Vivian Kjelland

Prevalence and genotypes of Borrelia burgdorferi sensu lato infection in Ixodes ricinus ticks in southern Norway.

Abstract From April to October 2007, host-seeking Ixodes ricinus ticks were collected from 4 locations in southern Norway: Farsund, Mandal, Søgne and Tromøy. Two hundred and ten larvae, 1130 nymphs and 449 adults were investigated for infection with Borrelia burgdorferi sensu lato (s.l.) by real-time polymerase chain reaction (PCR) amplification of the 16S rRNA gene. The total percentage of B. burgdorferi s.l. in nymphal and adult ticks was determined to be 31.3% in Farsund, 25.2% in Mandal, 22.3% in Søgne and 22.1% in Tromøy. Larvae were pooled in groups of 10 before analysis, and Borrelia infection was detected in 1 of the 21 larvae pools. B. burgdorferi s.l. were genotyped by melting curve analysis after real-time PCR amplification of the hbb gene, or by direct sequencing of the PCR amplicon generated from the rrs (16S)–rrl (23S) intergenetic spacer. The most prevalent B. burgdorferi genospecies identified were B. afzelii (61.6%), followed by B. garinii (23.4%) and B. burgdorferi sensu stricto (10.6%). B. valaisiana (4.5%) was identified in Norwegian ticks for the first time. Mixed infections were observed in 0.3% of the infected ticks. A higher prevalence of B. burgdorferi s.l. was found in the present study than what has been reported in previous Nordic studies.

Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from migratory birds in Southern Norway

BackgroundBorrelia burgdorferi sensu lato (s.l.) are the causative agent for Lyme borreliosis (LB), the most common tick-borne disease in the northern hemisphere. Birds are considered important in the global dispersal of ticks and tick-borne pathogens through their migration. The present study is the first description of B. burgdorferi prevalence and genotypes in Ixodes ricinus ticks feeding on birds during spring and autumn migration in Norway.Methods6538 migratory birds were captured and examined for ticks at Lista Bird Observatory during the spring and the autumn migration in 2008. 822 immature I. ricinus ticks were collected from 215 infested birds. Ticks were investigated for infection with B. burgdorferi s.l. by real-time PCR amplification of the 16S rRNA gene, and B. burgdorferi s.l. were thereafter genotyped by melting curve analysis after real-time PCR amplification of the hbb gene, or by direct sequencing of the PCR amplicon generated from the rrs (16S)-rrl (23S) intergenetic spacer.ResultsB. burgdorferi s.l. were detected in 4.4% of the ticks. The most prevalent B. burgdorferi genospecies identified were B. garinii (77.8%), followed by B.valaisiana (11.1%), B. afzelii (8.3%) and B. burgdorferi sensu stricto (2.8%).ConclusionInfection rate in ticks and genospecies composition were similar in spring and autumn migration, however, the prevalence of ticks on birds was higher during spring migration. The study supports the notion that birds are important in the dispersal of ticks, and that they may be partly responsible for the heterogeneous distribution of B. burgdorferi s.l. in Europe.

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Ixodes ricinus ticks in Brønnøysund in northern Norway.

Ticks are important vectors of disease for both humans and animals. In Europe, Lyme borreliosis is the most abundant tick-borne human disease, whereas anaplasmosis, or tick-borne fever, is the most widespread tick-borne infection in domestic animals. However, knowledge about the prevalence of the causative disease agents in questing ticks in the northern range of their distribution in Norway is missing. Ixodes ricinus ticks were therefore collected by flagging vegetation in Brønnøysund, an area near the Arctic Circle in Norway where ticks have been abundant for decades. Ticks were analysed for infection with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by real-time PCR amplification of the 16S rRNA gene of B. burgdorferi and the msp2 gene of A. phagocytophilum. B. burgdorferi s.l. were subsequently genotyped by conducting a melt curve analysis of the PCR-amplified hbb gene or by directly sequencing the PCR-amplified rrs (16S)-rrl (23S) intergenetic spacer. A. phagocytophilum was genotyped by msp2 gene sequencing. B. burgdorferi s.l. isolates were detected in 11.3% (15/133) of the nymphal ticks and in 33.3% (29/87) of the adult ticks. Of the 44 Borrelia-infected ticks collected, B. afzelii was identified in 42 ticks (95.5%), whereas B. garinii was detected in only 2 ticks (4.5%). A. phagocytophilum was detected in 0.8% of nymphal ticks (1/133) and in 4.6% of adult ticks (4/87). Mixed infections of more than one B. burgdorferi genospecies were not observed. One adult tick was co-infected with B. afzelii and A. phagocytophilum.

