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Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidiousBorreliaspecies - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth ofBorreliawould be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 degrees C for 2.5 years and BSK-II stored at -20 degrees C for 11 years). Growth of differentBorrelia afzelii,Borrelia garinii,Borrelia lusitaniaeandBorrelia valaisianastrains was assessed during 2 weeks of incubation at 33 degrees C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 degrees C (median final cell number of 1.5 x 10(6)/mL) than in BSK-II at -20 degrees C (median final cell number of 7.75 x 10(6)/mL) and in fresh BSK-H media (median final cell number of 8.95 x 10(6)/mL). Duration of storage of media had no impact onBorreliamorphology and motility. Our results indicate that temperature of -20 degrees C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth ofBorreliaand may be employed for cultivation.

Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum, was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia. Divlje kanide su prepoznate kao važni domaćini rezervoari za nekoliko vektorima prenosivih zoonotskih patogena i predstavljaju dobre sentinel vrste za epidemiološki nadzor. Identifikacija domaćina rezervoara za pojedine zoonotske patogene na određenom području omogućava primenu odgovarajućih mera kako bi se sprečila pojava zoonoza kod ljudi i domaćih životinja. Cilj ove studije bio je da se predstavi uloga divljih kanida u epidemiologiji zoonoza koje se prenose vektorima u Srbiji na temelju prethodno objavljenih radova i da se dostupni podaci prodiskutuju sa epidemiološkog stanovišta. Na osnovu dosadašnjih molekularnih istraživanja u Srbiji, divlje kanide su prepoznate kao potencijalni rezervoari za nekoliko krpeljima prenosivih zoonotskih patogena. Kod šakala je potvrđeno prisustvo Anaplasma phagocytophilum, dok je prisustvo Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii i Borrelia lusitaniae molekularnim metodama potvrđeno kod lisica. Rezultati pokazuju da su divlje kanide potencijalni domaćini rezervoari i izvor zoonotskih patogena u Srbiji i da predstavljaju dobre domaćine za više vrsta krpelja koji su potvrđeni kompetentni vektori pojedinih patogena. Potrebno je kontinuirano epidemiološko praćenje bolesti uzrokovanih patogenima čije je prisustvo prethodno potvrđeno kod divljih kanida. Takođe, potrebna su dodatna istraživanja kako bi se otkrila uloga drugih divljih i domaćih životinja u epidemiologiji zoonoza koje se prenose vektorima u našoj zemlji.

Introduction: Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis, is the most prevalent tick-borne pathogen in Europe, where it is mainly transmitted by Ixodes ricinus. This tick also circulates Borrelia miyamotoi, a member of the relapsing fever group of species. Objectives: A longitudinal study was performed to assess the prevalence of Borrelia spp. in questing ticks. Relationships between Borrelia prevalence in ticks and some variables such as tick development stage, sampling area, and questing tick density were assessed; in addition, the distribution of B. burgdorferi s.l. was analyzed to establish the periods of acarological risk. Methods: Ticks were collected monthly by flagging in three different ecological areas (coast, plateau, and mountain) in north-western Spain during a 2-year study. Borrelia DNA was detected by PCR, targeting the flagellin (fla) gene. Positive samples were also characterized at the rrfA-rrlB intergenic spacer region and the glycerophosphodiester phosphodiesterase (GlpQ) gene. Results: B. burgdorferi s.l. DNA was detected in 11.84% of I. ricinus. Five Lyme Borrelia species were identified (Borrelia afzelii, B. burgdorferi sensu stricto, Borrelia garinii, Borrelia lusitaniae, and Borrelia valaisiana). One single relapsing fever species (B. miyamotoi) was detected (0.85%). Questing Ixodes frontalis, Ixodes acuminatus, Dermacentor reticulatus, and Dermacentor marginatus yield negative results. The prevalence of B. burgdorferi s.l. was significantly higher in female ticks and in the mountain area. In addition, a seasonal pattern in the B. burgdorferi s.l. prevalence distribution throughout the study was not detected. Conclusions: The detection of a noticeable prevalence of B. burgdorferi s.l. in questing I. ricinus suggests a high acarological risk, especially in mountain area. There is no evidence of a relationship between the prevalence of B. burgdorferi s.l. and the density of questing ticks, nor traces of a seasonal pattern in the values of prevalence in ticks.

Spirochetes from the Borrelia burgdorferi sensu lato (s.l.). (Spirochaetales: Spirochaetaceae) species complex, including the causative agents of Lyme borreliosis, have been isolated from ticks, vertebrate reservoirs and humans. Previous analyses based on direct molecular detection in ticks indicated a considerable diversity of B. burgdorferi s.l. complex in Serbia. The present study aimed (a) to isolate borrelia strains from Serbia; (b) to determine their genotypic characteristics; and (c) to establish a collection of viable B. burgdorferi s.l. strains for further biological, ecological and genetic studies. For the present study, 231 adult Ixodes ricinus (Ixodida: Ixodidae) ticks from 16 ecologically different localities in Serbia were individually processed to cultivate B. burgdorferi s.l. This led to the isolation of 36 strains. A hbb gene quantitative real-time polymerase chain reaction (PCR) based on melting temperature determination and ospA gene sequencing were used to genotype the isolated spirochetes. The species identified based on the hbb gene real-time PCR were: Borrelia lusitaniae (44.4%), Borrelia afzelii (36.1%), Borrelia garinii (13.9%) and Borrelia valaisiana (5.6%), whereas the ospA sequence analysis revealed the occurrence of Borrelia bavariensis. This is the first report of the isolation of B. lusitaniae, B. garinii, B. bavariensis and B. valaisiana strains in Serbia.

In the present study, the effectiveness of six antimicrobial agents have been tested against 24 borrelia strains isolated from Ixodes ricinus ticks (11 Borrelia lusitaniae, eight Borrelia afzelii, three Borrelia garinii and two Borrelia valaisiana) and one B. lusitaniae strain isolated from human skin. The minimum inhibitory concentration range of antimicrobial agents was as follows: amoxicillin, 0.125–2 mg/L; doxycycline, 0.125–1 mg/L, ceftriaxone, 0.016–0.063 mg/L; cefuroxime, 0.063–1 mg/L; azithromycin, 0.0017–0.11 mg/L; amikacin 32–512 mg/L. Potentially pathogenic B. lusitaniae and B. valaisiana species were more susceptible to amoxicillin and azithromycin than pathogenic B. afzelii and B. garinii (P < 0.05); B. garinii, B. lusitaniae and B. valaisiana were more susceptible to doxycycline than B. afzelii (P < 0.05) while all species showed same susceptibility to ceftriaxone and cefuroxime (P > 0.05). This study is the first report on in vitro susceptibility of isolates from Serbia to antimicrobial agents and the first report on susceptibility of larger number of isolates of potentially pathogenic species B. lusitaniae. We showed that antimicrobial agents in vitro inhibit growth of borrelia strains very effectively, indicating the potential of their equally beneficial use in the treatment of Lyme borreliosis.

