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Multiple sequence alignment of target, primer and probe sequences. Dots indicated identity to the reference sequence. Hyphens indicate regions where no sequence information is available. The following IUPAC ambiguity codes are used: Y = T/C; R = A/G; M = A/C; S = G/C; H = A/C/T; K = G/T; D = A/G/T; B = C/T/G. Variant positions are indicated by lower case letters in the consensus and primer/probe sequences. Variants accommodated by G:T basepairing or degeneracy of the primer/probe sequence are highlighted in green in the target sequence. Variants not so accommodated are highlighted in red. Primer and probe sequences are highlighted in blue. Unless otherwise noted, sequences are written 5’ – 3’. (Color figure online)

Multiple sequence alignment of target, primer and probe sequences. Dots indicated identity to the reference sequence. Hyphens indicate regions where no sequence information is available. The following IUPAC ambiguity codes are used: Y = T/C; R = A/G; M = A/C; S = G/C; H = A/C/T; K = G/T; D = A/G/T; B = C/T/G. Variant positions are indicated by lower case letters in the consensus and primer/probe sequences. Variants accommodated by G:T basepairing or degeneracy of the primer/probe sequence are highlighted in green in the target sequence. Variants not so accommodated are highlighted in red. Primer and probe sequences are highlighted in blue. Unless otherwise noted, sequences are written 5’ – 3’. (Color figure online)

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A consensus TaqMan real-time PCR test targeting the chromosomal flaB gene of Borrelia burgdorferi sensu lato was constructed. The test was compared with a recently published generic Light Upon eXtension (LUX) 16S rRNA real-time PCR test (Wilhelmsson et al. in J Clin Microbiol 48:4169-4176, 2010) on material consisting of 242 Ixodes ricinus ticks co...

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Context 1
... primers. Primer and probe design was performed using Primer Express (Applied Biosystems, Foster City, CA) with B. burgdorferi X16833 as input sequence. Variant positions were accommodated by use of the noncanonical G:T basepair, a G/C degeneracy in the forward primer and two probe sequences. Primers, probes and target sequences are shown in Fig. 1 and Table 1. It should be noted that no sequence information for the target region is available for B. americana, B. bissettii, B. californiensis, B. carolinensis, B. spielmanii, B. kurtzenbachii or B. yangtze (Stanek and Reiter 2011). Primers were obtained from Genscript Corporation (Piscataway, NJ). Taq- Man MGB probes labelled with ...
Context 2
... rRNA PCR test is a broad-specificity test designed to detect all Borrelia spp, including the relapsing fever Borreliae. The flaB PCR test is designed to detect B. burg- dorferi s.l. species. On the basis of sequence mismatches (see Fig. 1) the flaB test may have reduced sensitivity for B. sinica, B. lusitaniae, and certain variants of B. burgdorferi sensu stricto and B. valaisiana, while sequence information for the target region is unavailable for B. americana, B. bissettii, B. californiensis, B. carolinensis, B. kurtzenbachii, B. spielmanii, and B. yangtze; and ...

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... [17][18] The geographical distribution of I. ricinus in Norway has been examined in several studies. 2,[19][20][21][22][23] Both Tambs-Lyche (1943) and Mehl (1983) found I. ricinus to be mainly distributed in the coastal areas of Norway, from the southeastern border to Sweden, along the southern and western coastline, up to Nordland County at ~66°N. [19][20] The density of ticks varies between locations, even when separated by short distances. ...
... northeast) of the hitherto established distribution limit of I. ricinus in Norway, the vast majority of these are engorged females. [22][23] Migratory birds may deposit engorged larvae or nymphs in areas where temperatures permit development to the next stage but not completion of the life cycle. Thus, such records do not constitute evidence for established and sustainable tick populations as this requires the presence of all the active stages (larvae, nymphs, and adults) in a locality for at least two consecutive seasons. ...
