Borrelia miyamotoi is a spirochete transmitted by several ixodid tick species. It causes a relapsing fever in humans and is currently considered as an emerging pathogen. In Europe, B. miyamotoi seems to occur at low prevalence in Ixodes ricinus ticks but has a wide distribution. Here we report the first detection of B. miyamotoi in Ixodes ricinus ticks collected in two independent studies conducted in 2016 in the north-eastern and north-western Alps, Italy

Capelli, Gioia

Krawczyk, Aleksandra Iwona

Mastrorilli, Eleonora

Milani, Adelaide

Montarsi, Fabrizio

Ravagnan, Silvia

Rossi, Luca

Sprong, Hein

Tomassone, Laura

Parasites & Vectors

Abstract Background Borrelia miyamotoi is a spirochete transmitted by several ixodid tick species. It causes a relapsing fever in humans and is currently considered as an emerging pathogen. In Europe, B. miyamotoi seems to occur at low prevalence in Ixodes ricinus ticks but has a wide distribution. Here we report the first detection of B. miyamotoi in Ixodes ricinus ticks collected in two independent studies conducted in 2016 in the north-eastern and north-western Alps, Italy. Results Three out of 405 nymphs (0.74%) tested positive for Borrelia miyamotoi. In particular, B. miyamotoi was found in 2/365 nymphs in the western and in 1/40 nymphs in the eastern alpine area. These are the first findings of B. miyamotoi in Italy. Conclusions Exposure to B. miyamotoi and risk of human infection may occur through tick bites in northern Italy. Relapsing fever caused by Borrelia miyamotoi has not yet been reported in Italy, but misdiagnoses with tick-borne encephalitis, human granulocytic anaplasmosis or other relapsing fever can occur. Our findings suggest that B. miyamotoi should be considered in the differential diagnosis of febrile patients originating from Lyme borreliosis endemic regions. The distribution of this pathogen and its relevance to public health need further investigation

Silvia Ravagnan

Laura Tomassone

Fabrizio Montarsi

Aleksandra Iwona Krawczyk

Eleonora Mastrorilli

Hein Sprong

Adelaide Milani

Luca Rossi

Gioia Capelli

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First detection of Borrelia miyamotoi in Ixodes ricinus ticks from northern Italy