We previously isolated and cultivated the novel Rickettsia raoultii strain Jongejan. This prompted us to ask whether this strain is unique or more widely present in Austria. To assess this issue, we retrospectively screened ticks collected from dogs in 2008. Of these collected ticks, we randomly selected 75 (47 females and 28 males) Dermacentor reticulatus, 44 (21 females, 7 males, and 16 nymphs) Haemaphysalis concinna, and 55 (52 females and 3 males) ticks of the Ixodes ricinus complex. Subsequently, these ticks were individually screened for the presence of tick-borne pathogens using the reverse line blot hybridization assay. In our current study, we detected DNA from the following microbes in D. reticulatus: Anaplasma phagocytophilum, Borrelia lusitaniae, Borrelia spielmanii, Borrelia valaisiana, and R. raoultii, all of which were R. raoultii strain Jongejan. In H. concinna, we found DNA of a Babesia sp., Rickettsia helvetica, and an organism closely related to Theileria capreoli. Lastly, I. ricinus was positive for Anaplasma phagocytophilum, Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii/Borrelia bavariensis, B. lusitaniae, B. spielmanii, B. valaisiana, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, and Theileria (Babesia) microti DNA. The detection of DNA of the Babesia sp. and an organism closely related to Theileria capreoli, both found in H. concinna ticks, is novel for Austria.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., Populations of vector-borne pathogens are shaped by the distribution and movement of vector and reservoir hosts. To study what impact host and vector association have on tick-borne pathogens, we investigated the population structure of Borrelia lusitaniae using multilocus sequence typing (MLST). Novel sequences were acquired from questing ticks collected in multiple North African and European locations and were supplemented by publicly available sequences at the Borrelia Pubmlst database (accessed on 11 February 2020). Population structure of B. lusitaniae was inferred using clustering and network analyses. Maximum likelihood phylogenies for two molecular tick markers (the mitochondrial 16S rRNA locus and a nuclear locus, Tick-receptor of outer surface protein A, trospA) were used to confirm the morphological species identification of collected ticks. Our results confirmed that B. lusitaniae does indeed form two distinguishable populations: one containing mostly European samples and the other mostly Portuguese and North African samples. Of interest, Portuguese samples clustered largely based on being from north (European) or south (North African) of the river Targus. As two different Ixodes species (i.e., I. ricinus and I. inopinatus) may vector Borrelia in these regions, reference samples were included for I. inopinatus but did not form monophyletic clades in either tree, suggesting some misidentification. Even so, the trospA phylogeny showed a monophyletic clade containing tick samples from Northern Africa and Portugal south of the river Tagus suggesting a population division in Ixodes on this locus. The pattern mirrored the clustering of B. lusitaniae samples, suggesting a potential co-evolution between tick and Borrelia populations that deserve further investigation., This research was financially supported by the Slovak Research and Development Agency (grant number APVV-16-0463), by the Fundação para a Ciência e a Tecnologia by the transitory norm contract DL57/2016/CP1370/CT89 to Ana Cláudia Norte and MARE (MARE-UID/MAR/04292/2020), and by the National Institute of Health Doutor Ricardo Jorge, Lisbon, Portugal. The National Reference Center for Borrelia was supported by the Robert-Koch Institute, Berlin, Germany.

Citizen science may be described as a research involving communities and individuals, other than scientists. Following this approach, along with the evidence of a high prevalence of Rickettsia spp. in Dermacentor marginatus from wild boars in hunting areas of southern Italy, this study aimed to assess the occurrence of tick-borne pathogens (TBPs) in ticks collected from hunters and their hunting dogs. From October 2020 to May 2021, ticks were collected from wild boar hunters (n = 347) and their dogs (n = 422) in regions of southern Italy (i.e., Apulia, Basilicata, Calabria, Campania and Sicily). All ticks were morphologically identified, classified according to gender, feeding status, host, geographic origin, and molecularly screened for zoonotic bacteria. Adult ticks (n = 411) were collected from hunters (i.e., n = 29; 8.4%; mean of 1.6 ticks for person) and dogs (i.e., n = 200; 47.4%; mean of 1.8 ticks for animal) and identified at species level as D. marginatus (n = 240, 58.4%), Rhipicephalus sanguineus sensu lato (n = 135, 32.8%), Rhipicephalus turanicus (n = 27, 6.6%) and Ixodes ricinus (n = 9, 2.2%). Overall, 45 ticks (i.e., 10.9%, 95% CI: 8.3-14.3) tested positive for at least one tick-borne agent, being Rickettsia slovaca the most frequent species (n = 37, 9.0%), followed by Rickettsia raoultii, Rickettsia aeschlimannii, Rickettsia monacensis, Coxiella burnetii, Borrelia lusitaniae and Candidatus Midichloria mitochondrii (n = 2, 0.5% each). Data herein presented demonstrate a relevant risk of exposure to TBPs for hunters and hunting dogs during the hunting activities. Therefore, the role of hunters to monitor the circulation of ticks in rural areas may be considered an effective example of the citizen science approach, supporting the cooperation toward private and public health stakeholders.

International audience; BackgroundThe tick midgut and salivary glands represent the primary organs for pathogen acquisition and transmission, respectively. Specifically, the midgut is the first organ to have contact with pathogens during the blood meal uptake, while salivary glands along with their secretions play a crucial role in pathogen transmission to the host. Currently there is little data about pathogen composition and prevalence in Ixodes ricinus midgut and salivary glands. The present study investigated the presence of 32 pathogen species in the midgut and salivary glands of unfed I. ricinus males and females using high-throughput microfluidic real-time PCR. Such an approach is important for enriching the knowledge about pathogen distribution in distinct tick organs which should lead to a better understanding I. ricinus-borne disease epidemiology.ResultsBorrelia lusitaniae, Borrelia spielmanii and Borrelia garinii, were detected in both midgut and salivary glands suggesting that the migration of these pathogens between these two organs might not be triggered by the blood meal. In contrast, Borrelia afzelii was detected only in the tick midgut. Anaplasma phagocytophilum and Rickettsia helvetica were the most frequently detected in ticks and were found in both males and females in the midgut and salivary glands. In contrast, Rickettsia felis was only detected in salivary glands. Finally, Borrelia miyamotoi and Babesia venatorum were detected only in males in both midguts and salivary glands. Among all collected ticks, between 10-21% of organs were co-infected. The most common bacterial co-infections in male and female midgut and salivary glands were Rickettsia helvetica+Anaplasma phagocytophilum and Rickettsia helvetica+Borrelia lusitaniae, respectively.ConclusionsAnalysing tick-borne pathogen (TBP) presence in specific tick organs enabled us to (i) highlight contrasting results with well-established transmission mechanism postulates; (ii) venture new hypotheses concerning pathogen location and migration from midgut to salivary glands; and (iii) suggest other potential associations between pathogens not previously detected at the scale of the whole tick. This work highlights the importance of considering all tick scales (i.e. whole ticks vs organs) to study TBP ecology and represents another step towards improved understanding of TBP transmission.