Borrelia miyamotoi is widespread in Ixodes ricinus ticks in southern Norway.

From April to October 2007, host-seeking Ixodes ricinus ticks were collected from four locations in southern Norway; Farsund, Mandal, Søgne and Tromøy, respectively. Larvae (n=210), nymphs (n=1130) and adults (n=449) were investigated for infection with Borrelia miyamotoi by real-time polymerase chain reaction (PCR) amplification of part of the 16S rRNA gene. Results were verified by direct sequencing of the PCR amplicon generated from the rrs (16S)-rrl (23S) intergenetic spacer. B. miyamotoi was detected at all sites and throughout the period of questing activity, with infection prevalence (≤1.26%) similar to what has been seen in other European countries. Detection of the relapsing fever spirochete at all locations indicates a wide distribution in southern Norway. This is the first report of B. miyamotoi prevalence in ticks collected from Norway. As not much is known about the spatiotemporal dynamics of this relatively recently discovered pathogen, the conclusions of this study significantly add to the knowledge regarding B. miyamotoi in this region.

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks collected from moose (Alces alces) and roe deer (Capreolus capreolus) in southern Norway

As part of a larger survey, ears from 18 roe deer (Capreolus capreolus) and 52 moose (Alces alces) shot in the 2 southernmost counties in Norway were collected and examined for Ixodes ricinus ticks. Seventy-two adult ticks, 595 nymphs, and 267 larvae from the roe deer, and 182 adult ticks, 433 nymphs, and 70 larvae from the moose were investigated for infection with Borrelia burgdorferi sensu lato (s.l.). The results showed the presence of B. burgdorferi s.l. DNA in 2.9% of the nymphs collected from roe deer and in 4.4% of the nymphs and 6.0% of the adults collected from moose. The spirochetes were not detected in adult ticks from roe deer, or in larvae feeding on roe deer or moose. In comparison, the mean infection prevalences in questing I. ricinus collected from the same geographical area were 0.5% infection in larvae, 24.5% in nymphs, and 26.9% in adults. The most prevalent B. burgdorferi genospecies identified in ticks collected from roe deer was B. afzelii (76.5%), followed by B. garinii (17.6%), and B. burgdorferi sensu stricto (5.9%). Only B. afzelii (76.7%) and B. garinii (23.3%) were detected in ticks collected from moose. The present study indicates a lower prevalence of B. burgdorferi infection in I. ricinus ticks feeding on roe deer and moose compared to questing ticks. This is the first study to report B. burgdorferi s.l. prevalence in ticks removed from cervids in Norway.

Tick-borne bacteria in Ixodes ricinus collected in southern Norway evaluated by a commercial kit and established real-time PCR protocols

Ticks are important vectors of human pathogens. The knowledge of disease causing agents harboured by ticks in Norway is limited. The focus of this study was (a) to detect the bacteria of medical importance in ticks collected from the vegetation at locations in the southern part of the country and (b) to evaluate a novel commercially available multiplex PCR based method by comparing results with conventional established real-time PCR protocols. Borrelia burgdorferi sensu lato was confirmed to be the most prevalent pathogen detected (31%) among one hundred individually analysed adult ticks. Borrelia miyamotoi, a spirochete associated with relapsing fever, was detected in one sample. Anaplasma phagocytophilum was found in 4% of the ticks, followed by Rickettsia helvetica which was detected in one sample. Similar pathogen prevalence was also detected in 500 ticks analysed in pools. This is the first report of the spotted fever group Rickettsia in Norway. Francisella tularensis, Bartonella species or Coxiella burnetti was not detected. However, due to the low number of ticks analysed, the possible presence of these pathogens in the region cannot be ruled out. All isolates were screened by at least two different molecular methods for each bacterial target; one commercially available multiplex PCR based tick-borne bacteria flow chip system (Master Diagnostica) and corresponding real-time PCR protocols. The comparison of methods verified that most findings were detected by both methods (71 Borrelia, 15 Anaplasma and 2 Rickettsia), whereas two additional Borrelia and Anaplasma infected samples were detected by the real-time protocols.