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In Norway, tick-borne encephalitis (TBE) has been a mandatory notifiable disease since 1975 (Norwegian Surveillance system for communicable diseases, MSIS). According to ECDCs classification, coastal areas in southern Norway (counties of Agder, and Vestfold and Telemark) are endemic for TBE. Further, Viken County (former Østfold, Akershus and Buskerud), and western and northern Norway to Brønnøy municipality is imperiled.
... [17][18] The geographical distribution of I. ricinus in Norway has been examined in several studies. 2,[19][20][21][22][23] Both Tambs-Lyche (1943) and Mehl (1983) found I. ricinus to be mainly distributed in the coastal areas of Norway, from the southeastern border to Sweden, along the southern and western coastline, up to Nordland County at ~66°N. [19][20] The density of ticks varies between locations, even when separated by short distances. ...
... northeast) of the hitherto established distribution limit of I. ricinus in Norway, the vast majority of these are engorged females. [22][23] Migratory birds may deposit engorged larvae or nymphs in areas where temperatures permit development to the next stage but not completion of the life cycle. Thus, such records do not constitute evidence for established and sustainable tick populations as this requires the presence of all the active stages (larvae, nymphs, and adults) in a locality for at least two consecutive seasons. ...
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In Norway, tick-borne encephalitis (TBE) has been a mandatory notifiable disease since 1975 (Norwegian Surveillance system for communicable diseases, MSIS).1 According to ECDCs classification, coastal areas in southern Norway (counties of Agder, and Vestfold and Telemark) are endemic for TBE. Further, Viken County (former Østfold, Akershus and Buskerud), and western and northern Norway to Brønnøy municipality is imperiled.2-9
... In Norway, I. ricinus is distributed along the coast as far north as the Arctic Circle [23][24][25][26]. Regarding tick borne diseases, disseminated Lyme borreliosis and TBE are mandatory reportable to the Norwegian Surveillance System for Communicable Diseases (MSIS). ...
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Background: Tick-borne encephalitis (TBE) constitutes a public health concern in Europe. Certain coastal municipalities in southern Norway are considered TBE risk areas and in the last two years, there have been increasing numbers of TBE cases. Since the majority of infections are claimed to be asymptomatic, the aim of the current study was to assess the seroprevalence of antibodies to tick-borne encephalitis virus (TBEV) among unvaccinated adults living in a TBE endemic area in Norway. Methods: One thousand one hundred and twenty-three blood donors living in Vestfold and Telemark county were included and associated sera were analysed for TBEV IgG antibodies. Information regarding tick bites, previous flavivirus exposure and knowledge regarding TBE and TBE prevention were obtained through a questionnaire. Results: Fifty-eight samples were reactive by ELISA, of which 21 (36.2%) were confirmed by a TBEV-specific serum neutralization test. Of the 21 blood donors with neutralizing TBEV antibodies detected, 17 reported previous TBE vaccination. Thus, only four blood donors (0.4%) had TBEV neutralizing antibodies consistent with previously undergone TBEV infection. Regarding TBE awareness, half of the blood donors were familiar with TBE, but only 35% were aware of a preventive TBE vaccine. Conclusions: Our study indicates low prevalence of subclinical TBEV infections among blood donors living in Vestfold and Telemark county and there is a lack of awareness among general public.
... They can be located on the chromosome or on plasmid DNA. The most frequent chromosomal targets that have been reported in clinical studies are flagellin (26,164,(180)(181)(182), 16S rRNA gene (180,(183)(184)(185), the gene codifying for the 66 kDa protein (26,56,184,185), while the most used plasmid target is OspA (56,180,183,(186)(187)(188), which has been also reported to be more stable after degradation of spirochetes (178). At present the major concern in Borrelia diagnosis by PCR is the lack of standardization of the protocols and analyzed targets (167, 177,178). ...