International audience; Tick-borne pathogens (TBPs) pose a major threat to human health in Europe and the whole northern hemisphere. Despite a high prevalence of TBPs in Ixodes ricinus ticks, knowledge on the incidence of tick-borne diseases in humans infested by this tick species is limited. This study was conducted in the year 2019 on patients who presented themselves to the Pasteur Institute Novi Sad with tick infestations. Ticks (n = 31) feeding on human (n = 30) and blood samples from the same individuals were collected by physicians and a microfluidic real-time high-throughput PCR system was used to test the genomic DNA of the samples for the presence of 27 bacterial and eight parasitic microorganisms in Serbia. Except for one Rhipicephalus sanguineus s.l. adult male tick, all ticks infesting humans were morphologically identified as I. ricinus. A high proportion of ticks (74 %, 23/31) were infected with at least one of the tested TB microorganisms, being Rickettsia helvetica (54 %, 17/31) the most common pathogen, but Borrelia afzelii (9 %, 3/31), Anaplasma phagocytophilum (6 %, 2/31), Borrelia miyamotoi (6 %, 2/31), and Francisella like-endosymbiont (6 %, 2/31), Borrelia valaisiana (3 %, 1/31), Borrelia lusitaniae (3 %, 1/31), Rickettsia felis (3 %, 1/31) and Rickettsia aeschlimannii (3 %, 1/31) were also identified. Despite the high infection rate of TBPs in ticks, only two human blood samples (6 %, 2/30) tested positive for the presence of TBPs, one patient (code H12, 67 years old female) was diagnosed with Borrelia spp. and the other patient was diagnosed (code H17, 71 years old female) with R. felis infection. The tick infesting patient H12 tested positive for B. afzelii, and R. helvetica and the tick infesting patient H17 tested positive for R. felis. Upon clinical examination, both patients were diagnosed with erythema migrans. No additional discomfort was reported by the patient and no additional pathology was observed by the physician. We concluded that humans bitten by I. ricinus in Serbia are exposed to a diverse array of TBPs with clinical impact in the Serbian cohort studied.

Made available in DSpace on 2019-10-04T12:34:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-15 Global-Doc program (University of Bari) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) BackgroundBorrelia burgdorferi (sensu lato) is a complex containing pathogenic bacteria of which some species, such as Borrelia lusitaniae, use birds, small mammals and reptiles as reservoirs. In Italy, the bacteria have been detected in reptilian and avian reservoirs in the northern and central regions.ResultsHere, 211 reptiles from three orders [Squamata (Sauria with seven species in five families and Ophidia with 11 species in three families), Crocodylia (one family and two species), and Testudines (two families and two species)] were examined for ectoparasites and molecular detection of B. burgdorferi (s.l.) in three different sites of southern Italy, an area for which no information was previously available on the occurrence of borreliosis in animals and humans. Borrelia lusitaniae was molecularly detected in larvae and nymphs (11.6%) of Ixodes ricinus infesting lizards (i.e. Podarcis muralis, Podarcis siculus and Lacerta bilineata) and in 12.3% blood samples of P. siculus. Finally, B. lusitaniae and Borrelia garinii were detected in 5.1% (32/630) of questing I. ricinus.ConclusionsThese results show the circulation of B. lusitaniae in southern Italy and suggest that P. siculus could play a role as a reservoir, representing a potential medical threat to humans living in or visiting these localities. Univ Bari, Dept Vet Med, I-70010 Valenzano, BA, Italy Univ Sao Paulo, Fac Vet Med, BR-05508270 Sao Paulo, Brazil Butantan Inst, BR-05503900 Sao Paulo, Brazil Univ Estadual Paulista, Dept Vet Pathol, BR-14884900 Jaboticabal, Brazil Fundacao Oswaldo Cruz, Aggeu Magalhaes Inst, BR-50670420 Recife, PE, Brazil Univ Estadual Paulista, Dept Vet Pathol, BR-14884900 Jaboticabal, Brazil CNPq: 142409/2015-4

Headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL) is a rare headache syndrome included in the Classification of Headache of the International Headache Society as a “headache attributed to non-infectious inflammatory intracranial disease.” We report one 15-year-old patient with clinical history and cerebrospinal fluid findings compatible with the diagnosis of HaNDL in whom Borrelia lusitaniae was identified in cerebrospinal fluid by polymerase chain reaction. info:eu-repo/semantics/publishedVersion

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.

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe-based quantitative real-time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co-infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co-infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia-positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.

Introduced Siberian chipmunks Eutamias sibiricus have been reported to be important reservoirs for human Lyme disease, as they may host high numbers of hard ticks carrying Borrelia spp. and other pathogens. In the present study, we assessed the prevalence of Borrelia spp. and other pathogenic bacteria in ectoparasite arthropod species infesting Siberian chipmunks and coexisting native small rodents. Small rodents were trapped with Sherman traps in Veneto (NE Italy), where the largest Italian populations of chipmunks occur. A total of 14 individual ticks were found on 223 rodents, with 6 more ticks obtained from stored dead chipmunks from the same study area. Ectoparasites were screened for pathogens by molecular analyses including species-specific PCR amplifications. Rickettsia monacensis, Borrelia lusitaniae, and Anaplasma platys were present in the parasites of both native rodents and introduced chipmunks. The present findings suggest a role for the invasive species E. sibiricus in the maintenance of the Ixodes ricinus life cycle, which may result in the modification of the transmission dynamics of tick-borne pathogens. Moreover, the presence of Rickettsia in urban populations of chipmunks may represent a serious risk for human health and should be investigated further.

This study was financially supported by the Portuguese Foundation for Science and Technology (SFRH/BPD/62898/2009) and INSA/DDI project.