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SHORT REPORT Open AccessFirst detection of Borrelia miyamotoi inIxodes ricinus ticks from northern ItalySilvia Ravagnan1†, Laura Tomassone2†, Fabrizio Montarsi1, Aleksandra Iwona Krawczyk3, Eleonora Mastrorilli1,Hein Sprong3, Adelaide Milani1, Luca Rossi2 and Gioia Capelli1*AbstractBackground: Borrelia miyamotoi is a spirochete transmitted by several ixodid tick species. It causes a relapsing fever inhumans and is currently considered as an emerging pathogen. In Europe, B. miyamotoi seems to occur at low prevalencein Ixodes ricinus ticks but has a wide distribution. Here we report the first detection of B. miyamotoi in Ixodes ricinus tickscollected in two independent studies conducted in 2016 in the north-eastern and north-western Alps, Italy.Results: Three out of 405 nymphs (0.74%) tested positive for Borrelia miyamotoi. In particular, B. miyamotoi was found in2/365 nymphs in the western and in 1/40 nymphs in the eastern alpine area. These are the first findings of B. miyamotoiin Italy.Conclusions: Exposure to B. miyamotoi and risk of human infection may occur through tick bites in northern Italy.Relapsing fever caused by Borrelia miyamotoi has not yet been reported in Italy, but misdiagnoses with tick-borneencephalitis, human granulocytic anaplasmosis or other relapsing fever can occur. Our findings suggest that B.miyamotoi should be considered in the differential diagnosis of febrile patients originating from Lyme borreliosisendemic regions. The distribution of this pathogen and its relevance to public health need further investigation.Keywords: Borrelia miyamotoi, Ixodes ricinus, Zoonosis, Northern ItalyBackgroundBorrelia miyamotoi is a spirochete transmitted to vertebratehosts by the same hard ticks that transmit Borrelia burgdor-feri (sensu lato), the agent of Lyme disease, namely Ixodesricinus and I. persulcatus in Europe, I. persulcatus, I. ovatusand I. pavlovskyi in Asia, and I. scapularis and I. pacificusin the USA [1]. Borrelia miyamotoi was first identified in1994 in ticks from Japan [2] and is currently considered anemerging pathogen affecting humans, in whom it can causean infection similar to a relapsing fever [1, 3, 4]. Three typesof B. miyamotoi are currently recognized: American, Asian(Siberian) and European.In Europe, wild rodents are reservoir hosts for B. miyamo-toi [5–7]. The pathogen has been shown to be widespread[5, 8], and occurs in I. ricinus at a low prevalence, with thehighest frequency registered to date in Hungary (4.8%) [9].Despite the increasing number of human cases in therecent years [5], our knowledge on the distribution, ecologyand epidemiology of B. miyamotoi is limited. To date, B.miyamotoi had not been detected in humans or in I. ricinusticks in Italy, probably because it had not been searchedfor. In fact, in northern Italy, as in other European coun-tries, I. ricinus is the tick species that most frequently biteshumans [10–12].The Italian Alps in northern Italy, offer favourable envir-onmental conditions for the survival and proliferation of I.ricinus [13], which were found to be infected by severaltick-borne pathogens (TBPs), namely B. burgdorferi (s.l.),spotted fever group rickettsiae, “Candidatus Neoehrlichiamikurensis”, Anaplasma phagocytophilum, tick-borne en-cephalitis flavivirus, and Babesia spp. [14–20]. The easternAlps have been considered a hot-spot for tick-bornehuman infections for a long time [21, 22], but the invasionof I. ricinus and associated pathogens in the western area isa more recent phenomenon [17].In this short note, we report the first detection of B.miyamotoi in I. ricinus ticks collected in two independentstudies conducted in 2016 in the north-eastern and north-western Alps of Italy.* Correspondence: gcapelli@izsvenezie.it†Equal contributors1Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, ItalyFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Ravagnan et al. Parasites & Vectors  (2018) 11:130 https://doi.org/10.1186/s13071-018-2713-zMethodsTicks were collected by standard dragging, using a 1 m2white flannel cloth. In the north-western alpine area, 45sites at different altitudes [range 950–1880 m above sealevel (masl)] were monitored in a regional natural park inSusa Valley, Turin Province (http://www.parchialpicozie.it/).Here, tick bites are increasingly reported by people visitingthe protected area and cases of Lyme disease have recentlybeen reported. The aim of the study was to explore the di-versity, abundance and distribution of ticks within the parkand determine the prevalence of