Surveillance of tick-borne encephalitis in emerging risk areas in southern Sweden: a retrospective case finding study.

Tick-borne encephalitis (TBE) is an emerging infection causing CNS infection of various severity. Good knowledge of the incidence in the population and defined risk areas is important in risk communication and vaccination recommendations. The aim of this study was to investigate potential underreporting by retrospectively diagnose TBE among patients with viral CNS infections of unknown etiology in a region with emerging risk areas for TBE, and define variables associated with performed TBE serology at the time of infection. Epidemiological data and microbiological diagnostics of cases with viral CNS infection of unknown etiology treated at departments of infectious diseases and pediatrics in Skåne County during 2000-2012 were investigated. Analyses to evaluate variables associated with performed TBE serology at the time of infection were performed. Retrospective TBE serology was performed on stored blood samples when available. TBE serology was already performed at the time of CNS infection in 193 out of 761 cases. Department, type of clinical manifestation, time period of illness, and whether Borrelia serology had been performed were independent variables associated with having had TBE serology performed or not at the time of illness. Only one of 137 cases, where samples could be retrospectively analyzed for TBE, turned out positive. This study shows a low frequency of TBE sampling among patients with meningoencephalitis in a region with emerging risk for TBE. A higher awareness of TBE as differential diagnosis could contribute to earlier detection of new risk areas and adequate preventive advice to the public.

Cytokine responses of immunosuppressed and immunocompetent patients with Neoehrlichia mikurensis infection.

PURPOSE: The tick-borne bacterium Neoehrlichia mikurensis causes the infectious disease neoehrlichiosis in humans. Vascular endothelium is one of the target cells of the infection. Neoehrlichiosis patients with compromised B cell immunity present with more severe inflammation than immunocompetent patients. The aim of this study was to compare the cytokine profiles of immunocompetent and immunosuppressed patients with neoehrlichiosis. METHODS: Blood samples from Swedish and Norwegian immunosuppressed (N = 30) and immunocompetent (N = 16) patients with neoehrlichiosis were analyzed for the levels of 30 cytokines, using a multiplex cytokine assay and ELISA. A gender-matched healthy control group (N = 14) was analyzed in parallel. Data were analyzed using the multivariate method OPLS-DA. RESULTS: The multiplex cytokine analyses generated more cytokine results than did the uniplex ELISA analyses. Multivariate analysis of the multiplex cytokine results established that increased levels of FGF2, GM-CSF, CXCL10, and IFN-γ were associated with immunosuppressed patients, whereas increased levels of IL-15 and VEGF were associated with immunocompetent neoehrlichiosis patients. When multivariate analysis findings were confirmed with uniplex ELISA, it was found that both groups of patients had similarly elevated levels of VEGF, FGF2 and IFN-γ. In contrast, the immunosuppressed patients had clearly elevated levels of CXCL10, CXCL13 and BAFF, whereas the immunocompetent patients had the same levels as healthy controls. CONCLUSION: Pro-angiogenic and type 1 cytokines were produced as part of the host response of neoehrlichiosis independent of immune status, whereas immunosuppressed neoehrlichiosis patients produced cytokines required for B cell-mediated defense.

Lyme neuroborreliosis in Swedish children-PCR as a complementary diagnostic method for detection of Borrelia burgdorferi sensu lato in cerebrospinal fluid.

The aim of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for the detection of Borrelia burgdorferi s.l. in CSF of Swedish children with LNB. This study was performed retrospectively on CSF and serum samples collected from children evaluated for LNB (n = 233) and controls with other specific neurological disorders (n = 59) in a Swedish Lyme endemic area. For anti-Borrelia antibody index, the IDEIA Lyme Neuroborreliosis kit (Oxoid) was used. Two in-house real-time PCR assays targeting the 16S rRNA gene were evaluated (TaqMan® and LUX™). Among patients classified as LNB cases (n = 102), five children (5%) were Borrelia PCR-positive in CSF with the TaqMan® assay. In the Non-LNB group (n = 131), one patient was Borrelia PCR positive with the TaqMan® assay. Among controls (n = 59), all CSF samples were PCR negative. When amplifying and sequencing ospA, we found B. garinii (n = 2), B. afzelii (n = 2), B. bavariensis (n = 1), and one untypable (n = 1). With the LUX™ technology, all CSF samples were PCR negative. The TaqMan® assay could detect only few cases (n = 6) of B. burgdorferi s.l. in CSF among children with LNB and the sensitivity was very low (5%). However, using larger CSF volumes and centrifugation of samples, the PCR technique could still be useful as a complementary diagnostic method when evaluating LNB. Furthermore, detection of spirochete DNA in clinical matrices, including CSF, is the method of choice for studying epidemiological aspects of LNB, a tick-borne emerging disease.