The first report of members of the spirochete genus Borrelia in the seabird tick, Ixodes uriae , and seabird colonies occurred during the early 1990s. Since then, Borrelia spp. have been detected in these ticks and seabird colonies around the world. To date, the primary species detected has been Borrelia garinii , with rare occurrences of Borrelia burgdorferi sensu stricto and Borrelia lusitaniae. During our research on Borrelia and I. uriae in seabird colonies of Newfoundland and Labrador, Canada, we have identified Borrelia bavariensis in I. uriae . To our knowledge, B. bavariensis has previously been found only in the Eurasian tick species Ixodes persulcatus and Ixodes ricinus , and it was believed to be a rodent-specific Borrelia ecotype. We found B. bavariensis within I. uriae from three seabird colonies over three calendar years. We also reanalyzed B. garinii sequences collected from I. uriae from Eurasian seabird colonies and determined that sequences from two Russian seabird colonies likely also represent B. bavariensis . The Canadian B. bavariensis sequences from I. uriae analyzed in this study cluster with previously described sequences from Asia. Overall, this is an important discovery that illustrates and expands the range of hosts and vectors for B. bavariensis , and it raises questions regarding the possible mechanisms of pathogen dispersal from Asia to North America. IMPORTANCE To our knowledge, this is the first documentation of B. bavariensis outside Eurasia. Additionally, the bacterium was found in a marine ecosystem involving the seabird tick I. uriae and its associated seabird hosts. This indicates that the epizootiology of B. bavariensis transmission is much different from what had been described, with this species previously believed to be a rodent-specific ecotype, and it indicates that this pathogen is established, or establishing, much more widely.

Introduction Borrelia miyamotoi is a tick-borne pathogen belonging to the relapsing fever group. It had not been reported from Spain, but its wide distribution and the presence of the tick-vector (Ixodes ricinus) made us suspect its circulation. The aim of this study was to investigate the presence of Borrelia spp. in I. ricinus in Spain. Methods A total of 652 I. ricinus nymphs collected in northern Spain were processed. The DNA was extracted using incubations with ammonium hydroxide. Borrelia spp. DNA was amplified using Borrelia-specific PCR assays (glpQ, 16S rRNA and flagellin genes). Results B. miyamotoi was amplified in 4 specimens, and Borrelia burgdorferi sensu lato in 27 (8 Borrelia afzelii, 7 Borrelia garinii, 8 Borrelia lusitaniae, 3 Borrelia valaisiana and 1 B. burgdorferi sensu stricto). Conclusion B. miyamotoi should be considered in the differential diagnoses of patients with confirmed or suspected tick-bite in Spanish endemic areas for Lyme disease.

Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia , Rickettsiae , Anaplasma / Ehrlichia (including “ Candidatus Neoehrlichia”), Babesia , and Coxiella . The pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferi sensu lato complex, in 142 ticks (25.6%). Borrelia afzelii (80/142) was the most frequently detected species, followed by Borrelia burgdorferi sensu stricto (38/142) and Borrelia valaisiana (36/142). Borrelia garinii/Borrelia bavariensis , Borrelia lusitaniae , and Borrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. “ Candidatus Neoehrlichia mikurensis,” Babesia spp. ( B. venatorum , B. divergens , B. microti ), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence of Babesia spp. and “ Candidatus Neoehrlichia mikurensis” showed a significant positive correlation. We also compared different PCR-RLBs for detection of Borrelia burgdorferi sensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging. IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.

Spirochetes belonging to the Borrelia burgdoferi sensu lato (sl) group cause Lyme Borreliosis (LB), which is the most commonly reported vector-borne zoonosis in Europe. B. burgdorferi sl is maintained in nature in a complex cycle involving Ixodes ricinus ticks and several species of vertebrate hosts. The transmission dynamics of B. burgdorferi sl is complicated by the varying competence of animals for different genospecies of spirochetes that, in turn, vary in their capability of causing disease. In this study, a set of difference equations simplifying the complex interaction between vectors and their hosts (competent and not for Borrelia) is built to gain insights into conditions underlying the dominance of B. lusitaniae (transmitted by lizards to susceptible ticks) and the maintenance of B. afzelii (transmitted by wild rodents) observed in a study area in Tuscany, Italy. Findings, in agreement with field observations, highlight the existence of a threshold for the fraction of larvae feeding on rodents below which the persistence of B. afzelii is not possible. Furthermore, thresholds change as nonlinear functions of the expected number of nymph bites on mice, and the transmission and recovery probabilities. In conclusion, our model provided an insight into mechanisms underlying the relative frequency of different Borrelia genospecies, as observed in field studies., 14 pages, 3 figures, to be published in Theoretical Population Biology

In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., “ Candidatus Neoehrlichia mikurensis,” and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., “ Candidatus Neoehrlichia mikurensis,” and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and “ Candidatus Neoehrlichia mikurensis” were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia - and A. phagocytophilum -infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii , Borrelia lusitaniae , and Rickettsia monacensis , were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica , were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.

This meta-analysis of reports examining ticks throughout the Western Palearctic region indicates a distinct geographic pattern for Borrelia burgdorferi sensu lato prevalence in questing nymphal Ixodes ricinus ticks. The greatest prevalence was reported between the 5°E and 25°E longitudes based on an analysis of 123 collection points with 37,940 nymphal tick specimens (87.43% of total nymphs; 56.35% of total ticks in the set of reports over the target area). Climatic traits, such as temperature and vegetation stress, and their seasonality correlated with Borrelia prevalence in questing ticks. The greatest prevalence was associated with mild winter, high summer, and low seasonal amplitude of temperatures within the range of the tick vector, higher vegetation indices in the May-June period, and well-connected vegetation patches below a threshold at which rates suddenly drop. Classification of the target territory using a qualitative risk index derived from the abiotic variables produced an indicator of the probability of finding infected ticks in the Western Palearctic region. No specific temporal trends were detected in the reported prevalence. The ranges of the different B. burgdorferi sensu lato genospecies showed a pattern of high biodiversity between 4°W and 20°E, partially overlapping the area of highest prevalence in ticks. Borrelia afzelii and Borrelia garinii are the dominant species in central Europe (east of ∼25°E), but B. garinii may appear alone at southern latitudes and Borrelia lusitaniae is the main indicator species for meridional territories.

