquartile range. Statistical analyses were performed and graphs were drawn using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). p-values ≤ 0.05 were considered significant.

Results

Ticks collected

A total of 2,154 ticks, including 15 adult males (1%), 496 adult females (23%), 1,510 nymphs (70%), and 87 larvae (4%) were collected (Table 1). Forty-six (2%) ticks were so damaged that neither life stage nor species could be determined. The remaining ticks were morphologically determined as I. ricinus. Ixodes mitochondrial 16S rRNA was detected by PCR in all damaged ticks; these may be assumed to be I. ricinus, as this is the only Ixodes tick known to bite humans in Sweden.

RegionaNo. (%) of ticks examinedbNo.(%) of positive tickscNo. (%) of ticks containing Borrelia species determined by nucleotide sequencingd
B. aB. gB. vB. bB. mB. sB. lMixedUT
Total2154 (100)556 (26)279 (50)109 (19)35 (7)21 (4)11 (2)6 (1)2 (1)2 (1)91 (15)
Adult male15 (1)5 (33)2 (40)1 (20)1 (20)000001(20)
Adult female496 (23)175 (36)68 (38)44 (25)16 (9)11 (6)5 (3)1 (1)1 (1)1 (1)28 (16)
Nymph1510 (70)369 (25)208 (56)62 (17)18 (5)9 (2)6 (2)5 (1)1 (1)1 (1)59 (15)
Larva87 (4)0000000000
NDe46 (2)7 (15)1 (14)2 (29)01 (14)00003 (43)
A541 (25)133 (25)68 (51)24 (18)8 (6)7 (5)1 (1)01 (1)1 (1)23 (17)
Adult male1 (1)0000000000
Adult female115 (21)41 (36)16 (39)11 (27)4 (10)3 (7)0001 (2)6 (15)
Nymph411 (76)92 (23)52 (57)13 (14)4 (4)4 (4)1 (1)01 (1)017 (19)
Larva14 (2)0000000000
B771 (37)236 (31)123 (52)41 (17)12 (5)9 (4)8 (3)1 (1)1 (1)041 (17)
Adult male9 (1)5 (56)2 (40)1 (20)1 (20)000001 (20)
Adult female254 (33)99 (39)42 (42)22 (22)7 (8)5 (5)4 (4)01 (1)018 (18)
Nymph503 (65)132 (26)79 (60)18 (13)4 (3)4 (3)4 (3)1 (1)0022 (17)
Larva5 (1)0000000000
C19 (1)2 (11)1 (50)01 (50)000000
Adult male00000000000
Adult female9 (47)0000000000
Nymph9 (47)2 (22)1 (50)01 (50)000000
Larva1 (6)0000000000
D777 (37)178 (23)86 (48)42 (24)14 (8)4 (2)2 (1)5 (3)01(1)24 (13)
Adult male5 (1)0000000000
Adult female118 (15)35 (30)10 (28)11 (30)5 (14)3 (8)1 (3)1 (3)004 (11)
Nymph587 (74)143 (24)76 (53)31 (21)9 (6)1 (1)1 (1)4 (3)01 (1)20 (14)
Larva67 (8)0000000000

Table 1. Borrelia prevalence and species in ticks, collected from tick-bitten individuals in different regions

aRegions: A, Southernmost Sweden; B, South Central Sweden; C, Northern Sweden; D, Åland Islands
bPercent and numbers in shaded rows indicate proportion of the total number of ticks examined, all other percent and numbers indicate proportion within respective region
cNumber of ticks yielding a positive outcome by real-time PCR analysis
dAbbreviations: B. a, B. afzelii; B. g, B. garinii; B. v, B. valaisiana; B. b, B. burgdorferi sensu stricto; B. m, B. miyamotoi; B. s, B. spielmanii; B. l, B. lusitaniae; UT, untypeable
eND, developmental stage could not be determined due to damages. ND-ticks are only presented in the column showing total collected ticks
CSV
Download CSV

Tick life stage distribution differed between the study regions (Table 1). A higher proportion of adult ticks were collected in Sweden (21%; Southernmost Sweden, 33%; South Central Sweden) than in the Åland Islands (15%, p < 0.001, respectively). Seventy-seven percent of larvae (67 of 87) were collected from the Åland Islands. Due to the low number of ticks collected from Northern Sweden (n = 19), these ticks were excluded from further statistical analysis.

Every fourth tick contained Borrelia

Overall, 26% of the collected ticks (556 of 2,154) contained Borrelia (Table 1). Adult female ticks had a higher Borrelia prevalence (36%) compared to nymphs (25%, p < 0.001). Borrelia was not detected in any of the 87 larvae.