TBPs. Borrelia miyamotoiwas searched by a qPCR targeting a fragment of the flagellingene [23] in a sample of 365 individually tested nymphs.In the north-eastern alpine area, ticks were collected ineight sites located at different altitudes (range 324–1050masl) in three areas recognized to be endemic for TBPs[16], namely Verona, Belluno and Udine provinces. Theaim of this study was to characterize the microbiota ofcollected I. ricinus, using targeted amplicon sequencing(16S rDNA). Individual adults (17) and 10 pools of 4nymphs each (n = 40) were examined. The high-throughput sequencing analysis identified a sequencebelonging to B. miyamotoi. The presence of B. miyamotoiwas then confirmed by a specific real time PCR targetingthe glpQ gene [24].To harmonize the results and for sequencing, one posi-tive sample from the north-western area and the positivesample from the north-eastern area were further amplifiedby a traditional PCR targeting ~900 bp of the glpQ gene[23]. For the second positive sample from the north-western area the extracted DNA was insufficient for furtheramplification.The PCR products of glpQ gene were sequenced withboth forward and reverse primers using a 16-capillary ABIPRISM 3130xl Genetic Analyzer (Applied Biosystem, FosterCity, CA, USA) and compared with representativesequences available in GenBank using the Basic LocalAlignment Search Tool (BLAST). Phylogenetic analyseswere carried out using the neighbour-joining (N-J) method,with 1000 bootstrap replicates implemented in the MEGA6 programme [25].Confidence intervals for prevalence have been calcu-lated with the free software WinEpi available from:http://www.winepi.net/uk/index.htm.ResultsOverall, three out of 405 I. ricinus nymphs (0.74%; 95%CI: 0.15–2.1%) tested positive for B. miyamotoi. In detail,B. miyamotoi was found in 2/365 nymphs (0.5%; 95% CI:0.07–1.96%) originating from the western area and in 1/40nymphs (2.5%; 95% CI: 0.06–13.1%) from the eastern area.The two sequences obtained, one from the westernarea and one from the eastern area, on BLAST analysisboth showed 100% identity with B. miyamotoi from theNetherlands (GenBank: AB824855) and 98% identitywith B. miyamotoi from Japan (GenBank: CP004217).The longest sequence (888 bp) of this study was depos-ited in GenBank (MG451835) and used to perform thephylogenetic analysis (Fig. 1). The Italian sequence wasidentical (100% identity) to the sequences from theFig. 1 Phylogenetic tree of glpQ gene (625 bp) of Borrelia miyamotoi. Sequence dataset was analyzed using MEGA 6, the neighbour-joining (NJ)method, and bootstrap analysis (1000 replicates) based on the ClustalW algorithm. Significant bootstrapping values (> 70%) are shown on thenodes. Borrelia lonestari was used as an outgroup. The sequence generated in this study (MG451835) is indicated by a black circleRavagnan et al. Parasites & Vectors  (2018) 11:130 Page 2 of 4Netherlands, Slovakia and Hungary, and clustered withinthe European type.DiscussionOur findings are the first report of B. miyamotoi in I.ricinus ticks in Italy and indicate the presence of thepathogen across the Alpine arch. This is not surprising,since the pathogen has been reported to be widespreadin questing I. ricinus in Europe and was also recentlyreported in southern European countries, i.e. Portugal[26], France [7] and Spain [27]. Our low prevalence isconsistent with findings throughout Europe, where 1.8%of questing Ixodes ticks were found on average to beinfected [8].However, further study is needed to better define thedistribution and prevalence of this pathogen in thevector and reservoir hosts in the Alps and other Italianregions where I. ricinus is present.The eastern alpine area investigated in this study is ahot-spot of TBPs in Italy. Specifically, it accounts for themajority of human cases of Lyme borreliosis and tick-borneencephalitis [21, 22]. However, the risk of B. burgdorferi(s.l.) and other tick-borne infections is currently increasingin areas previously deemed unsuitable for Ixodes ticks, suchas city parks [28], the Po River plain [29] and the westernAlps [17].Although no human cases of B. miyamotoi were unam-biguously identified in Italy, misdiagnosis may haveoccurred at the time of other tick-borne infections causingfever. Indeed, consistent with the low prevalence in ticks, B.miyamotoi has been infrequently found in clinical humancases throughout Europe [5]. Relapsing fever, Lyme disease-like symptoms as skin rash, and human granulocyticanaplasmosis-like symptoms have been reported in humanpatients infected by B. miyamotoi [3, 30, 31].ConclusionsBorrelia