Tick-transmitted co-infections among erythema migrans patients in a general practice setting in Norway: a clinical and laboratory follow-up study.

BACKGROUND: Erythema migrans (EM) is the most common manifestation of Lyme borreliosis. Here, we examined EM patients in Norwegian general practice to find the proportion exposed to tick-transmitted microorganisms other than Borrelia, and the impact of co-infection on the clinical manifestations and disease duration. METHODS: Skin biopsies from 139/188 EM patients were analyzed using PCR for Neoehrlichia mikurensis, Rickettsia spp., Anaplasma phagocytophilum and Babesia spp. Follow-up sera from 135/188 patients were analyzed for spotted fever group (SFG) Rickettsia, A. phagocytophilum and Babesia microti antibodies, and tested with PCR if positive. Day 0 sera from patients with fever (8/188) or EM duration of ≥ 21 days (69/188) were analyzed, using PCR, for A. phagocytophilum, Rickettsia spp., Babesia spp. and N. mikurensis. Day 14 sera were tested for TBEV IgG. RESULTS: We detected no microorganisms in the skin biopsies nor in the sera of patients with fever or prolonged EM duration. Serological signs of exposure against SFG Rickettsia and A. phagocytophilum were detected in 11/135 and 8/135, respectively. Three patients exhibited both SFG Rickettsia and A. phagocytophilum antibodies, albeit negative PCR. No antibodies were detected against B. microti. 2/187 had TBEV antibodies without prior immunization. There was no significant increase in clinical symptoms or disease duration in patients with possible co-infection. CONCLUSIONS: Co-infection with N. mikurensis, A. phagocytophilum, SFG Rickettsia, Babesia spp. and TBEV is uncommon in Norwegian EM patients. Despite detecting antibodies against SFG Rickettsia and A. phagocytophilum in some patients, no clinical implications could be demonstrated.

Validating a common tick survey method: cloth-dragging and line transects.

Cloth-dragging is the most widely-used method for collecting and counting ticks, but there are few studies of its reliability. By using cloth-dragging, we applied a replicated line transects survey method, in two areas in Sweden with different Ixodes ricinus tick-densities (low at Grimsö and high at Bogesund) to evaluate developmental stage specific repeatability, agreement and precision in estimates of tick abundance. 'Repeatability' was expressed as the Intraclass Correlation Coefficient (ICC), 'agreement' with the Total Deviation Index (TDI) and 'precision' by the coefficient of variation (CV) for a given dragging distance. Repeatability (ICC) and agreement (TDI) were higher for the most abundant instar (nymphs) and in the area of higher abundance. At Bogesund tick counts were higher than at Grimsö and so also repeatability, with fair to substantial ICC estimates between 0.22 and 0.75, and TDI ranged between 1 and 44.5 counts of difference (thus high to moderate agreement). At Grimsö, ICC was poor to moderate and ranged between 0 and 0.59, whereas TDI remained low with estimates lower or equal to 1 count (thus high agreement). Despite a 100-fold lower abundance at Grimsö, the same level of precision for nymphs could be achieved with a 70% increase of dragging effort. We conclude that the cloth-dragging technique is useful for surveying ticks' and primarily to estimate abundance of the nymphal stage, whereas it rarely will be recommended for larvae and adults.

Rejection of the name Borreliella and all proposed species comb. nov. placed therein.

Rejection (nomen rejiciendum) of the name Borreliella and all new combinations therein is being requested on grounds of risk to human health and patient safety (Principle 1, subprinciple 2 and Rule 56a) and violation to aim for stability of names, to avoid useless creation of names (Principle 1, subprinciple 1 and 3) and that names should not be changed without sufficient reason (Principle 9 of the International Code of Nomenclature of Prokaryotes).