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... Now, however, I. ricinus is regularly reported far outside (i.e. northeast) of the hitherto established distribution limit of I. ricinus in Norway, the vast majority as engorged female ticks (Jenkins et al., 2012;Hvidsten et al., 2014). These findings do not confirm the presence of sustainable populations of ticks, which requires the presence of all the active stages (larvae, nymphs and adults) in a locality for at least two consecutive seasons (Anonymous, 1991;Piesman, 1991). ...
... Several articles from 'Flått i Nord' covering the period 2009-2017 determined the occurrence of various pathogens in I. ricinus (Jenkins et al., 2012;Hvidsten et al., 2014;Henningsson et al., 2015;Hvidsten et al., 2015Hvidsten et al., , 2017Larsson et al., 2018). The results presented here are based on field research in the years 2010-2018, with the main objective being determination of the current distribution limit of I. ricinus, with particular reference to climate change, by means of cloth-dragging, capturing small mammals and collecting ticks from pets, and also to provide a climate-based explanation, the GSL, for the data. ...
... Reverse transcribed total nucleic acid from the ticks was extracted, purified and isolated automatically using physical and chemical methods, as previously described (Jenkins et al., 2012). All cDNA samples from the ticks were individually assayed using a Light Upon eXtension (LUX) real-time PCR targeting the 16S rRNA gene of Borrelia spp. ...
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In north-western Europe, the common tick, Ixodes ricinus, is widely established, its distribution appears to be increasing and the spread of tick-borne diseases is of increasing concern. The project ‘Flått i Nord’ (Ticks in northern Norway) commenced in spring 2009 with the intention of studying the tick’s distribution and that of its pathogens in northern Norway. Several methods were used: cloth-dragging, collecting from trapped small mammals, and collecting from pets. Since 2010, the occurrence of ticks in the region of northern Norway was determined directly by cloth-dragging 167 times in 109 separate locations between the latitudes of 64 °N and 70 °N (included seven locations in the northern part of Trøndelag County). The northernmost location of a permanent I. ricinus population was found to be Nordøyvågen (66.2204 °N, 12.59 °E) on the Island of Dønna. In a sample of 518 nymphal and adult ticks, the Borrelia prevalence collected close to this distribution limit varied but was low (1–15 %) compared with the locations in Trøndelag, south of the study area (15–27 %). Five specimens (1 %) were positive for Rickettsia helvetica. The length of the vegetation growing season (GSL) can be used as an approximate index for the presence of established populations of I. ricinus. The present study suggests that the threshold GSL for tick establishment is about 170 days, because the median GSL from 1991 to 2015 was 174–184 days at sites with permanent tick populations, showing a clear increase compared with the period 1961–1990. This apparent manifestation of climate change could explain the northward extension of the range of I. ricinus.
... In Norway, I. ricinus ticks are mainly distributed along the coastline from Østfold county in the southeast up to near the Arctic Circle (66°33′47.5″N) in Nordland county (Hvidsten et al., 2014;Jenkins et al., 2012;Mehl, 1983;Soleng et al., 2018;Tambs-Lyche, 1943). TBEV has been documented in ticks, where I.ricinus is abundant (Andreassen et al., 2012;Paulsen et al., 2015;Soleng et al., 2018). ...
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Tick‐borne encephalitis virus (TBEV) is the causative agent of tick‐borne encephalitis (TBE). TBEV is one of the most important neurological pathogens transmitted by tick bites in Europe. The objectives of this study were to investigate the seroprevalence of TBE antibodies in cervids in Norway and the possible emergence of new foci, and furthermore to evaluate if cervids can function as sentinel animals for the distribution of TBEV in the country. Serum samples from 286 moose, 148 roe deer, 140 red deer and 83 reindeer from all over Norway were collected and screened for TBE immunoglobulin G (IgG) antibodies with a modified commercial enzyme‐linked immunosorbent assay (ELISA) and confirmed by TBEV serum neutralisation test (SNT). The overall seroprevalence against the TBEV complex in the cervid specimens from Norway was 4.6%. The highest number of seropositive cervids was found in south‐eastern Norway, but seropositive cervids were also detected in southern‐ and central Norway. Antibodies against TBEV detected by SNT were present in 9.4% of the moose samples, 1.4% in red deer, 0.7% in roe deer, and nil in reindeer. The majority of the positive samples in our study originated from areas where human cases of TBE have been reported in Norway. The study is the first comprehensive screening of cervid species in Norway for antibodies to TBEV, and shows that cervids are useful sentinel animals to indicate TBEV occurrence, as supplement to studies in ticks. Furthermore, the results indicate that TBEV might be spreading northwards in Norway. This information may be of relevance for public health considerations and supports previous findings of TBEV in ticks in Norway.