Ten years after the discovery of spirochaetes as agents of Lyme disease in 1982 in the USA, three genomic species had diverged from the phenotypically heterogeneous strains of Borrelia burgdorferi isolated in North America and Europe: Borrelia afzelii, B. burgdorferi sensu stricto (further B. burgdorferi), and Borrelia garinii. Whereas B. burgdorferi remained the only human pathogen in North America, all three species are aetiological agents of Lyme borreliosis in Europe. Another seven genospecies were described in the 1990s, including species from Asia (Borrelia japonica, Borrelia turdi, and B. tanukii), North America (Borrelia andersonii), Europe (Borrelia lusitaniae and Borrelia valaisiana), and from Europe and Asia (Borrelia bissettii). Another eight species were delineated in the years up to 2010: Borrelia sinica (Asia), Borrelia spielmanii (Europe), Borrelia yangtze (Asia), Borrelia californiensis, Borrelia americana, Borrelia carolinensis (North America), Borrelia bavariensis (Europe), and Borrelia kurtenbachii (North America). Of these 18 genomic species B. afzelii, B. burgdorferi and B. garinii are the confirmed agents of localized, disseminated and chronic manifestations of Lyme borreliosis, whereas B. spielmanii has been detected in early skin disease, and B. bissettii and B. valaisiana have been detected in specimens from single cases of Lyme borreliosis. The clinical role of B. lusitaniae remains to be substantiated.

Spirochetes belonging to the Borrelia burgdorferi sensu lato complex differ in resistance to complement-mediated killing by human serum. Here, we characterize complement sensitivity of a panel of B. lusitaniae isolates derived from ticks collected in Germany and Portugal as well as one patient-derived isolate, PoHL. All isolates are highly susceptible to complement-mediated lysis in human serum and activate complement predominantly by the alternative pathway, leading to an increased deposition of complement components C3, C6, and the terminal complement complex. Interestingly, serum-sensitive B. lusitaniae isolates were able to bind immune regulator factor H (CFH), and some strains also bound CFH-related protein 1 (CFHR1) and CFHR2. Moreover, CFH bound to the surface of B. lusitaniae was inefficient in mediating C3b conversion. Furthermore, the identification and characterization of a potential CFH-binding protein, OspE, revealed that this molecule possesses a significantly reduced binding capacity for CFH compared to that of CFH-binding OspE paralogs expressed by various serum-resistant Borrelia species. This finding suggests that a reduced binding capability of CFH is associated with an increased serum sensitivity of B. lusitaniae to human complement.

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host-vector-pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host-vector-pathogen system--green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia-Anaplasma coinfection rates.

To investigate the involvement of lizard species in the natural cycle of Borrelia burgdorferi sensu lato (s.l.) in Hungary, a total of 186 reptiles belonging to three species--126 green lizards (Lacerta viridis), 40 Balkan wall lizards (Podarcis taurica), and 20 sand lizards (Lacerta agilis)--were captured in 2007 and 2008. All ticks removed from the lizards were Ixodes ricinus, either larvae (324/472; 68.6%) or nymphs (148/472; 31.4%). More than half (66/126; 52.4%) of L. viridis individuals were infested, and the prevalence of tick infestation on both the other two species was 35% each. All 472 I. ricinus ticks and tissue samples collected from 134 collar scales and 62 toe clips of lizards were further analyzed for the presence of B. burgdorferi s.l. with polymerase chain reaction. The amplification of B. burgdorferi s.l. DNA was successful in 8% (n = 92) of L. viridis, 9% (n = 32) of P. taurica, and 10% (n = 10) of L. agilis tissue samples. Restriction fragment length polymorphism genotyping identified the species Borrelia lusitaniae in all tested lizard samples. Prevalence of B. burgdorferi s.l. in ticks collected from L. viridis, P. taurica, and L. agilis was 8%, 2%, and 0%, respectively. Most of the infected ticks carried B. lusitaniae (74% of genotyped positives); however, Borrelia afzelii (5%) and B. burgdorferi sensu stricto (21%) were detected in ticks removed from green lizards and Balkan wall lizards, respectively. We conclude that lizards, particularly L. viridis, can be important hosts for I. ricinus larvae and nymphs; thus, they can be regarded as reservoirs of these important pathogen vectors. The role of green lizards has been confirmed, and the implication of Balkan wall lizards is suggested in the natural cycle of B. lusitaniae at our study site.

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. However, the DNA of Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii and Borrelia bissettii has already been detected in samples of human origin, or the spirochetes were isolated from the patients with symptoms of LB. Molecular analysis of 12 selected serum samples collected in the regional hospital confirmed the presence of B. bissettii DNA in cases of single and multiple infection in patients with symptomatic borreliosis or chronic borrelial infection. The presence of B. bissettii as a single strain in patients provides strong support of the fact that B. bissettii might be a causative agent of the disease. After the first isolation of B. bissettii from the samples of human origin in Slovenia, following the detection of this species in cardiac valve tissue of the patient with endocarditis and aortic valve stenosis in the Czech Republic, here we present additional molecular data supporting the involvement of B. bissettii in LB in Europe.

Nymphal Ixodes ricinus ticks (n=180) were collected from three different areas in the Netherlands to investigate the effect of forest composition on tick-associated microbial communities. Sampled habitats differed in thickness of leaf litter and humus layers and vegetation associations and were located near Amsterdam (Beech-Oak), Ede (Birch-Oak) and Veldhoven (Birch-Oak). Analysis of nine 16S rRNA gene clone libraries made from individual ticks showed nearest matches with presumed pathogens Candidatus Neoehrlichia mikurensis and Rickettsia australis and arthropod endosymbionts Wolbachia pipientis and Candidatus Midichloria mitochondrii. Total bacterial species diversity (Shannon index) and Borrelia species infections were determined in I. ricinus by, respectively, PCR-denaturing gradient gel-electrophoresis and PCR-reverse line blot with probes specific for Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia ruski. Bacterial diversity differed significantly per area and was lowest in Ede. In contrast, Borrelia species-infected ticks were more abundant in Ede, Candidatus Neoehrlichia mikurensis-infected ticks in Ede and Veldhoven, and R. australis-infected ticks in Amsterdam. Borrelia afzelii was the most common Borrelia species found in all three areas. Bacterial tick diversity was influenced by local differences in forest structure, which is proposed to modulate animal populations that are commonly parasitized by I. ricinus.

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.

A potential role of seabirds in spreading Lyme disease (LB) spirochetes over large spatial scales was suggested more than 10 years ago when Borrelia garinii was observed in marine birds of both hemispheres. Since then, there have been few studies examining the diversity of Borrelia spp. circulating in seabirds, or the potential interaction between terrestrial and marine disease cycles. To explore these aspects, we tested 402 Ixodes uriae ticks collected from five colonial seabird species by amplification of the flaB gene. Both the average prevalence (26.0% +/- 3.9) and diversity of LB spirochetes was high. Phylogenetic analyses grouped marine isolates in two main clades: one associated with B. garinii and another with B. lusitaniae, a genospecies typically associated with lizards. One sequence also clustered most closely with B. burgdorferi! sensu stricto. Prevalence in ticks varied both among seabird species within colonies and among colonies. However, there was no clear association between different Borrelia isolates and a given seabird host species. Our findings indicate that LB spirochetes circulating in the marine system are more diverse than previously described and support the hypothesis that seabirds may be an important component in the global epidemiology and evolution of Lyme disease. Future work should help determine the extent to which isolates are shared between marine and terrestrial systems.

