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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.

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.

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.

© 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.

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

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.

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.

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.

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.

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.

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.

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.