The Borrelia prevalence in ticks collected from South Central Sweden (31%) was higher compared to Southernmost Sweden (25%, p < 0.05), and to Åland Islands (23%, p < 0.01). Only 11% (2 of 19) of the ticks collected from Northern Sweden contained Borrelia. The Borrelia prevalence in adult ticks collected from South Central Sweden was higher (40%, 104 of 263) compared to adult ticks collected from Åland Islands (28%, 35 of 123, p < 0.05). No other significant differences in the Borrelia prevalence in adult ticks between the regions were noticed. The Borrelia prevalence in nymphal ticks collected from the four regions varied between 22% and 26%, but did not differ significantly (Table 1). No significant seasonal variations (24% - 29%) in the prevalence of Borrelia infection in ticks were noticed (data not shown).

High diversity of Borrelia species found in the ticks

Seven different Borrelia species were identified by sequence analysis of the 5S-23S and 16S-23S IGS (Table 1). B. afzelii was the predominant species and was detected in 50% of all ticks containing Borrelia, followed by B. garinii (19%), B. valaisiana (7%), B. burgdorferi ss (4%), B. miyamotoi (2%), B. spielmanii (1%), B. lusitaniae (1%), mixed infection of Borrelia species (1%), and 15% were untypeable. A similar descending order in distribution of Borrelia species was observed in all regions, except in Northern Sweden where only two (of 19) ticks were Borrelia infected. Among the ticks with mixed infection of Borrelia species (n = 2), an adult female tick contained B. garinii and B. valaisiana and a nymphal tick contained two strains of B. burgdorferi ss.

Of all samples that were determined to Borrelia species, B. afzelii was more prevalent in nymphs (67%, 208 of 309) than in adult ticks (46%, 70 of 150, p < 0.001) (Figure 2). B. garinii and B. valaisiana showed the opposite pattern and were more prevalent in adult ticks (30%, 45 of 150, and 11%, 17 of 150, respectively) than in nymphs (20%, 62 of 309, and 6%, 18 of 309, respectively) (p < 0.05). No significant regional or seasonal differences in prevalence of Borrelia species were detected.

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Figure 2. Distribution of Borrelia species in nymphs (N) and adults (A).

The percentage of positive ticks per typeable species is given. The numbers of infected ticks examined are indicated above the bars. Abbreviations: B. a, B. afzelii; B. g, B. garinii; B. v, B. valaisiana; B. b, B. burgdorferi sensu stricto; B. m, B. miyamotoi; B. s, B. spielmanii; B. l, B. lusitaniae.

http://dx.doi.org/10.1371/journal.pone.0081433.g002

The Borrelia load in the ticks differed according to Borrelia species

According to the LUX real-time PCR assay, the Borrelia load per tick ranged from 1.0 × 10° to 1.8 × 106 cells per tick (Figure 3) with a median of 1.6 × 103. No significant difference in Borrelia load between adult ticks and nymphs was observed (medians 1.5 × 103, and 1.7 × 103, respectively) (data not shown). This was also the case for adults and nymphs with estimated feeding times less than 24 hours (medians 6.3× 103, and 3.2 × 103, respectively) (data not shown).

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Figure 3. Borrelia species plotted against the number of Borrelia cells per tick.

Horizontal lines indicate the median, with upper and lower quartiles. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Abbreviations: B. a, B. afzelii; B. g, B. garinii; B. v, B. valaisiana; B. b, B. burgdorferi sensu stricto; B. m, B. miyamotoi; B. s, B. spielmanii; B. l, B. lusitaniae.

http://dx.doi.org/10.1371/journal.pone.0081433.g003

Ticks infected with B. miyamotoi had a significantly higher Borrelia load (median of 2.1 × 105 cells per tick) compared to ticks infected with B. afzelii (median 4.5 × 103, p < 0.01), B. garinii (median 2.7 × 103, p < 0.001), B. valaisiana (median 1.0 × 103, p < 0.001), and B. burgdorferi ss (median 2.0 × 103, p < 0.05) (Figure 3). No other significant differences in Borrelia load between species were noticed. Ticks infected with untypeable Borrelia, had a significantly lower Borrelia load (median of 1.2 × 101 cells per tick) compared to ticks infected with typeable Borrelia species (p < 0.001) (data not shown). The Borrelia load in the ticks with mixed infection of Borrelia species contained 6.6 × 102 and 6.0 × 101 cells per tick, respectively.