Serological diagnostics of Lyme borreliosis: comparison of assays in twelve clinical laboratories in Northern Europe.

Lyme borreliosis (LB), caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, is the most common tick-borne infection in Europe. Laboratory diagnosis of LB is mainly based on the patients' medical history, clinical signs and symptoms in combination with detection of Borrelia-specific antibodies where indirect enzyme-linked-immunosorbent assay (ELISA) is the most widely used technique. The objective of the study was to evaluate and compare the diagnostic accuracy (sensitivities and specificities) of serological tests that are currently in use for diagnosis of LB in clinical laboratories in Northern Europe, by use of a large serum panel. The panel consisted of 195 serum samples from well-characterized and classified patients under investigation for clinically suspected LB (n = 59) including patients with Lyme neuroborreliosis, Lyme arthritis, acrodermatitis chronica atrophicans, erythema migrans or other diseases (n = 112). A total of 201 serum samples from healthy blood donors were also included. The panel (396 serum samples altogether) was sent to 12 clinical laboratories (using five different ELISA methods) as blinded for group affiliation and the laboratories were asked to perform serological analysis according to their routine procedure. The results from the study demonstrated high diagnostic concordance between the laboratories using the same diagnostic assay and lower diagnostic concordance between laboratories using different diagnostic assays. For IgG, the results were in general rather homogenous and showed an average sensitivity of 88% (range 85-91%) compared to IgM which showed lower average sensitivity of 59% (range 50-67%) and more heterogeneous results between assays and laboratories.

Alkhurma Hemorrhagic Fever Virus RNA in Hyalomma rufipes Ticks Infesting Migratory Birds, Europe and Asia Minor.

Alkhurma hemorrhagic fever virus RNA was detected in immature Hyalomma rufipes ticks infesting northward migratory birds caught in the North Mediterranean Basin. This finding suggests a role for birds in the ecology of the Alkhurma hemorrhagic fever virus and a potential mechanism for dissemination to novel regions. Increased surveillance is warranted.

Human Tick-Borne Encephalitis and Characterization of Virus from Biting Tick.

We report a case of human tick-borne encephalitis (TBE) in which the TBE virus was isolated from the biting tick. Viral growth and sequence were characterized and compared with those of a reference strain. Virus isolation from ticks from patients with TBE may offer a new approach for studies of epidemiology and pathogenicity.

Norovirus Dynamics in Wastewater Discharges and in the Recipient Drinking Water Source: Long-Term Monitoring and Hydrodynamic Modeling.

Norovirus (NoV) that enters drinking water sources with wastewater discharges is a common cause of waterborne outbreaks. The impact of wastewater treatment plants (WWTPs) on the river Göta älv (Sweden) was studied using monitoring and hydrodynamic modeling. The concentrations of NoV genogroups (GG) I and II in samples collected at WWTPs and drinking water intakes (source water) during one year were quantified using duplex real-time reverse-transcription polymerase chain reaction. The mean (standard deviation) NoV GGI and GGII genome concentrations were 6.2 (1.4) and 6.8 (1.8) in incoming wastewater and 5.3 (1.4) and 5.9 (1.4) log10 genome equivalents (g.e.) L-1 in treated wastewater, respectively. The reduction at the WWTPs varied between 0.4 and 1.1 log10 units. In source water, the concentration ranged from below the detection limit to 3.8 log10 g.e. L-1. NoV GGII was detected in both wastewater and source water more frequently during the cold than the warm period of the year. The spread of NoV in the river was simulated using a three-dimensional hydrodynamic model. The modeling results indicated that the NoV GGI and GGII genome concentrations in source water may occasionally be up to 2.8 and 1.9 log10 units higher, respectively, than the concentrations measured during the monitoring project.

Infections with Candidatus Neoehrlichia mikurensis and Cytokine Responses in 2 Persons Bitten by Ticks, Sweden.

