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Both early localized and late disseminated forms of Lyme borreliosis are caused by Borrelia burgdorferi senso lato. Differentiating between the spirochetes that only cause localized skin infection from those that cause disseminated infection, and tracing the group of medically-important spirochetes to a specific vertebrate host species, are two critical issues in disease risk assessment and management. Borrelia burgdorferi senso lato isolates from Lyme borreliosis cases with distinct clinical manifestations (erythema migrans, neuroborreliosis, acrodermatitis chronica atrophicans, and Lyme arthritis) and isolates from Ixodes ricinus ticks feeding on rodents, birds and hedgehogs were typed to the genospecies level by sequencing part of the intergenic spacer region. In-depth molecular typing was performed by sequencing eight additional loci with different characteristics (plasmid-bound, regulatory, and housekeeping genes). The most abundant genospecies and genotypes in the clinical isolates were identified by using odds ratio as a measure of dominance. Borrelia afzelii was the most common genospecies in acrodermatitis patients and engorged ticks from rodents. Borrelia burgdorferi senso stricto was widespread in erythema migrans patients. Borrelia bavariensis was widespread in neuroborreliosis patients and in ticks from hedgehogs, but rare in erythema migrans patients. Borrelia garinii was the dominant genospecies in ticks feeding on birds. Spirochetes in ticks feeding on hedgehogs were overrepresented in genotypes of the plasmid gene ospC from spirochetes in erythema migrans patients. Spirochetes in ticks feeding on hedgehogs were overrepresented in genotypes of ospA from spirochetes in acrodermatitis patients. Spirochetes from ticks feeding on birds were overrepresented in genotypes of the plasmid and regulatory genes dbpA, rpoN and rpoS from spirochetes in neuroborreliosis patients. Overall, the analyses of our datasets support the existence of at least three transmission pathways from an enzootic cycle to a clinical manifestation of Lyme borreliosis. Based on the observations with these nine loci, it seems to be justified to consider the population structure of B. burgdorferi senso lato as being predominantly clonal.

In this study we used typing based on the eight multilocus sequence typing scheme housekeeping genes (MLST) and 5S-23S rDNA intergenic spacer (IGS) to explore the population structure of Borrelia burgdorferi sensu lato isolates from patients with Lyme borreliosis (LB) and to test the association between the B. burgdorferi s.l. sequence types (ST) and the clinical manifestations they cause in humans. Isolates of B. burgdorferi from 183 LB cases across Europe, with distinct clinical manifestations, and 257 Ixodes ricinus lysates from The Netherlands, were analyzed for this study alone. For completeness, we incorporated in our analysis also 335 European B. burgdorferi s.l. MLST profiles retrieved from literature. Borrelia afzelii and Borrelia bavariensis were associated with human cases of LB while Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana were associated with questing I. ricinus ticks. B. afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. The samples in our study belonged to 251 different STs, of which 94 are newly described, adding to the overall picture of the genetic diversity of Borrelia genospecies. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and Borrelia spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). We found six multilocus sequence types that were significantly associated to clinical manifestations in humans and five IGS haplotypes that were associated with the human LB cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs.

The aim of this study was to investigate the natural cycle of the new human pathogenic bacteria Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in Southern Hungary. We collected rodents with live-traps (2010-2013) and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all the ectoparasites were removed and stored in 70% alcohol. We found relatively low overall prevalence of tick infestation (8%). Samples were analysed for A. phagocytophilum and Candidatus N. mikurensis with multiplex quantitative real-time PCR targeting a part of major surface protein 2 (msp2) and the heat shock protein groEL genes, respectively. The overall prevalence in tissue samples was 6.6% (skin) and 5.1% (spleen) for A. phagocytophilum and 1.7% (skin) and 3.4% (spleen) for Candidatus N. mikurensis. Candidatus N. mikurensis was only detected in Apodemus flavicollis and Apodemus agrarius, while A. phagocytophilum was found in A. flavicollis, A. agrarius, Myodes glareolus, Microtus arvalis and Mus musculus samples. Prevalence of A. phagocytophilum in skin samples of A. flavicollis was significantly higher than prevalence of N. mikurensis (p