... Ticks were collected from vegetation by flag-dragging [32] or from dogs and cats brought to veterinary clinics [33]. DNA was extracted by (1) manual disruption and protease digestion [2], (2) mechanical disruption, automated total nucleic acid extraction and reverse transcription of total nucleic acid [33], (3) digestion with ammonium hydroxide [34,35] or (4) phenol-chloroform extraction [36]. ...
... Ticks were collected from vegetation by flag-dragging [32] or from dogs and cats brought to veterinary clinics [33]. DNA was extracted by (1) manual disruption and protease digestion [2], (2) mechanical disruption, automated total nucleic acid extraction and reverse transcription of total nucleic acid [33], (3) digestion with ammonium hydroxide [34,35] or (4) phenol-chloroform extraction [36]. Table 1 describes the tick collections, the instar distribution and the method used for DNA extraction. ...
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Background: Candidatus Neoehrlichia mikurensis is an emerging tick-borne pathogen. It is widely distributed in Ixodes ricinus ticks in Europe, but knowledge of its distribution in Norway, where I. ricinus reaches its northern limit, is limited. In this study we have developed a real time PCR test for Ca. N. mikurensis and used it to investigate the distribution of Ca. N. mikurensis in Norway. Results: Real time PCR targeting the groEL gene was developed and shown to be highly sensitive. It was used to detect Ca. N. mikurensis in 1651 I. ricinus nymphs and adults collected from twelve locations in Norway, from the eastern Oslo Fjord in the south to near the Arctic Circle in the north. The overall prevalence was 6.5% and varied locally between 0 and 16%. Prevalence in adults and nymphs was similar, suggesting that ticks acquire Ca. N. mikurensis predominantly during their first blood meal. In addition, 123 larvae were investigated; Ca. N. mikurensis was not found in larvae, suggesting that transovarial transmission is rare or absent. Sequence analysis suggests that a single variant dominates in Norway. Conclusions: Ca. N. mikurensis is widespread and common in ticks in Norway and reaches up to their northern limit near the Arctic Circle. Ticks appear to acquire Ca. N. mikurensis during their first blood meal. No evidence for transovarial transmission was found.
... A multi-source analysis by Jore et al. (2011) suggests that tick populations in Norway have undergone recent latitudinal and altitudinal range shifts. 23 Jenkins et al. (2012) found few ticks attached to dogs and cats in the region north of 66°N, while Hvidsten et al. (2014) confirmed these findings and stated that further studies are needed in order to clarify if tick populations are established north of the Arctic Circle. 4,5 According to Soleng 6 This is a region with holiday cabins and outdoor recreation areas for both local inhabitants and tourists, and it is known for high temperatures during spring and summer. ...
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Ixodes ricinus ticks are mainly distributed along the Norwegian coastline from Østfold County in the southeast up approximately to 66°N in Nordland County.
... The procedure for extraction of total nucleic acid and cDNA synthesis has been described previously [18]. In brief, a lysis buffer containing β-mercaptoethanol was added into the tubes that contained the ticks. ...