The prevalence of Candidatus Neoehrlichia mikurensis infection was determined in 102 persons bitten by ticks in Sweden. Two infected women had erythematous rashes; 1 was co-infected with a Borrelia sp., and the other showed seroconversion for Anaplasma phagocytophilum. Both patients had increased levels of Neoehrlichia DNA and serum cytokines for several months.

The NeBoP score - a clinical prediction test for evaluation of children with Lyme Neuroborreliosis in Europe.

BACKGROUND: The diagnosis of Lyme neuroborreliosis (LNB) in Europe is based on clinical symptoms and laboratory data, such as pleocytosis and anti-Borrelia antibodies in serum and CSF according to guidelines. However, the decision to start antibiotic treatment on admission cannot be based on Borrelia serology since results are not available at the time of lumbar puncture. Therefore, an early prediction test would be useful in clinical practice. The aim of the study was to develop and evaluate a clinical prediction test for children with LNB in a relevant European setting. METHOD: Clinical and laboratory data were collected retrospectively from a cohort of children being evaluated for LNB in Southeast Sweden. A clinical neuroborreliosis prediction test, the NeBoP score, was designed to differentiate between a high and a low risk of having LNB. The NeBoP score was then prospectively validated in a cohort of children being evaluated for LNB in Central and Southeast Sweden (n = 190) and controls with other specific diagnoses (n = 49). RESULTS: The sensitivity of the NeBoP score was 90 % (CI 95 %; 82-99 %) and the specificity was 90 % (CI 95 %; 85-96 %). Thus, the diagnostic accuracy (i.e. how the test correctly discriminates patients from controls) was 90 % and the area under the curve in a ROC analysis was 0.95. The positive predictive value (PPV) was 0.83 (CI 95 %; 0.75-0.93) and the negative predictive value (NPV) was 0.95 (CI 95 %; 0.90-0.99). CONCLUSION: The overall diagnostic performance of the NeBoP score is high (90 %) and the test is suggested to be useful for decision-making about early antibiotic treatment in children being evaluated for LNB in European Lyme endemic areas.

Coinfection of Babesia and Borrelia in the Tick Ixodes ricinus-A Neglected Public Health Issue in Europe?

Ixodes ricinus nymphs and adults removed from humans, and larvae and nymphs from birds, have been analysed for infection with Babesia species and Borrelia species previously in separately published studies. Here, we use the same data set to explore the coinfection pattern of Babesia and Borrelia species in the ticks. We also provide an overview of the ecology and potential public health importance in Sweden of I. ricinus infected both with zoonotic Babesia and Borrelia species. Among 1952 nymphs and adult ticks removed from humans, 3.1% were PCR-positive for Babesia spp. Of these Babesia-positive ticks, 43% were simultaneously Borrelia-positive. Among 1046 immatures of I. ricinus removed from birds, 2.5% were Babesia-positive, of which 38% were coinfected with Borrelia species. This study shows that in I. ricinus infesting humans or birds in Sweden, potentially zoonotic Babesia protozoa sometimes co-occur with human-pathogenic Borrelia spp. Diagnostic tests for Babesia spp. infection are rarely performed in Europe, and the medical significance of this pathogen in Europe could be underestimated.

Anaplasma phagocytophilum in Ticks Blood-Feeding on Migratory Birds in Sweden.

Migratory birds play a dual role as potential reservoirs of tick-borne pathogens, and potential dispersers of pathogen-containing ticks during their migratory journeys. Ixodes ricinus, a prevalent tick species in Northern and Western Europe, serves as a primary vector for Anaplasma phagocytophilum-a bacterium with implications for human and animal health. There is limited information available regarding A. phagocytophilum in birds. Our investigation focused on A. phagocytophilum prevalence in ticks collected from migratory birds in southeastern Sweden. The identification of ticks involved both molecular analyses for species determination and morphological classification to ascertain the developmental stage. The presence of A. phagocytophilum was determined using real-time PCR. Of the 1115 ticks analyzed from 4601 birds, 0.9% (n = 10), including I. ricinus and Ixodes frontalis, tested positive for A. phagocytophilum. Notably, common blackbirds (Turdus merula) yielded the highest number of A. phagocytophilum-infected ticks. The findings suggest that A. phagocytophilum is present in a small proportion of ticks infesting migratory birds in southeastern Sweden. Consequently, the role of birds as hosts for ticks infected with A. phagocytophilum appears to be low, suggesting that birds seem to play a minor indirect role in the geographic dispersal of A. phagocytophilum.