Background Borrelia miyamotoi causes systemic febrile illness and is transmitted by the same tick species that transmits Borrelia burgdorferi sensu lato and tick-borne encephalitis virus. We describe a serological test using a fragment of glycerophosphodiester phosphodiesterase (GlpQ) as an antigen, and determined its performance in well-defined patient categories. Methods Serum of patients with PCR-confirmed Borrelia miyamotoi disease (BMD), Lyme borreliosis (LB), tick-borne encephalitis (TBE), and healthy blood donors (HBD) were collected in Udmurt Republic, Russia. Sera of BMD and LB patients were collected at hospital admission, one week, one month and one year after admission. Results The levels of IgM and IgG anti-GlpQ antibodies, determined as optical density values in Luminex bead-based assays, were significantly higher in the BMD patient group than in LB patients, TBE patients or HBD group (all p Conclusions By using a strict cut-off value, it was possible to exclude B. miyamotoi infection in LB and TBE patients and to serologically confirm B. miyamotoi infection in 44% to 94% of the PCR-positive BMD patients (95% confidence interval). Thus, sensitive serological assays should not solely rely on rGlpQ, to support the diagnosis of acute BMD.

SUMMARYThis paper reports the first detection ofBorrelia miyamotoiin UKIxodes ricinusticks. It also reports on the presence and infection rates ofI. ricinusfor a number of other tick-borne pathogens of public health importance. Ticks from seven regions in southern England were screened forB. miyamotoi, Borrelia burgdorferi sensu lato(s.l.),Anaplasma phagocytophilumandNeoehrlichia mikurensisusing qPCR. A total of 954I. ricinusticks were tested, 40 were positive forB. burgdorferi s.l., 22 positive forA. phagocytophilumand three positive forB. miyamotoi, with noN. mikurensisdetected. The three positiveB. miyamotoiticks came from three geographically distinct areas, suggesting a widespread distribution, and from two separate years, suggesting some degree of endemicity. Understanding the prevalence ofBorreliaand other tick-borne pathogens in ticks is crucial for locating high-risk areas of disease transmission.

Background European hedgehogs (Erinaceus europaeus) are urban dwellers and host both Ixodes ricinus and Ixodes hexagonus. These ticks transmit several zoonotic pathogens like Borrelia burgdorferi (sensu lato), Anaplasma phagocytophilum, Rickettsia helvetica, Borrelia miyamotoi and “Candidatus Neoehrlichia mikurensis”. It is unclear to what extent hedgehogs in (sub) urban areas contribute to the presence of infected ticks in these areas, which subsequently pose a risk for acquiring a tick-borne disease. Therefore, it is important to investigate to what extent hedgehogs contribute to the enzootic cycle of these tick-borne pathogens, and to shed more light at the mechanisms of the transmission cycles involving hedgehogs and both ixodid tick species. Methods Engorged ticks from hedgehogs were collected from (sub) urban areas via rehabilitating centres in Belgium. Ticks were screened individually for presence of Borrelia burgdorferi (sensu lato), Borrelia miyamotoi, Anaplasma phagocytophilum, Rickettsia helvetica and “Candidatus Neoehrlichia mikurensis” using PCR-based methods. Infection rates of the different pathogens in ticks were calculated and compared to infection rates in questing ticks. Results Both Ixodes hexagonus (n = 1132) and Ixodes ricinus (n = 73) of all life stages were found on the 54 investigated hedgehogs. Only a few hedgehogs carried most of the ticks, with 6 of the 54 hedgehogs carrying more than half of all ticks (624/1205). Borrelia miyamotoi, A. phagocytophilum, R. helvetica and B. burgdorferi genospecies (Borrelia afzelii, Borrelia bavariensis and Borrelia spielmanii) were detected in both I. hexagonus and I. ricinus. Anaplasma phagocytophilum, R. helvetica, B. afzelii, B. bavariensis and B. spielmanii were found significantly more in engorged ticks in comparison to questing I. ricinus. Conclusions European hedgehogs seem to contribute to the spread and transmission of tick-borne pathogens in urban areas. The relatively high prevalence of B. bavariensis, B. spielmanii, B. afzelii, A. phagocytophilum and R. helvetica in engorged ticks suggests that hedgehogs contribute to their enzootic cycles in (sub) urban areas. The extent to which hedgehogs can independently maintain these agents in natural cycles, and the role of other hosts (rodents and birds) remain to be investigated. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2065-0) contains supplementary material, which is available to authorized users.