miyamotoi has been shown to be a geographic-ally widespread pathogen occurring at low prevalence inI. ricinus ticks in northern Italy. The focus on Borreliagenotypes causing Lyme disease has likely delayed thisemerging pathogen being found in humans and vectors,and the use of specific tests or a metagenomic approachwere pivotal to its discovery. Our results are intended tocontribute to raising awarness of this pathogen amongstpeople in charge of TBP surveillance as well as inmedical doctors, since unrecognized human B. miyamo-toi infection may occur in areas endemic for I. ricinus.AbbreviationsTBP: Tick-borne pathogen; masl: Meters above sea level; PCR: Polymerasechain reaction; glpQ: Glycerophosphodiester phosphodiesterase gene;qPCR: Real time PCRAcknowledgementsThe authors thank Graziana da Rold, Elena Porcellato, Stefania Cazzin andRyanne Jaarsma for their technical help. Many thanks go to the personnel ofthe natural park ‘Gran Bosco di Salbertrand’ and to the students who helpedto collect in ticks. The English language was revised by Joanne Fleming. Thework was done under the umbrella of EurNegVec COST Action TD1303.Publication of this paper has been sponsored by Bayer Animal Health in theframework of the 13th CVBD World Forum Symposium.FundingThe study performed in the north-eastern area was funded by the ItalianMinistry of Health (project code RF-2010-2314989).Availability of data and materialsAll the data concerning Borrelia miyamotoi are presented within the text. Thefull set of data concerning the two independent studies in north-western andnorth-eastern Italy will be published separately. A representative sequence wassubmitted to the GenBank database under the accession number MG451835.Authors’ contributionsSR and LT wrote the paper. SR, GC, LT, HS and LR conceived the studies. FMand LT performed the tick collection and identification. SR, LT and AIKperformed the molecular test. EM analysed the metagenomic data. GC andLR revised the text; all the authors contributed to the final version of themanuscript. All authors read and approved the final manuscript.Ethics approval and consent to participateNot applicable.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy.2Dipartimento Scienze Veterinarie, Università degli Studi di Torino, Grugliasco,Italy. 3National Institute of Public Health and Environment (RIVM), Bilthoven,The Netherlands.Received: 9 November 2017 Accepted: 15 February 2018References1. Krause PJ, Fish D, Narasimhan S, Barbour AG. Borrelia miyamotoi infection innature and in humans. Clin Microbiol Infect. 2015;21:631–9.2. Fukunaga M, Takahasci Y, Tsuruta Y, Matsushita O, Ralph D, McClelland M, etal. Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolatedfrom the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan.Int J Syst Bacteriol. 1995;45:804–10.3. Platonov AE, Karan LS, Kolyasnikova NM, Makhneva NA, Toporkova MG,Maleev VV, et al. Humans infected with relapsing fever spirochete Borreliamiyamotoi, Russia. Emerg Infect Dis. 2011;17:1816–23.4. Telford SR, Goethert HK, Molloy PJ, Berardi VP, Chowdri HR, Gugliotta JL, etal. Borrelia miyamotoi disease. Clin Lab Med. 2015;35:867–82.5. Siński E, Welc-Falęciak R, Zajkowska J. Borrelia miyamotoi: A human tick-borne relapsing fever spirochete in Europe and its potential impact onpublic health. Adv Med Sci. 2016;61:255–60.6. Burri C, Schumann O, Schumann C, Gern L. Are Apodemus spp. mice andMyodes glareolus reservoirs for Borrelia miyamotoi, Candidatus Neoehrlichiamikurensis, Rickettsia helvetica, R. monacensis and Anaplasmaphagocytophilum? Ticks Tick Borne Dis. 2014;5:245–51.7. Cosson J-F, Michelet L, Chotte J, Le Naour E, Cote M, Devillers E, et al.Genetic characterization of the human relapsing fever spirochete Borreliamiyamotoi in vectors and animal reservoirs of Lyme disease spirochetes inFrance. Parasit Vectors. 2014;7:233.Ravagnan et al. Parasites & Vectors  (2018) 11:130 Page 3 of 48. Wagemakers A, Staarink PJ, Sprong H, Hovius JWR. Borrelia miyamotoi: awidespread tick-borne relapsing fever spirochete. Trends Parasitol. 