How to Detect Antibodies Against Babesia divergens in Human Blood Samples.

Background: Today only indirect fluorescent antibody assays (IFAs) are commercially available to detect antibodies against Babesia divergens in humans. IFA is subjective and requires highly experienced staff. We have therefore developed an enzyme-linked immunosorbent assay (ELISA)-based method for measuring anti-B. divergens immunoglobulin G antibodies in human blood samples. Methods: Crude merozoite extract from in vitro cultures of a new B. divergens isolate was used in ELISA to detect antibodies in different sets of samples: Borrelia burgdorferi-positive samples, healthy individuals, tick-bitten individuals including follow-up samples 3 months later, positive control samples from patients with an active Babesia infection, and samples from malaria-endemic regions. As a reference, IFA was used to detect antibodies in the tick-bitten samples. Western blot was used to evaluate reactions against specific bands in extracts with/without parasites. Results: Using IFA as the reference method, the sensitivity and specificity of the ELISA were 86% (12/14) and 100% (52/52). There was a very high correlation (r = -0.84; P = .0004) between IFA dilution factors and ELISA absorbances among the samples classified as positive. Five percent of the B. burgdorferi-positive samples were judged as weakly positive and 5% as strongly positive in our ELISA. Western blot showed that the immunodominant antigens (∼120 kDa) were from merozoites and not from erythrocytes. Conclusions: This ELISA can detect antibodies directed against B. divergens, and it can be a useful and easy assay to handle compared with IFA. The ELISA can also measure high and low levels of antibodies, which could give insight into the recency of a B. divergens infection.

Tick-borne diseases in the North Sea region-A comprehensive overview and recommendations for diagnostics and treatment.

As part of the NorthTick project, co-funded by the European Union through the European Regional Development Fund and the North Sea Region Programme, specialists in the field of tick-borne diseases from seven North Sea countries co-operated with patient organisations and governmental health care institutions to provide this comprehensive overview of diagnostics and treatment recommendations in the region for Lyme borreliosis, Borrelia miyamotoi infection, tick-borne encephalitis, human granulocytic anaplasmosis, rickettsiosis, neoehrlichiosis and babesiosis. The main conclusion is that the recommendations in these northern countries are essentially the same, with very few differences. This overview presents the current diagnostics and provides useful clinical guidance.

Low level of antimicrobial resistance in Escherichia coli among Swedish nursing home residents.

BACKGROUND: Screening for bacterial colonization and antimicrobial resistance (AMR) among a defined population could aid in the identification of at-risk populations and provide targets for antibiotic stewardship and infection control programmes. METHODS: Two hundred and sixty-eight participants at 11 Swedish nursing homes underwent serial screening for colonization with Escherichia coli between March 2008 and September 2010. Seventy-two of the 268 participants (27%) were male. The median age was 85 y. Samples were collected from urine, the rectal mucosa, the groin, and active skin lesions. RESULTS: Two hundred and nine of 268 participants (78%) were colonized with E. coli at any body site/fluid. The specific colonization rates were 81% (rectum), 48% (urine), 30% (groin), 59% (unknown), and 13% (skin lesion). An antibiotic-resistant E. coli isolate was identified in 18% of all participants regardless of colonization status; all together, 87 resistant isolates were detected. Only 1 participant carried isolates with resistance to third-generation cephalosporins (cefotaxime and ceftazidime). CONCLUSIONS: The presence of resistance was generally low, and the greater part of the resistant cases was connected with 3 common antibiotics: ampicillin, trimethoprim/sulfamethoxazole, and ciprofloxacin. In spite of generally increasing resistance against third-generation cephalosporins in E. coli in Sweden, this study does not implicate residence at a Swedish nursing home as a risk factor for the acquisition of expressed cephalosporin resistance.

Winter activity of Ixodes ricinus in Sweden.