The Borrelia load in adult female ticks differed according to estimated time of tick feeding

When the Borrelia load, quantified in adult females (n = 156), and in nymphs (n = 350), was plotted against their respective feeding-time (hours), a significant correlation between the Borrelia load in adult females with estimated time of tick feeding was found (Spearman correlation = -0.25, p < 0.01). When the estimated time of tick feeding was stratified into intervals, we noticed a significantly lower Borrelia load in the adult females after 36 hours of feeding (Figure 4A). This was the case for both B. afzelii and B. garinii (Figure 5). For the remaining Borrelia species numbers were too small to allow analysis. No significant correlation between the Borrelia load and time of nymphal tick-feeding was observed (Figure 4B).

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Figure 4. Tick feeding-time (hours) plotted against the number of Borrelia cells in A. adult female ticks, and B. nymphal ticks.

Horizontal lines indicate the median, with upper and lower quartiles. *, p < 0.05.

http://dx.doi.org/10.1371/journal.pone.0081433.g004

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Figure 5. Tick feeding-time (hours) plotted against the number of Borrelia cells in ticks of different stages.

Adult female ticks and nymphs infected with B. afzelii or B. garinii. Horizontal lines indicate the median, with upper and lower quartiles. *, p < 0.05; ***, p < 0.001.

http://dx.doi.org/10.1371/journal.pone.0081433.g005

Discussion

This study detected that 26% of the ticks that had bitten humans in four regions of Sweden and Åland Islands were Borrelia infected. This is considerably higher than the overall mean prevalence of Borrelia infection in ticks in Europe (13.6%) [10]. In agreement with previous reports [10], adult ticks were more often infected than nymphs, which is probably related to the higher number of blood meals ingested by the adult ticks. None of the larvae was infected with Borrelia, which is in agreement with previous results indicating that species belonging to the B. burgdorferi sl complex lack a trans-ovarial transmission route [29].

The regions of Åland Islands and Southernmost Sweden are estimated to have different incidences of LB. However, the tick populations collected from these regions exhibited only minor differences in the prevalence of Borrelia; therefore, it appears insufficient to use the Borrelia prevalence in a tick population as the only indicator to reflect the incidence of LB among humans. This indicates that other factors such as tick density, human behavior, exposure to ticks and frequency of tick bites may influence the incidence of LB in a region. Alternatively, since LB is not a mandatory notifiable disease in these regions, we have to rely on estimates of the LB incidences, which may be fraught with some uncertainty; it is possible that there could be a similar incidence of LB in both Åland Islands and Southernmost Sweden. Interestingly, the prevalence of Borrelia infected adult ticks collected from South Central Sweden was significantly higher (40%) compared to the prevalence found in adult ticks collected from the Åland Islands (28%). A high prevalence of Borrelia in a tick population could be due to a long season of tick activity and a low mortality rate for ticks, as well as a high access to Borrelia reservoir hosts [30]. However, the reason for the high prevalence of Borrelia infected ticks in South Central Sweden is unknown and needs to be further investigated.

Seven Borrelia species were detected. B. afzelii was the dominating species, which has frequently been associated to have rodent populations as reservoir hosts [31,32]. Thereafter followed B. garinii and B. valaisiana; two species that have been linked to avian populations [31]. Interestingly, B. afzelii was significantly more prevalent in nymphs than in adult ticks, while B. garinii and B. valaisiana showed the opposite pattern, indicating different host preferences of ticks according to their life stages. Nymphs are developed from larvae, which frequently feed on small rodents [33], and adult ticks are developed from nymphs, which frequently feed on birds [34]. B. burgdorferi ss, a species that has the ability to persist in a wide range of vertebrates [9], was also detected. However, to estimate the absolute contribution of a reservoir host to the prevalence of Borrelia infection in a tick population, one must first accurately identify the host, which could be achieved by analyzing the ticks’ previous blood-meal [35]. In the present study, B. miyamotoi, B. lusitaniae, and, for the first time in ticks collected from Sweden and Åland Islands, B. spielmanii were all detected in a low number of ticks and were geographically restricted. There could be many reasons for this, e.g. these species might have a narrow spectrum of reservoir hosts in the studied areas or they might have lower transmission efficiency from reservoir hosts to ticks. Only 1% of all ticks contained a mixed Borrelia infection, which corroborates previous reports from the Nordic countries Sweden and Norway [2,36], but is lower than the average percentage (13%) reported in a meta-analysis of European studies [10]. The reason for the lower occurrence of mixed Borrelia infection in ticks from the Nordic countries is unknown, but it may be due to elimination of existing Borrelia infections during tick feeding [8,9]. We also detected Borrelia that could not be determined to species with the use of the IGS sequences, probably due to the low number of Borrelia cells found in those ticks.