To the Editor: Candidatus Neoehrlichia mikurensis is a member of the order Rickettsiales, family Anaplasmataceae (1). Manifestations of infection with these bacteria are atypical and severe and include cough, nausea, vomiting, anemia, headache, pulmonary infiltration, malaise, myalgia, arthralgia, fatigue, recurrent fever for ≤8 months, and/or death (2–5). Candidatus N. mikurensis has been detected in Ixodes ovatus, I. persulcatus, and Haemaphysalis concinna ticks in Asia (1,5). Candidatus N. mikurensis has been identified as one of the most prevalent pathogenic agents in I. ricinus ticks throughout Europe (2,3,6). Rodents of diverse species and geographic origins have been shown to carry these bacteria, but transmission experiments have not been conducted to unambiguously identify natural vertebrate reservoirs (1–3,5–7). This emerging tickborne pathogen has been detected mainly in immunocompromised patients in Sweden (n = 1), Switzerland (n = 3), Germany (n = 2), and the Czech Republic (n = 2) and in immunocompetent patients in China (n = 7) (2–5). Anaplasma phagocytophilum is an obligate, intracellular, tickborne bacterium of the family Anaplasmataceae and causes granulocytic anaplasmosis in humans and domestic animals. In Europe, I. ricinus ticks are its major vector, and red deer, roe deer, rodents, and European hedgehogs (Erinaceus europaeus) are suspected reservoir hosts (8). Northern white-breasted hedgehogs (Erinaceus roumanicus) are urban-dwelling mammals (order Eulipotyphla, family Erinaceidae) that serve as major maintenance hosts for the 3 stages of I. ricinus ticks (9). However, E. roumanicus hedgehogs have not been studied for their ability to carry A. phagocytophilum. In addition, no suspected reservoirs other than rodents have been investigated for Candidatus N. mikurensis. The purpose of this study was to determine whether this hedgehog is a potential reservoir of these 2 bacteria. We conducted an ecoepidemiologic study during 2009–2011 to obtain information about ticks and tickborne pathogens of urban hedgehogs in a park on Margaret Island in central Budapest, Hungary (9). Ear tissue samples were obtained from hedgehogs anesthetized with intramuscular ketamine (5 mg/kg) and dexmedetomidine (50 µg/kg). DNA was extracted from samples by using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) or the Miniprep Express Matrix protocol (MP Biomedicals, Santa Ana, CA, USA). We used quantitative real-time PCRs that partially amplify the heat shock protein gene (groEL) of Candidatus N. mikurensis and the merozoite surface protein 2 gene (msp2) of A. phagocytophilum (3). PCR was performed in a 20-μL volume containing iQ Multiplex Powermix (Bio-Rad Laboratories, Hercules, CA, USA) in a LightCycler 480 Real-Time PCR System (F. Hoffmann-La Roche, Basel, Switzerland). Final PCR concentrations were 1× iQ Powermix, 250 nmol/L of primers ApMSP2F and ApMSP2R, 125 nmol/L of probe ApMSP2P-FAM, 250 nmol/L of primers NMikGroEL-F2a and NMikGroEL-R2b, 250 nmol/L of probe NMikGroEL-P2a-RED, and 3 μL of template DNA. To confirm quantitative PCR results, we performed conventional PCRs in a Px2 Thermal Cycler (Thermo Electron Corporation, Waltham, MA, USA) on selected PCR-positive samples for both pathogens (3). Sequences obtained were submitted to GenBank under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"KF803997","term_id":"576637860","term_text":"KF803997"}}KF803997 (groEL gene of Candidatus N. mikurensis) and {"type":"entrez-nucleotide","attrs":{"text":"KF803998","term_id":"576637863","term_text":"KF803998"}}KF803998 (groEL gene of A. phagocytophilum). Candidatus N. mikurensis was detected in 2 (2.3%) of 88 hedgehog tissue samples. Formerly, rodents were the only wild mammals found to act as potential reservoirs for this pathogen. Results of studies that attempted to detect these bacteria in common shrews (Sorex araneus), greater white-toothed shrews (Crocidura russula) (2,3), or common moles (Talpa europaea) (2) were negative. However, our results indicate that northern white-breasted hedgehogs might be a non-rodent reservoir for Candidatus N. mikurensis. The low pathogen prevalence observed in this urban hedgehog population compared with that in rodents in other locations (2,3) might be caused by use of skin samples. Skin samples from rodents showed only 1.1% positivity in a study in Germany; however, average prevalence of Candidatus N. mikurensis in transudate, spleen, kidney, and liver samples from the same animals was 37.8%–51.1% (2). Although we did not test other organs, we hypothesize that prevalence of Candidatus N. mikurensis infection urban hedgehogs is probably >2.3%. We detected A. phagocytophilum in 67 (76.1%) of 88 urban hedgehogs. This prevalence was similar to that found among European hedgehogs in Germany (8). I. ricinus ticks are more common than I. hexagonus ticks in this urban hedgehog population (9). Thus, I. ricinus ticks can acquire these bacteria when feeding on hedgehogs and the risk for human infection with A. phagocytophilum in this park in Budapest is relatively high. Neoehrlichiosis and granulocytic anaplasmosis have not been diagnosed in humans in Hungary. This finding is probably caused by diagnostic difficulties rather than absence of these pathogens in the environment. Infection with Candidatus N. mikurensis and A. phagocytophilum cause predominantly noncharacteristic symptoms. Laboratory cultivation and serologic detection of Candidatus N. mikurensis has not been successful, and this pathogen has not been identified in blood smears. Thus, accurate diagnosis of suspected cases requires suitable molecular methods. Parks can be considered points of contact for reservoir animals, pathogens, ticks, and humans. Our results indicate that E. roumanicus hedgehogs play a role in urban ecoepidemiology of ≥2 emerging human pathogens. To better understand the urban cycle of these pathogens, potential reservoir hosts, ticks collected from these hosts, and vegetation in parks should be investigated.