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Background “Candidatus Neoehrlichia mikurensis” is a gram-negative bacterium belonging to the family Anaplasmataceae that, in Europe, is transmitted by Ixodes ricinus ticks. “Candidatus N. mikurensis” can cause a severe systemic inflammatory syndrome, neoehrlichiosis, mostly in persons with other underlying diseases. To date, “Ca. N. mikurensis” has been found in ticks in different countries in Asia and Europe, but never as far north as at the Arctic Circle. Methods A total of 1104 I. ricinus ticks collected from vegetation and from animals in northern Norway (64–68°N) were analysed for the prevalence of “Ca. N. mikurensis”. Of them, 495 ticks were collected from vegetation by flagging and 609 ticks were collected from dogs and cats. Total nucleic acid extracted from the ticks were converted to cDNA and analyzed with real-time PCR targeting the 16S rRNA gene of “Ca. N. mikurensis”. Positive samples were further analysed by nested PCR and sequencing. Results “Candidatus N. mikurensis” was detected in 11.2% of all collected I. ricinus ticks in northern Norway. The prevalence differed between ticks collected from vegetation (18.2%; 90/495) compared to ticks collected from dogs and cats (5.6%; 34/609). The ticks from dogs and cats were collected in Brønnøy area and seven additional districts further north. The prevalence of “Ca. N. mikurensis” in these ticks differed between geographical localities, with the highest prevalence in the Brønnøy area. Conclusions The detection of “Ca. N. mikurensis” in I. ricinus ticks from the Arctic Circle in northern Norway indicates potential risk for tick-bitten humans at this latitude to be infected with “Ca. N. mikurensis”.
... In Europe, B. garinii is the main cause of Lyme neuroborreliosis, while B. afzelii is mostly associated with skin manifestations [41,42]. Among the identified genospecies, B. valaisiana was detected in 6.4% of ticks, which is in accordance with the findings in the neighboring countries (6% in Norway, Sweden and Estonia) [30,43,44]. Even though, B. valaisiana is not often detected in I. persulcatus, it has been shown that in the sympatric areas of I. ricinus and I. persulcatus, B. valaisiana may exchange vectors and can also be found in I. persulcatus [30,45]. ...
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Background Almost 3500 tick samples, originally collected via a nationwide citizen science campaign in 2015, were screened to reveal the prevalence and distribution of a wide spectrum of established and putative tick-borne pathogens vectored by Ixodes ricinus and I. persulcatus in Finland. The unique geographical distribution of these two tick species in Finland allowed us to compare pathogen occurrence between an I. ricinus-dominated area (southern Finland), an I. persulcatus-dominated area (northern Finland), and a sympatric area (central Finland). Results Of the analysed ticks, almost 30% carried at least one pathogen and 2% carried more than one pathogen. A higher overall prevalence of tick-borne pathogens was observed in I. ricinus than in I. persulcatus: 30.0% (604/2014) versus 24.0% (348/1451), respectively. In addition, I. ricinus were more frequently co-infected than I. persulcatus: 2.4% (49/2014) versus 0.8% (12/1451), respectively. Causative agents of Lyme borreliosis, i.e. bacterial genospecies in Borrelia burgdorferi (sensu lato) group, were the most prevalent pathogens (overall 17%). “Candidatus Rickettsia tarasevichiae” was found for the first time in I. ricinus ticks and in Finnish ticks in general. Moreover, Babesia divergens, B. venatorum and “Candidatus Neoehrlichia mikurensis” were reported for the first time from the Finnish mainland. Conclusions The present study provides valuable information on the prevalence and geographical distribution of various tick-borne pathogens in I. ricinus and I. persulcatus ticks in Finland. Moreover, this comprehensive subset of ticks revealed the presence of rare and potentially dangerous pathogens. The highest prevalence of infected ticks was in the I. ricinus-dominated area in southern Finland, while the prevalence was essentially equal in sympatric and I. persulcatus-dominated areas. However, the highest infection rates for both species were in areas of their dominance, either in south or north Finland.