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      Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing

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          Abstract

          Background

          Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.

          Methodology/Principal Findings

          We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.

          Conclusions/Significance

          Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

          Author Summary

          Diseases transmitted by ticks have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world, both in humans and animals. The emergence or re-emergence of tick-borne diseases is increasingly becoming a problem as the geographical distribution of several tick species is expanding, as well as the numbers of potential or known tick-borne pathogens are constantly evolving. It is thus necessary to know which microorganisms circulate within communities of this major vector to ensure adequate epidemiological surveillance. In this study, we evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce, without a priori, an inventory of both predicted and non-expected parasites carried by Ixodes ricinus, the most prevalent human biting tick in France. Our findings suggest that NGS strategies could be used to produce an inventory of live parasites residing in ticks from a selected area, thereby expanding our knowledge base of tick-associated parasites.

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          Most cited references35

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          Babesiosis: recent insights into an ancient disease.

          Ever since the discovery of parasitic inclusions in erythrocytes of cattle in Romania by Victor Babes at the end of the 19th century, newly recognised babesial pathogens continue to emerge around the world and the substantial public health impact of babesiosis on livestock and man is ongoing. Babesia are transmitted by ixodid ticks and infection of the host causes a host-mediated pathology and erythrocyte lysis, resulting in anemia, hyperbilirubinuria, hemoglobinuria, and possibly organ failure. Recently obtained molecular data, particularly for the 18S rRNA gene, has contributed significantly to a better understanding of the sometimes puzzling phylogenetic situation of the genus Babesia and new information has been added to help determine the taxonomic position of many species. Moreover, it seems that owing to higher medical awareness the number of reported cases in humans is rising steadily. Hitherto unknown zoonotic babesias are now being reported from geographical areas where babesiosis was not known to occur and the growing numbers of immunocompromised individuals suggest that the frequency of cases will continue to rise. This review covers recent insights into human babesiosis with regard to phylogeny, diagnostics and treatment in order to provide new information on well known as well as recently discovered parasites with zoonotic potential.
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            Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe

            In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.
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              Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland.

              We have designed and performed a new PCR method based on the 18S rRNA in order to individuate the presence and the identity of Babesia parasites. Out of 1159 Ixodes ricinus (Acari: Ixodidae) ticks collected in four areas of Switzerland, nine were found to contain Babesia DNA. Sequencing of the short amplicon obtained (411-452 bp) allowed the identification of three human pathogenic species: Babesia microti, B. divergens, for the first time in Switzerland, Babesia sp. EU1. We also report coinfections with B. sp. EU1-Borrelia burgdorferi sensu stricto and Babesia sp. EU1-B. afzelii.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                March 2014
                27 March 2014
                : 8
                : 3
                : e2753
                Affiliations
                [1 ]USC INRA Bartonella-tiques, UMR BIPAR ENVA-ANSES, Maisons-Alfort, France
                [2 ]PathoQuest SAS, Paris, France
                [3 ]Ecole Nationale Vétérinaire d'Alfort, UMR 1161 Virologie ENVA, INRA, ANSES, Maisons-Alfort, France
                [4 ]Institut Pasteur, Laboratory of Pathogen Discovery, Paris, France
                University of Texas Medical Branch, United States of America
                Author notes

                I have read the journal's policy and have the following conflicts: CH and JC are employees and ME is the chairman of PathoQuest, a spin-off of Institut Pasteur. This does not alter our adherence to all PLOS NTDs policies on sharing data and materials.

                Conceived and designed the experiments: SB SM MVT ME. Performed the experiments: LM JC CH. Analyzed the data: SB LM. Contributed reagents/materials/analysis tools: SB LM SM JC CH ME. Wrote the paper: SB LM.

                Article
                PNTD-D-13-00092
                10.1371/journal.pntd.0002753
                3967966
                24675738
                5d37b317-f22b-4b02-9c2f-118c4b4eb779
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 January 2013
                : 11 February 2014
                Page count
                Pages: 6
                Funding
                This study was partially funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext 097 ( http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and don't necessarily reflect the views of the European Commission. Financial aid was also provided by the DIM Ile de France, the CoVetLab partner institutes, the ANSES, the Animal Health department of INRA and the INRA Metaprogram MEM and the PathoID project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Veterinary Science
                Medicine and Health Sciences

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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