Background Borrelia miyamotoi, the newly discovered human pathogenic relapsing fever spirochete, and Borrelia burgdorferi sensu lato are maintained in natural rodent populations. The aim of this study was to investigate the natural cycle of B. miyamotoi and B. burgdorferi s.l. in a forest habitat with intensive hunting, forestry work and recreational activity in Southern Hungary. Methods We collected rodents with modified Sherman-traps during 2010–2013 and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all ectoparasites were removed and stored. Samples were screened for pathogens with multiplex quantitative real-time polymerase chain reaction (qPCR) targeting a part of flagellin gene, then analysed with conventional PCRs and sequencing. Results 177 spleen and 348 skin samples of six rodent species were individually analysed. Prevalence in rodent tissue samples was 0.2 % (skin) and 0.5 % (spleen) for B. miyamotoi and 6.6 % (skin) and 2.2 % (spleen) for B. burgdorferi s.l. Relapsing fever spirochetes were detected in Apodemus flavicollis males, B. burgdorferi s.l. in Apodemus spp. and Myodes glareolus samples. Borrelia miyamotoi was detected in one questing Ixodes ricinus nymph and B. burgdorferi s.l in nymphs and adults. In the ticks removed from rodents DNA amplification of both pathogens was successful from I. ricinus larvae (B. miyamotoi 5.6 %, B. burgdorferi s.l. 11.1 %) and one out of five nymphs while from Ixodes acuminatus larvae, and nymph only B. burgdorferi s.l. DNA was amplified. Sequencing revealed B. lusitaniae in a questing I. ricinus nymph and altogether 17 B. afzelii were identified in other samples. Two Dermacentor marginatus engorged larva pools originating from uninfected hosts were also infected with B. afzelii. Conclusions This is the first report of B. miyamotoi occurrence in a natural population of A. flavicollis as well as in Hungary. We provide new data about circulation of B. burgdorferi s.l. in rodent and tick communities including the role of I. acuminatus ticks in the endophilic pathogen cycle. Our results highlight the possible risk of infection with relapsing fever and Lyme borreliosis spirochetes in forest habitats especially in the high-risk groups of hunters, forestry workers and hikers.