BACKGROUND: In Europe, Ixodes ricinus (Acari: Ixodidae) is the most widespread and abundant tick species, acting as a vector for several microorganisms of medical and veterinary importance. In Northern and Central Europe, the tick has a bimodal activity pattern consisting of a peak in spring to the beginning of summer and a second peak at the end of summer. However, several findings of ticks on animals during winter have been reported, which raises the question of whether this is an overwintering strategy or whether ticks are active during winter in Scandinavia. The objectives of our study were to determine (i) whether ticks were active and finding hosts during winter, (ii) whether they parasitize their hosts, and (iii) what climatic factors-i.e., temperature, snow depth and precipitation-govern tick winter activity. METHODS: Throughout three winter seasons, we examined wild-living and free-ranging roe deer (Capreolus capreolus) for ticks on 332 occasions. In total, 140 individual roe deer were captured in two climatically contrasting sites in south-central Sweden, Grimsö and the Bogesund research area, respectively. We re-examined individual roe deer up to 10 times within the same winter or approximately once a week (mean 10 days, median 7 days between re-examinations) and recorded the absence or presence of ticks on the animals, and tested to what extent meteorological factors affected tick activity. To determine the attachment day, we used the coxal/scutal index of 18 nymphs and 47 female ticks. RESULTS: In total, 243 I. ricinus were collected from 301 roe deer captures between 14 December and 28 February at the Bogesund study site during three subsequent years (2013/2014-2015/2016). We found attached ticks every third to every second examination (32%, 48% and 32% of the examinations, respectively). However, we collected only three I. ricinus females from 31 roe deer captures at the Grimsö study site between 17 December 2015 and 26 February 2016. At the Bogesund study site, based on 192 captures of previously examined deer, we collected 121 ticks, and ticks were found at 33%, 48% and 26% of the examinations during the respective winters. The probability of finding an attached tick on a roe deer at a temperature of -5 °C was > 8% ± 5 (SE), and that probability increased to almost 20% ± 7 (SE) if the air temperature increased to 5 °C. CONCLUSIONS: To the best of our knowledge, this is the first time that winter-active nymphs and female ticks have been documented to attach and feed on roe deer during winter (December to February) in Scandinavia. The main weather conditions regulating winter activity for females were temperature and precipitation, and the lowest estimated air temperature for finding an active tick was well below 5 °C. The behaviour of winter-active and blood-feeding ticks was documented over several winters and in two contrasting areas, implying that it is a common phenomenon that should be investigated more thoroughly, since it may have important consequences for the epidemiology of tick-borne pathogens.

Candidatus Rickettsia Vini DNA in Ticks Collected from Nest Burrows of the European Sand Martin (Riparia riparia) in Sweden.

Background: Birds can cross geographical and environmental barriers and thereby facilitate dispersal of tick-borne pathogens both as carriers of infected ticks and as reservoirs of pathogenic microorganisms. Ixodes lividus (Ixodida: Ixodidae) is an endophilic tick in the Palearctic region that is highly specialized on its host, the European sand martin Riparia riparia. The purpose of this study was to determine whether I. lividus ticks sampled from sand martin nests in Sweden carry vector-borne pathogens. Materials and Methods: Fed ticks were collected in the autumns of 2017 and 2019 from the nests of a European sand martin colony in southern Sweden. Ticks were identified morphologically to developmental stage and species and were tested for tick-borne pathogens using PCR-based methods. Results: None of the 41 ticks tested positive for five tick-borne pathogens including Borrelia spp., tick-borne encephalitis virus (TBEV), Neoehrlichia mikurensis, Anaplasma phagocytophilum, and Babesia spp. Thirty-seven (13 females, 23 nymphs and 1 larva) of the 41 ticks tested positive for the gltA gene of Rickettsia spp. The sequences of the 17 kDa and gltA genes were most closely related to Candidatus Rickettsia vini. Conclusion: Our study confirms other reports that I. lividus ticks associated with the European sand martin have high infection prevalence of Ca. R. vini.

Characterization of the bacterial microbiome of Swedish ticks through 16S rRNA amplicon sequencing of whole ticks and of individual tick organs.

BACKGROUND: The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. METHODS: In the present study the microbial flora of whole Ixodes ricinus, I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. RESULTS: The most abundant bacteria belonged to the group of Proteobacteria (Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus-associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. CONCLUSIONS: These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models.