Even though no significant differences in prevalence of Borrelia species between the different regions were noticed, higher proportions of B. garinii infected adult ticks and nymphs were found in Åland Islands, compared to the regions in Sweden (Table 1). Further, 80% (5 of 6) of the ticks infected with B. spielmanii were collected from the Åland Islands. Notably, 70% (8 of 11) of the ticks infected with the RF-causing B. miyamotoi were collected from the region in South Central Sweden (Table 1), where clinicians should be advised to be observant for the signs and symptoms of RF among patients. In Northern Sweden, the prevalence of Borrelia in ticks was low (11%). However, this figure is uncertain since few ticks (n = 19) were collected from this region. The low number of ticks collected probably reflect the low abundance of the ticks in this region [37].

The number of Borrelia cells in the infected ticks ranged from fewer than ten to more than a million. The ticks infected with B. miyamotoi had a significantly higher Borrelia load per tick compared to the ticks infected with other Borrelia species (Figure 3). The reason for this is unknown but the ticks infected with B. miyamotoi could have obtained a higher load of this species from their previous blood meal; reservoir hosts for B. miyamotoi have shown to contain a higher load of B. miyamotoi in their blood compared to other Borrelia species [38]. Another explanation for the high load of Borrelia cells found in the B. miyamotoi infected ticks could be that this species has a high survival rate during the tick molting process. However, this has never been investigated. Nevertheless, a high load of Borrelia in a feeding tick could facilitate a transmission of Borrelia spirochetes from tick to host [19].

We examined how the Borrelia load was influenced by the duration of tick feeding. After 36 hours of feeding, adult female ticks infected with B. afzelii and B. garinii contained a significantly lower Borrelia load compared to ticks with a shorter duration of feeding than 36 hours. The reason for this is unknown, but one could speculate that the lower Borrelia loads, observed in adult female ticks after 36 hours of feeding, reflect a transmission of these Borrelia species from tick to human. De Silva et al. (1995) observed that B. burgdorferi ss in Ixodes nymphs migrated from the gut to the salivary glands of the tick approximately 36 hours after they started to feed on mice [39]. However, to conclude that the present observation was a process of transmission, one would have to investigate the number of Borrelia cells an infected tick transmit when feeding on humans. When we first observed these differences in the Borrelia load, we questioned whether the lower Borrelia loads quantified in the adult female ticks that had fed for more than 36 hours represented PCR inhibition due to the greater volume of blood present. However, when evaluating the real-time PCR assay on fully engorged ticks spiked with Borrelia bacteria, no signs of inhibition were noticed. On the other hand, three days may have elapsed between sampling and freezing of ticks at -70 °C. This delay may have led to a higher degradation of Borrelia bacteria in ticks that had ingested a greater volume of blood, thus a higher level of innate immune cells and elements of the complement cascade. Compared to adult female ticks, nymphs ingest a smaller volume of blood. In addition, we did not observe a significant reduction of the Borrelia load in nymphs that had fed for more than 36 hours.

This study, which was conducted during two subsequent seasons, represents a “snapshot” of the prevalence and the diversity of Borrelia species in ticks that have fed on humans. However, to detect data trends in temporal and spatial shifts regarding these parameters, long-term studies are needed. A longitudinal study can also reveal the effect of weather and climate on the distribution of LB. Further, to predict and prevent human risk of exposure to LB, it is important to identify the Borrelia reservoir hosts and their abundance and distribution. It is also important to further investigate what impact the Borrelia load in a feeding tick might have on the probability of infection in humans. This could be achieved by relating the Borrelia load in the ticks to the serological and clinical response of the bitten persons.

Supporting Information

Figure S1.

Comparison of quantification cycle (Cq) values for 16S rRNA (RTNA) and 16S rRNA gene (DNA) using LUX real-time PCR assay. Numbers of spiked Borrelia cells before total NA and DNA extraction are specified in the plot.

doi:10.1371/journal.pone.0081433.s001

(TIF)

Acknowledgments

We would like to thank all participants of the study as well as the staff at the PHCs involved in the TBD STING-study. We would also like to thank the TBD STING-study group, consisting of Clas Ahlm, Johan Berglund, Sten-Anders Carlsson, Christina Ekerfelt, Mats Haglund, Anna J Henningsson, Peter Nolskog, Dag Nyman, and Katarina Ornstein for all the valuable work and advice on the study design. A special thanks to Liselott Lindvall, Susanne Olausson, and Mari-Anne Åkeson for their continuous work with the study collection logistics. We would also like to thank Johan Nordgren and Andrew Jenkins for critical and constructive feedback on the manuscript.

Author Contributions

Conceived and designed the experiments: PW PL LF JE PF PEL. Performed the experiments: PW PL LF. Analyzed the data: PW PL LF JE PF PEL. Contributed reagents/materials/analysis tools: PF PEL. Wrote the manuscript: PW PL LF JE PF PEL.

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