Background European hedgehogs (Erinaceus europaeus) are hosts for Ixodes hexagonus and I. ricinus ticks, which are vectors for zoonotic microorganisms. In addition, hedgehogs may carry several enteric zoonoses as well. It is unclear to what extent a presence of pathogens in hedgehogs poses a risk to public health, as information on the presence of zoonotic agents in hedgehogs in urban areas is relatively scarce. Methods Engorged ticks and hedgehog faeces were collected from rehabilitating hedgehogs. Ticks were screened individually for presence of Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis using PCR-based assays. Faecal samples were screened for presence of Campylobacter, Salmonella, Giardia, Cryptosporidium, and extended-spectrum cephalosporin-resistant-Escherichia coli (ESC)-resistant E. coli, using both culture-based and PCR-based methods. Results Anaplasma phagocytophilum and Borrelia genospecies B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi sensu stricto were detected in both I. hexagonus and I. ricinus ticks. Despite their widespread distribution in the Netherlands, B. miyamotoi and Candidatus N. mikurensis were not detected in collected ticks. Analysis of hedgehog faecal samples revealed the presence of Salmonella enterica subspecies enterica and Campylobacter jejuni. In addition, ESC-resistant E. coli were observed in high prevalence in faecal samples, but no Shiga-toxin producing-E.coli were detected. Finally, potentially zoonotic protozoan parasites were observed in hedgehog faecal samples as well, including Giardia duodenalis assemblage A, Cryptosporidium parvum subtypes IIaA17G1R1 and IIcA5G3, and C. hominis subtype IbA10G2. Conclusions European hedgehogs in (sub)urban areas harbor a number of zoonotic agents, and therefore may contribute to the spread and transmission of zoonotic diseases. The relatively high prevalence of B. burgdorferi s.l. and A. phagocytophilum in engorged ticks, suggests that hedgehogs contribute to their enzootic cycles in (sub)urban areas. To what extent can hedgehogs maintain the enteric zoonotic agents in natural cycles, and the role of (spill-back from) humans remains to be investigated.

Borrelia turcica comprises the third major group of arthropod-transmitted borreliae and is phylogenetically divergent from other Borrelia groups. The novel group of Borrelia was initially isolated from Hyalomma aegyptium ticks in Turkey and it was recently found in blood and multiple organs of tortoises exported from Jordan to Japan. However, the ecology of these spirochetes and their development in ticks or the vertebrate hosts were not investigated in detail; our aims were to isolate the pathogen and to evaluate the possibility of transstadial transmission of Borrelia turcica by H. aegyptium ticks. Ticks were collected from Testudo graeca tortoises during the summer of 2013 from southeastern Romania. Engorged nymphs were successfully molted to the adult stage. Alive B. turcica was isolated from molted ticks by using Barbour-Stoenner-Kelly (BSK) II medium. Four pure cultures of spirochetes were obtained and analyzed by PCR and sequencing. Sequence analysis of glpQ, gyrB and flaB revealed 98%-100% similarities with B. turcica. H. aegyptium ticks collected from T. graeca tortoises were able to pass the infection with B. turcica via transstadial route, suggesting its vectorial capacity.