Host-seeking ticks were collected during monthly dragging sessions from November 2004 through October 2006 in Tuscany, central Italy. Normalized difference vegetation index (NDVI), which was calculated from Landsat ETM+ 7 remote sensing data recorded in August 2001, was significantly correlated with numbers of host-seeking immature Ixodes ricinus L. (Acari: Ixodidae) during periods of relatively low rainfall such as summer 2005 (Spearman’s ρ = 0.78, P < 0.001 for nymphs in July) and to a lower extent in spring–summer 2006. In spring 2005, when rainfall was relatively high, the correlation was weak and not statistically significant. Generalized estimating equations (GEEs), taking into account repeated sampling of the same dragging sites, were used to model the effects of NDVI and season on counts of host-seeking I. ricinus nymphs. Seasonal variations of the effect of NDVI yielded a significant NDVI-by-season interaction in the first year of the study (November 2004–October 2005), but not in the se...

Lizards and small rodents were live captured in Tuscany, central Italy, from May through August 2005. Prevalence of infestation by larval Ixodes ricinus L. (Acari: Ixodidae) and mean numbers of larvae per host were not significantly different for common wall lizards, Podarcis muralis Laurenti, and Apodemus spp. mice, whereas infestation levels by nymphs were significantly greater on lizards. Borrelia lusitaniae, which was previously shown to be dominant in host-seeking I. ricinus in the same study area, was detected by polymerase chain reaction (PCR) in 19.8% (95% confidence interval: 14.4, 26.0) of larval ticks and in 52.9% (27.8, 77.0) of nymphs that were collected from lizards. Moreover, 18.8% (7.2, 36.4) and 25.0 (3.2, 65.1) of lizards' tail biopsies and blood samples, respectively, were positive for B. lusitaniae. Conversely, attached ticks and ear biopsies from Apodemus spp. mice were PCR negative. Passerine birds belonging to 10 species were live captured in March 2005, and Borrelia valaisiana was detected in 57.1% (18.4, 90.1) of I. ricinus nymphs feeding on Eurasian blackbirds, Turdus merula L. Results of this study suggest that lizards play an important role as reservoirs for B. lusitanae and may affect the dominance of this genospecies in the Mediterranean area.

In the last decades, several studies have reported pathogenic species of Borrelia related to those that cause Tick-borne Relapsing Fever (RF), but unexpectedly suggesting their transmission by hard ticks, known vectors of Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species, rather than by soft ticks. This study was designed to update the presence of B. burgdorferi s.l. species in ticks from several districts of mainland Portugal, where Ixodes ricinus had been previously described. Ticks (a total of 2915 specimens) were collected in seven districts throughout the country, and analyzed using molecular methods. Three nested-PCR protocols, targeting the flagellin gene (flaB), the intergenic spacer region (IGS) located between 5S and 23S rRNA, and the glpQ gene, and a conventional PCR targeting the 16S rRNA, were used for Borrelia DNA detection. Borrelia DNA was detected in 3% of the ticks from Braga, Vila Real, Lisboa, Setubal, Evora and Faro districts. The obtained amplicons were sequenced and analyzed by BLASTn, and 15/63 (24%) matched with homologous sequences from Borrelia lusitaniae and 15/63 (24%) with B. garinii, being these the most prevalent species. DNA from B. burgdorferi sensu stricto (s.s.), B. valaisiana and B. afzelii were detected in 7/63 (11%), 6/63 (10%), and 2/63 (3%) of the specimens, respectively. Unexpectedly, DNA sequence (flaB) analysis from eight (13%) samples, two from Rhipicephalus sanguineus and six from Haemaphysalis punctata tick species, revealed high homology with RF-like Borrelia. Phylogenetic analyses obtained from three genetic markers (16S rRNA, flaB, and glpQ) confirmed their congruent inclusion in a strongly supported RF cluster, where they segregated in two subgroups which differ from the other Relapsing Fever species. Therefore, the results confirm the circulation of multiple species of B. burgdorferi s.l. over a wide geographic range, covering most of the Portuguese mainland territory. Surprisingly, the obtained data also revealed two putative Relapsing Fever-like Borrelia species in different species of hard ticks, possibly disclosing the circulation of novel RF-like Borrelia species with different associated tick vectors.

Lyme borreliosis is the most prevalent tick-borne disease in Europe. It is caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex and transmitted to humans by ticks of the genus Ixodes. Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana are the most common genospecies in Central Europe. In contrast, Borrelia lusitaniae predominates in Mediterranean countries such as Portugal, Morocco, and Tunisia. In Slovakia, its prevalence is low and restricted to only a few sites. The aim of our research was to study the expansion of ticks into higher altitudes in the ecosystem of the Mala Fatra mountains (north Slovakia) and their infection with B. burgdorferi s.l. pathogens. Questing ticks were collected by flagging in seven years (2004, 2006-2011) at three different altitudes: low (630-660 m above sea level (ASL)), intermediate (720-750 m ASL), and high (1040-1070 m ASL). Tick abundance was highest at the lowest altitude and lowest at the highest altitude. The average infection prevalence of B. burgdorferi s.l. in nymphs and adults was 16.8% and 36.2%, respectively. The number of infected ticks decreased from 38.5% at the lowest altitude to 4.4% at the highest altitude. B. lusitaniae was the most frequently found genospecies (>60% of the ticks found positive for B. burgdorferi s.l.) in all sites in all the studied years with the exception of 2008 when B. afzelii predominated (62%). Our study confirms the spread of Ixodes ricinus ticks to higher altitudes in Slovakia. The discovery that our mountain study sites were a natural foci of B. lusitaniae was unexpected because this genospecies is usually associated with lizards and xerothermic habitats.

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m 2 ). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m 2 . The highest tick density was found in the dune area (139 to 551/100 m 2 ). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma ( Ehrlichia/Anaplasma ) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae , Ehrlichia chaffeensis , and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma , which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.