2015;31:260–9.9. Szekeres S, Lügner J, Fingerle V, Margos G, Földvári G. Prevalence of Borreliamiyamotoi and Borrelia burgdorferi sensu lato in questing ticks from arecreational coniferous forest of East Saxony, Germany. Ticks Tick Borne Dis.2017;8:922–7.10. Otranto D, Dantas-Torres F, Giannelli A, Latrofa M, Cascio A, Cazzin S, et al.Ticks infesting humans in Italy and associated pathogens. Parasit Vectors.2014;7:328.11. Lindblom P, Wilhelmsson P, Fryland L, Sjöwall J, Haglund M, MatussekA, et al. Tick-borne encephalitis virus in ticks detached from humansand follow-up of serological and clinical response. Ticks Tick Borne Dis.2014;5:21–8.12. Matei IA, Kalmár Z, Lupşe M, D’Amico G, Ionică AM, Dumitrache MO, et al.The risk of exposure to rickettsial infections and human granulocyticanaplasmosis associated with Ixodes ricinus tick bites in humans in Romania:a multiannual study. Ticks Tick Borne Dis. 2017;8:375–8.13. Tagliapietra V, Rosà R, Arnoldi D, Cagnacci F, Capelli G, Montarsi F, etal. Saturation deficit and deer density affect questing activity and localabundance of Ixodes ricinus (Acari, Ixodidae) in Italy. Vet Parasitol. 2011;183:114–24.14. Carpi G, Bertolotti L, Rosati S, Rizzoli A. Prevalence and genetic variability oftick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy. JGen Virol. 2009;90:2877–83.15. Cassini R, Bonoli C, Montarsi F, Tessarin C, Marcer F, Galuppi R.Detection of Babesia EU1 in Ixodes ricinus ticks in northern Italy. VetParasitol. 2010;171:151–4.16. Capelli G, Ravagnan S, Montarsi F, Ciocchetta S, Cazzin S, Porcellato E, et al.Occurrence and identification of risk areas of Ixodes ricinus-bornepathogens: A cost-effectiveness analysis in north-eastern Italy. ParasitVectors. 2012;5:61.17. Pintore MD, Ceballos L, Iulini B, Tomassone L, Pautasso A, Corbellini D, et al.Detection of invasive Borrelia burgdorferi strains in north-eastern Piedmont,Italy. Zoonoses Public Health. 2015;62:365–74.18. Ragagli C, Mannelli A, Ambrogi C, Bisanzio D, Ceballos LA, Grego E, et al.Presence of host-seeking Ixodes ricinus and their infection with Borreliaburgdorferi sensu lato in the northern Apennines, Italy. Exp Appl Acarol.2016;69:167–78.19. Baráková I, Derdáková M, Selyemová D, Chvostáč M, Špitalská E, Rosso F, etal. Tick-borne pathogens and their reservoir hosts in northern Italy. TicksTick Borne Dis. 2017; https://doi.org/10.1016/j.ttbdis.2017.08.012.20. Zanet S, Trisciuoglio A, Bottero E, de Mera IG, Gortazar C, Carpignano M, etal. Piroplasmosis in wildlife: Babesia and Theileria affecting free-rangingungulates and carnivores in the Italian Alps. Parasit Vectors. 2014;7:70.21. Ciceroni L, Ciarrocchi S. Lyme disease in Italy, 1983–1996. New Microbiol.1998;21:407–18.22. Rezza G, Farchi F, Pezzotti P, Ruscio M, Lo Presti A, Ciccozzi M, et al. Tick-borne encephalitis in north-east Italy: a 14-year retrospective study, January2000 to December 2013. Euro Surveill. 2015;20:30034.23. Hovius JWR, de Wever B, Sohne M, Brouwer MC, Coumou J, Wagemakers A,et al. A case of meningoencephalitis by the relapsing fever spirochaeteBorrelia miyamotoi in Europe. Lancet. 2013;382:658.24. Vayssier-Taussat M, Moutailler S, Michelet L, Devillers E, Bonnet S,Cheval J, et al. Next generation sequencing uncovers unexpectedbacterial pathogens in ticks in western Europe. PLoS One. 2013;8:e81439.25. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6:Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol.2013;30:2725–9.26. Nunes M, Parreira R, Lopes N, Maia C, Carreira T, Sousa C, et al. Molecularidentification of Borrelia miyamotoi in Ixodes ricinus from Portugal. Vector-Borne Zoonot Dis. 2015;15:515–7.27. Palomar A, Portillo A, Santibáñez P, Santibáñez S, Oteo J. Borrelia miyamotoi:should it be considered for the diagnosis of tick-borne infectious diseases inSpain? Final Conf. Neglected Vectors Vector-Borne Dis. COST Action TD1303.Chania, Greece; 2017. p. 48.28. Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubálek Z, Földvári G, et al. Ixodesricinus and its transmitted pathogens in urban and peri-urban areas inEurope: new hazards and relevance for public health. Front Public Health.2014;2:251.29. Pistone D, Pajoro M, Fabbi M, Vicari N, Marone P, Genchi C, et al. Lymeborreliosis, Po River Valley, Italy. Emerg Infect Dis. 2010;16:1289–91.30. Boden K, Lobenstein S, Hermann B, Margos G, Fingerle V. Borreliamiyamotoi - associated neuroborreliosis in immunocompromisedperson. Emerg Infect Dis. 2016;22:1617–20.31. Jahfari S, Herremans T, Platonov AE, Kuiper H, Karan LS, Vasilieva O, etal. High seroprevalence of Borrelia miyamotoi antibodies in forestryworkers and individuals suspected of human granulocytic anaplasmosisin the Netherlands. New Microbe New Infect. 2014;2:144–9.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Ravagnan et al. Parasites & Vectors  (2018) 11:130 Page 4 of 4

First detection of Borrelia miyamotoi in Ixodes ricinus ticks from northern Italy.

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First detection of Borrelia miyamotoi in Ixodes ricinus ticks from northern Italy.

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