Background - Lipoptena cervi (Diptera: Hippoboscidae) is a hematophagous ectoparasite of cervids, which is considered to transmit pathogens between animals and occasionally to humans. The principal life stage that is able to parasitize new hosts is a winged ked that just emerged from a pupa. To facilitate efficient transmission of pathogens between hosts, vertical transmission from female deer keds to their offspring is necessary. We investigated vertical transmission of several vector-borne pathogens associated with cervids. Methods - Deer keds from several locations in Hungary were collected between 2009 and 2012. All life stages were represented: winged free-ranging adults, wingless adults collected from Capreolus capreolus and Cervus elaphus, developing larvae dissected from gravid females, and fully developed pupae. The presence of zoonotic pathogens was determined using qPCR or conventional PCR assays performed on DNA lysates. From the PCR-positive lysates, a gene fragment was amplified and sequenced for confirmation of pathogen presence, and/or pathogen species identification. Results - DNA of Bartonella schoenbuchensis was found in wingless males (2%) and females (2%) obtained from Cervus elaphus, dissected developing larvae (71%), and free-ranging winged males (2%) and females (11%). DNA of Anaplasma phagocytophilum and Rickettsia species was present in L. cervi adults, but not in immature stages. DNA of Candidatus Neoehrlichia mikurensis was absent in any of the life stages of L. cervi. Conclusions B. schoenbuchensis is transmitted from wingless adult females to developing larvae, making it very likely that L. cervi is a vector for B. schoenbuchensis. Lipoptena cervi is probably not a vector for A. phagocytophilum, Rickettsia species, and Candidatus N. mikurensis.

Substantial exposure to Borrelia miyamotoi occurs through bites from Ixodes ricinus ticks in the Netherlands, which also transmit Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. Direct evidence for B. miyamotoi infection in European populations is scarce. A flu-like illness with high fever, resembling human granulocytic anaplasmosis, has been attributed to B. miyamotoi infections in relatively small groups. Borrelia miyamotoi infections associated with chronic meningoencephalitis have also been described in case reports. Assuming that an IgG antibody response against B. miyamotoi antigens reflects (endured) infection, the seroprevalence in different risk groups was examined. Sera from nine out of ten confirmed B. miyamotoi infections from Russia were found to be positive with the recombinant antigen used, and no significant cross-reactivity was observed in secondary syphilis patients. The seroprevalence in blood donors was set at 2.0% (95% CI 0.4–5.7%). Elevated seroprevalences in individuals with serologically confirmed, 7.4% (2.0–17.9%), or unconfirmed, 8.6% (1.8–23%), Lyme neuroborreliosis were not significantly different from those in blood donors. The prevalence of anti-B. miyamotoi antibodies among forestry workers was 10% (5.3–16.8%) and in patients with serologically unconfirmed but suspected human granulocytic anaplasmosis was 14.6% (9.0–21.8%); these were significantly higher compared with the seroprevalence in blood donors. Our findings indicate that infections with B. miyamotoi occur in tick-exposed individuals in the Netherlands. In addition, B. miyamotoi infections should be considered in patients reporting tick bites and febrile illness with unresolved aetiology in the Netherlands, and other countries where I. ricinus ticks are endemic.