To investigate the reservoir role of the lizard Psammodromus algirus for the Lyme disease spirochete, 199 lizards were trapped from April to October 2003 in El Jouza, northwestern Tunisia. In this site, the infection rate of free-living Ixodes ricinus (L.) by Borrelia was evaluated by immunofluorescence as 34.6% for adult ticks and 12.5% for nymphs. Eighty percent of P. algirus (117/146) captured during this study were infested by I. ricinus, the predominant tick species collected from lizards. The intensity of tick infestation of this host by larvae and nymphs ranged from 0.14 to 7.07 and from 1.5 to 6.58, respectively. These immature stages of I. ricinus were found on lizards in spring and the beginning of summer, with a peak of intensity during June (10.16 immature ticks by lizard). Tissue cultures from lizards and xenodiagnosis with larval I. ricinus were used to assess the infection and the ability, respectively, of infected lizards to transmit Borrelia to naive ticks. Seventeen percent of xenodiagnostic ticks (40/229) acquired B. lusitaniae while feeding on P. algirus. Therefore, we demonstrated the ability of the lizards to sustain Borrelia infection and to infect attached ticks, and we proved that P. algirus is a reservoir host competent to transmit B. lusitaniae.

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii , one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards ( Lacerta agilis ) and common wall lizards ( Podarcis muralis ) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii , subadult ticks feeding on lizards became infected by B. lusitaniae . Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae .

Prevalence of infection by Borrelia burgdorferi s.l. and spotted fever group (SFG) rickettsiae was estimated in host-seeking ticks in an area in Tuscany, central Italy, where Lyme borreliosis was reported in a forestry worker. B. burgdorferi s.l. was identified by polymerase chain reaction in 16.7% (95% CI = 10.3, 24.8) of Ixodes ricinus (L.) nymphs and 39.6% (95% CI = 26.5, 54.0) of adults. Borrelia lusitaniae accounted for 82.9% of positive samples, followed by Borrelia garinii (9.8%), Borrelia afzelii (2.4%), and Borrelia burgdorferi s.s. (2.4%). One Rhipicephalus spp. adult was infected with B. garinii (prevalence = 8.3%; 95% CI = 0.21, 38.5). Prevalence of infection by SFG rickettsiae was 38.5% (95% CI = 26.7, 51.4) in I. ricinus nymphs, 34.6% (95% CI = 22.0, 49.1) in I. ricinus adults, and 50% (95% CI = 21.1, 78.9) in Rhipicephalus spp. adults. Phylogenetic analysis showed the similarity of B. lusitaniae strains that were identified in this study and of a strain that was previously isolated from a human patient in Portugal. Results of this study confirm the dominance of B. lusitaniae in areas in the Mediterranean basin and the infection by SFG rickettsiae in I. ricinus.

BACKGROUND: Borrelia lusitaniae was isolated from an Ixodes ricinus tick in Portugal in 1993 for the first time. Further, this borrelia genospecies has been found in ixodid ticks collected around the coasts of southern Portugal and North Africa. Its reservoir has not been defined yet. B. lusitaniae was isolated once until now from a patient with a long-standing and expanding skin disorder. PATIENT AND METHODS: A 46-year-old Portuguese woman presented with a skin lesion on the left thigh which had evolved slowly over ten years. The patient reported limb paraesthesias, cramps, chronic headaches, and cardiac rhythm disturbances. History of tick bites was negative, nor had the patient ever noticed a skin lesion comparable with erythema chronicum migrans. Skin biopsies were taken for histological evaluation, culture and DNA detection. Antibodies to borrelia were searched by indirect immunofluorescence assay and Western blot. RESULTS: A bilateral carpal tunnel syndrome and local synovitis was diagnosed. Dermato-histology was normal, serology was negative. Spirochaetal organisms were cultured from a skin biopsy and identified as B. lusitaniae. The patient improved after a 2-week course of intravenous ceftriaxone; the skin lesions did not expand further. CONCLUSIONS: This culture confirmed skin infection by B. lusitaniae in a patient from Portugal suggests an additional human pathogen out of the B. burgdorferi sensu lato complex in Europe, particularly in Portugal.

European genospecies of B. burgdorferi sensu lato were identified by examining unfed I. ricinus ticks collected from 10 locations in northwest Poland. Research was conducted using 3 methods: PCR amplification of the fla gene with the FLA1 and FLA2 primer set conserved in all European species of B. burgdorferi sensu lato, PCR-RFLP, and sequencing. There were 5 restriction patterns obtained in this study: 4 characteristic for genospecies B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. afzelii and 1 untypical restriction pattern type. PCR products for all restriction patterns were sequenced. Polish sequences of the fla gene for B. burgdorferi s.s., B. garinii, B. valaisiana, and B. afzelii were identical with sequences from GeneBank at 99.79%, 99.58%, 100% and 100% respectively. The fifth sequence demonstrated 99.79% identity with sequences of a B. lusitaniae PotiB2 isolate from Portugal, and also clusters with this strain. B. lusitaniae DNA in I. ricinus was detected in 3 out of 10 localities and constituted 5.9% of infected individuals with I. ricinus.

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii , because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.

In order to investigate the presence and prevalence of Lyme borreliosis (Lyme disease) Borrelia species, 312 unfed ticks were collected by flagging at a woodland area in Trakya, in the European side of Turkey, in May 2002. Twelve of 299 Ixodes ricinus ticks were infected with Borrelia spp., as determined by cultivation in BSK medium (prevalence rate 4.0 %). Ten pure cultures were subjected to further characterization by sequencing analysis of the 5S-23S rDNA intergenic spacer, 16S rDNA and flagellin gene. One isolate of Borrelia burgdorferi sensu stricto, two of Borrelia garinii (Eurasian type), two of Borrelia afzelii, four of Borrelia lusitaniae and one of Borrelia valaisiana were identified. However, no Asian-type B. garinii was found. Interestingly, all Borrelia species that are known to be carried by I. ricinus were discovered among the 10 isolates. These results provide the first evidence for the existence of the Lyme borreliosis agent in Turkey.

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii , B. garinii , and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii , and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana , whereas questing ticks were infected with B. afzelii , B. garinii , and B. valaisiana . Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii . This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

Borrelia burgdorferi sensu lato (s.l.) is maintained in nature by complex zoonotic transmission cycles, involving a large variety of vertebrates as hosts and hard ticks of the genus Ixodes as vectors. Recent studies suggest that the genospecies of B. burgdorferi s.l. and sometimes their subtypes are propagated by different spectra of hosts, mainly birds and rodents. In order to test the concept of host-association, we analysed the relationships between Borrelia genospecies, rodent hosts and I. ricinus ticks in an endemic focus of Lyme borreliosis in western Slovakia. Rodents and questing ticks were collected at a forested lowland locality near Bratislava. Tick infestation levels on rodents were determined, and spirochaete infections in ticks and in ear punch biopsies were analysed by PCR followed by genotyping. Mice were more heavily infested with ticks than bank voles, and a higher proportion of mice was infected with spirochaetes than voles. However, the infectivity of voles was much higher than that of mice. The vast majority of infections detected in the skin and in ticks feeding on the rodents represented B. afzelii. In contrast, more than half of all infections in questing ticks collected in the same region of Slovakia were identified as B. valaisiana and B. garinii. In conclusion, whilst the study reveals that mice and voles play different quantitative roles in the ecology of Lyme borreliosis, it demonstrates that B. afzelii is specifically maintained by European rodents, validating the concept of host-association of B. burgdorferi s.l.