BORRELIA BURGDORFERI SENSU LATO DETECTED IN SKIN OF NORWEGIAN MOUNTAIN HARES (LEPUS TIMIDUS) WITHOUT SIGNS OF DISSEMINATION

The mountain hare (Lepus timidus) population in southern Norway appears to be in decline. Necropsy and laboratory examinations of 36 hares found dead or diseased during 2007–2009 in Vest- and Aust-Agder counties showed that disease and deaths were attributed to multiple causes, with no specific etiology emerging as a cause for population decline. To investigate whether Borrelia burgdorferi sensu lato (s.l.) infection is associated with mortality in mountain hares, tissues and ticks collected from hares were investigated for infection with the spirochete. Borrelia burgdorferi s.l. DNA was not detected in samples from internal organs, whereas Borrelia afzelii, B. burgdorferi sensu stricto (s.s.), and the not-yet-defined Borrelia sp. SV1 were found in skin samples from hares and in adult and nymphal Ixodes ricinus feeding on hares. Only B. burgdorferi s.s. and Borrelia sp. SV1 were detected in larvae feeding on hares. Our results indicate that disseminated Borrelia infection in hares rarely occurs and, presumably, does not play a central role in the suspected population decline. The results also suggest that the mountain hare to some degree functions as a transmission host for B. burgdorferi s.s. and Borrelia sp. SV1.

Candidatus Neoehrlichia mikurensis and Borrelia burgdorferi sensu lato detected in the blood of Norwegian patients with erythema migrans

The most common tick-borne human disease in Norway is Lyme borreliosis. Ticks in Norway also harbour less known disease-causing agents such as Candidatus Neoehrlichia mikurensis, Borrelia miyamotoi and Rickettsia helvetica. However, human infections caused by these pathogens have never been described in Norway. The main aims of the study were to evaluate the contribution of several tick-borne bacterial agents, other than Borrelia burgdorferi sensu lato, to zoonotic diseases in Norway and to determine their clinical pictures. Blood samples from 70 symptomatic tick-bitten adults from the Agder counties in southern Norway were screened for seven tick-borne pathogens by using a commercial multiplex PCR-based method and by singleplex real-time PCR protocols. Most patients (65/70) presented with a rash clinically diagnosed as erythema migrans (EM). The most frequently detected pathogen DNA was from Ca. N. mikurensis and was found in the blood of 10% (7/70) of the patients. The Ca. N. mikurensis-infected patients presented with an EM-like rash as the only symptom. B. burgdorferi s.l. DNA was present in the blood of 4% (3/70) of the study participants. None had detectable Anaplasma phagocytophilum, B. miyamotoi, Rickettsia typhus group or spotted fever group, Francisella tularensis, Coxiella burnetii or Bartonella spp. DNA in the blood. The commercially available multiplex PCR bacteria flow chip system failed to identify half of the infected patients detected by corresponding real-time PCR protocols. The recovery of Ca. N. mikurensis DNA was higher in the pellet/plasma fraction of blood than from whole blood. To conclude, Ca. N. mikurensis appeared to be the etiological agent in patients with EM in a surprisingly large fraction of tick-bitten persons in the southern part of Norway.

Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood

Abstract Background A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group. Methods Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory. Results Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods. Conclusions The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

Does more favourable handling of the cerebrospinal fluid increase the diagnostic sensitivity of Borrelia burgdorferi sensu lato-specific PCR in Lyme neuroborreliosis?

Abstract Background: Tests for direct detection of Borrelia burgdorferi sensu lato (Bb) in Lyme neuroborreliosis (LNB) are needed. Detection of Bb DNA using PCR is promising, but clinical utility is hampered by low diagnostic sensitivity. We aimed to examine whether diagnostic sensitivity can be improved by the use of larger cerebrospinal fluid (CSF) volumes and faster handling of samples. Methods: Patients who underwent CSF examination for LNB were included. We collected two millilitres of CSF for PCR analysis, extracted DNA from the pellets within 24 h and analysed the eluate by two real-time PCR protocols (16S rRNA and OspA). Patients who fulfilled diagnostic criteria for LNB were classified as LNB cases and the rest as controls. Results: Bb DNA in CSF was detected by PCR in seven of 28 adults with LNB. Two were Bb antibody negative. No Bb DNA was detected in CSF from 137 controls. Diagnostic sensitivity was 25% and specificity 100%. There was a non-significant trend towards larger CSF sample volume, faster handling of the sample, shorter duration of symptoms, and higher CSF cell count in the PCR-positive cases. Conclusion: We did not find that optimized handling of CSF increased diagnostic sensitivity of PCR in adults with LNB. However, our case series is small and we hypothesize that the importance of these factors will be clarified in further studies with larger case series and altered study design. PCR for diagnosis of LNB may be useful in cases without Bb antibodies due to short duration of symptoms.