On April 1 2012, a 70-year-old patient came to our clinic reporting slow cognitive processing, memory defi cits, and a disturbed gait, all of which had gradually developed over several months and progressed during the last few weeks before the patient’s initial visit. He did not report fever, and he had not been outside the country for several years. He had recently been treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone), poly chemo therapy, and rituximab (last dose on Aug 2, 2011) for a stage 4 diff use large B cell lymphoma. His medical history also included Pneumocystis jirovecii pneumonia, un explained chronic diarrhoea, a splenectomy, extensive tick exposure, and two tick bites in the summer and fall before onset of symptoms. On neurological examination there was a distinct brady phrenia, and on cognitive assess ment, the patient scored 26 of 30 points on the mini mental state examination. Vital signs were normal and body tempera ture was 36·4°C. Cranial MRI showed no abnormalities, but two lumbar punctures showed cerebro spinal fl uid pleocytosis with raised protein values. The cause of this chronic meningitis was not identifi ed by wide-ranging micro biological, pathological, and haematological diag nostic testing (appen dix). A C6-immunofl uorescence assay for Borrelia burgdorferi in serum, but not in cerebro spinal fl uid, was weakly positive (index 1·8). However, a B burgdorferi IgG and IgM immuno blot were non-conclusive and nega tive, respec tively. A B burgdorferi sl qPCR in cerebrospinal fl uid was negative. Nonetheless, because of the absence of an alternative diagnosis and the progression of symptoms, on April 17, 2012, the patient was treated for a possible Lyme neuroborreliosis with once daily 2000 mg ceftriaxone intravenously for 2 weeks. Dur ing several weeks the patient fully recovered. At his last visit to the outpatient clinic in May, 2013, the patient did not have residual symptoms. Supported by the recent evidence of the presence of B miyamotoi in Ixodes ricinus ticks across Europe, the relation in time of the patient’s symptoms with the tick bites, and his immunocompromised status, we retrospectively considered B miyamotoi as the causative agent. We identifi ed motile spirochaetes in stored pre-treatment cerebrospinal fl uid by dark-fi eld microscopy (appendix). Additionally, a 16S rDNA pan-relapsing fever Borrelia quan titative (q)PCR and a qPCR targeting the B miyamotoi fl agellin gene was positive in two separate pre-treatment cerebrospinal fl uid samples and one pre-treatment blood sample (appendix). Notably, 2·2% of 352 I ricinus nymphal ticks from the vicinity of the patient’s recreational house in the dunes of Zandvoort, the Netherlands, proved to be positive for B miyamotoi by qPCR (appendix). Amplifi cation and sequencing of the glpQ and p66 genes confi rmed B miyamotoi as the causative agent and showed 100% identical sequences in ticks and the patient’s clinical samples (appendix). We were unable to culture the spiro chaetes in modifi ed Barbour-Stoenner-Kelly medium from stored blood and cerebrospinal fl uid samples. Finally, ELISA and Western blot did not show anti-GlpQ antibodies in blood and CSF. Relapsing fever is caused by various Borrelia species, which are predominantly transmitted by soft ticks. How ever, relapsing fever Borrelia species have also been identifi ed in hard ticks, including B miyamotoi in Ixodes ticks. B miyamotoi infection has been associated with systemic complaints, including malaise and fever, in case series. Recently, in the USA, B miyamotoi was shown to be able to cause meningoencephalitis in an immuno compromised patient. Physicians worldwide managing immunocompromised patients from Ixodes tick-endemic areas with a meningo encephalitis should consider B miyamotoi as a potential causative agent and should be aware that regular diagnostic tests for B burgdorferi will most probably overlook this diagnosis. Whether B miyamotoi is also able to cause neurological symptoms in non-immunocompromised patients requires further investigation.

Background Spotted fever group (SFG) rickettsiae have recently been identified for the first time in UK ticks. This included the findings of Rickettsia helvetica in Ixodes ricinus and Rickettsia raoultii in Dermacentor reticulatus. This paper further investigates the occurrence of SFG rickettsiae in additional geographically distinct populations of D. reticulatus, and for the first time, investigates the occurrence of SFG rickettsiae in UK populations of Haemaphysalis punctata ticks. Methods Questing D. reticulatus and H. punctata were collected at a number of sites in England and Wales. DNA from questing ticks was extracted by alkaline lysis and detection of rickettsiae DNA was performed, in addition to detection of A. phagocytophilum, N. mikurensis, C. burnetii and B. burgdorferi sensu lato. Results This paper builds on previous findings to include the detection of spotted fever Rickettsia which showed the highest homology to Rickettsia massiliae in Haemaphysalis punctata, as well as R. helvetica in D. reticulatus. The occurrence of SFG rickettsiae in D. reticulatus in the UK appears to be confined only to Welsh and Essex populations, with no evidence so far from Devon. Similarly, the occurrence of SFG rickettsiae in H. punctata appears confined to one of two farms known to be infested with this tick in North Kent, with no evidence so far from the Sussex populations. Anaplasma phagocytophilum, Neoehrlichia mikurensis, Coxiella burnetii and Borrelia burgdorferi sensu lato DNA was not detected in any of the ticks. Conclusion These two tick species are highly restricted in their distribution in England and Wales, but where they do occur they can be abundant. Following detection of these SFG rickettsiae in additional UK tick species, as well as I. ricinus, research should now be directed towards clarifying firstly the geographic distribution of SFG rickettsiae in UK ticks, and secondly to assess the prevalence rates in ticks, wild and domesticated animals and humans to identify the drivers for disease transmission and their public health significance.

Background Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis. Methods Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test. Results The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis. Conclusions Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.