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii , Borrelia garinii , Borrelia valaisiana , and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a ∼230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme Mse I and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

Free-living Ixodes ricinus ticks were collected from 12 different sites of canton Ticino, south of the Alps (Switzerland). Each tick was examined for the presence of Borrelia burgdorferi sensu lato (sl), the etiologic agent of Lyme borreliosis using direct fluorescent antibody assay, and isolation of the bacteria. Borreliae were characterized by PCR followed by RFLP. The abundance and infection rates of I. ricinus ticks varied greatly between the areas. Two localities were found free of Borrelia. The prevalence of infected ticks ranged from 5 to 19%. Most ticks (96%) were found infected by < 50 spirochetes. Three B. burgdorferi sl species were successfully isolated: B. garinii dominated, followed by B. lusitaniae and B. valaisiana. Additionally, a mixed infection with B. garinii and B. valaisiana was observed. The distribution of the various Borrelia species in the different areas was heterogeneous. This is the first report of the presence of B. lusitaniae in I. ricinus in Switzerland.

Lyme borreliosis is a multisystem disorder characterized by a wide spectrum of clinical manifestations. In Europe, the main aetiological agents are Borrelia afzelii, Borrelia garinii and B. burgdorferi sensu stricto, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported as rare or potential causes of human disease. In North America, B. burgdorferi sensu stricto is the only human pathogen. The most common clinical manifestation of Lyme borreliosis is a skin lesion, erythema migrans, which may resolve spontaneously without antibiotic treatment. However, the pathogen can spread to other tissues and organs, causing more severe manifestations that can involve a skin, nervous system, joints, and/or heart, requiring antibiotic therapy. The most commonly used antibiotics in the treatment of Lyme borreliosis are: amoxicillin, ceftriaxone, cefuroxime, doxycycline and azithromycin. Lajmska borelioza je multisistemska bolest koju karakteriše širok spektar kliničkih manifestacija. U Evropi je primarno prouzrokovana vrstama B. afzelii, B. garinii i B. burgdorferi sensu stricto, ali Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii se takođe navode kao retki ili potencijalni uzročnici oboljenja kod ljudi. U Severnoj Americi, kao jedini humani patogen, dokazana je vrsta B. burgdorferi sensu stricto. Najčešća klinička manifestacija lajmske borelioze je kožna lezija, erythema migrans, koja može da prođe spontano, bez terapije antibioticima. Međutim, patogen se može širiti do drugih delova kože i organa i tako prouzrokovati teže manifestacije kako na koži, tako i na nervnom sistemu, zglobovima i/ili srcu, pri čemu je terapija antibioticima neophodna. Najčešće korišćeni antibiotici u terapiji lajmske borelioze su: amoksicilin, ceftriakson, cefuroksim, doksiciklin i azitromicin.

Grazing animals are continuously exposed to tick bites. Consequently, one may expect that horses will become infected with the various pathogens carried by ticks including Borrelia burgdorferi sensu lato. Whether horses may develop clinical disease due to this pathogen is controversially discussed. We were interested to learn about the infection of horses with Borrelia burgdorferi sensu lato within one season by studying the dynamics of the humoral immune response in paired blood samples. The majority of horses examined were Lipizzaner from the stud farm in Piber/Steiermark, and from the Spanish Riding School in Vienna. Smaller groups of animals of different breeds were from stud farms in Karnten, Niederosterreich, Salzburg and Steiermark. Clinical status and medical history were obtained and blood was drawn at the beginning of the highest tick activity and nine months later in 1998. Immunoblot technique (Western blot) was used in order to determine the dynamics in the immune response patterns. As antigens served the genospecies Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia lusitaniae, and Borrelia valaisiana. 309 horses (age median 7 years, range 1/12 to 33 years) were seen at the first round. 186 of these animals (60.2%; median age 6 years, range 4/12 to 33 years) were re-examined in the second round. All animals were in normal health condition during both rounds of examination and blood sampling. Analysis of the immunoblot patterns was based on in-house-, Pko-, Pka2-, Pbi-, and European Union Concerted Action on Lyme Borreliosis (EUCALB) 2 & 3-criteria; analyses revealed a variety of positive results with different strains and criteria. Positive immunoblot results with 186 paired samples and B. afzelii as antigen, for example, ranged from 52 to about 91% in the first, and 53 to 93% in the second round. The age dependency analyses showed that the first infection with B. burgdorferi sensu lato occurs in the first year. Re-infection is characterised by appearance of additional bands. Continuously tick-exposed horses show a stable pattern of bands whilst in unexposed horses the number of bands decreases with age. In this study horses became repeatedly infected with B. burgdorferi sensu lato but, apparently, developed only rarely, if at all, clinical diseases. The infectious agent is predominantly B. afzelii. Antibodies to other borrelia genospecies are predominantly due to cross reactivity.

Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.

Lyme disease, caused by Borrelia burgdorferi sensu lato (1), is endemic to various areas of Italy (2). The main vector of Lyme disease in Italy is the hard tick (Ixodes ricinus), a species widespread in mountain regions populated by wild ungulates (2). Residents of these areas and forestry workers are at risk for Lyme disease (3). Heavily populated flat regions are not considered as risk areas. For example, I. ricinus ticks have never been reported in the flat areas of the Po River Valley in the Lombardy region, one of the most important industrial districts in Europe and an area of intensive agriculture and livestock breeding. Human population density is high; >6 million persons reside in Milano and surrounding counties. In areas of Italy to which I. ricinus ticks are known to be endemic, physicians have appropriate awareness of the risks from tick bite and Lyme disease; outside these areas, awareness probably is not adequate. In late spring 2008, a forestry worker at a natural park west of Milano in the Po River Valley was treated for cutaneous mycosis on the basis of an erythematous rash on an arm. In August 2008, this patient described this skin alteration to one of us (C.B.). Subsequent clinical examination and serologic analyses led to diagnosis of Lyme disease. Because of this case, we investigated different areas of the park for ticks; collected ticks were screened by PCR for Lyme borreliae. We conducted a retrospective analysis of forestry workers in the area; 2 workers reporting the appearance of erythematous rash in the previous months underwent serologic analyses.