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Rickettsia Vini N. Sp. (Rickettsiaceae) Infecting the Tick Ixodes Arboricola (Acari: Ixodidae) Marketa Novakova1,2* , Francisco B

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Rickettsia Vini N. Sp. (Rickettsiaceae) Infecting the Tick Ixodes Arboricola (Acari: Ixodidae) Marketa Novakova1,2* , Francisco B Novakova et al. Parasites & Vectors (2016) 9:469 DOI 10.1186/s13071-016-1742-8 RESEARCH Open Access Rickettsia vini n. sp. (Rickettsiaceae) infecting the tick Ixodes arboricola (Acari: Ixodidae) Marketa Novakova1,2* , Francisco B. Costa3, Frantisek Krause4, Ivan Literak1,2 and Marcelo B. Labruna3 Abstract Background: Recently, a new rickettsia named ‘Candidatus Rickettsia vini’ belonging to the spotted fever group has been molecularly detected in Ixodes arboricola ticks in Spain, the Czech Republic, Slovakia and Turkey, with prevalence reaching up to 100 %. The aim of this study was to isolate this rickettsia in pure culture, and to describe it as a new Rickettsia species. Methods: A total of 148 ornitophilic nidicolous ticks Ixodes arboricola were collected in a forest near Breclav (Czech Republic) and examined for rickettsiae. Shell vial technique was applied to isolate rickettsiae in Vero cells. Rickettsial isolation was confirmed by optical microscopy and sequencing of partial sequences of the rickettsial genes gltA, ompA, ompB, and htrA. Laboratory guinea pigs and chickens were used for experimental infestations and infections. Animal blood sera were tested by immunofluorescence assay employing crude antigens of various rickettsiae. Results: Rickettsia vini n. sp. was successfully isolated from three males of I. arboricola. Phylogenetic analysis of fragments of 1092, 590, 800, and 497 nucleotides of the gltA, ompA, ompB, and htrA genes, respectively, showed closest proximity of R. vini n. sp. to Rickettsia japonica and Rickettsia heilongjiangensis belonging to the spotted fever group. Experimental infection of guinea pigs and chickens with R. vini led to various levels of cross-reactions of R. vini-homologous antibodies with Rickettsia rickettsii, Rickettsia parkeri, ‘Candidatus Rickettsia amblyommii’, Rickettsia rhipicephali, Rickettsia bellii, and Rickettsia felis. Laboratory infestations by R. vini-infected I. arboricola larvae on chickens led to no seroconversion to R. vini n. sp., nor cross-reactions with R. rickettsii, R. parkeri, ‘Ca. R. amblyommii’, R. rhipicephali, R. bellii or R. felis. Conclusions: Our results suggest that R. vini n. sp. is possibly a tick endosymbiont, not pathogenic for guinea pigs and chickens. Regarding specific phenotypic characters and significant differences of DNA sequences in comparison to the most closely related species (R. japonica and R. heilongjiangensis), we propose to classify the isolate as a new species, Rickettsia vini. Keywords: Ticks, Ixodes arboricola, Tree-hole tick, Ixodidae, Ornitophilic ticks, Nidicolous ticks, Rickettsia vini, Rickettsiae, Spotted fever group, Czech Republic * Correspondence: [email protected] 1Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic 2CEITEC, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the 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. Novakova et al. Parasites & Vectors (2016) 9:469 Page 2 of 8 Background in immature stages of Ixodes ricinus [9]. I. arboricola tick is Rickettsiae are Gram-negative coccobacilli belonging to the widely distributed in the Palaearctic region. It lives in tree family Rickettsiaceae, order Rickettsiales in the alpha sub- holes and nest boxes where it feeds on hole-breeding birds. division of the class Proteobacteria. Rickettsia spp. have Although this tick species does not represent a primary risk small genomes (1.1–2.1 Mb) resulted from reductive evolu- for humans, it shares several host species and overlaps in tion caused by their obligate endosymbiotic relationship to feeding period with Ixodes ricinus [13]. Therefore, tick- eukaryotic cells [1]. Their host diversity is remarkably high. borne microorganisms, including ‘Ca. R. vini,’ could be po- Although all valid species are associated with arthropods, tentially bridged between these two tick species via co- novel genotypes have also been identified in annelids, feeding. Phylogenetic analysis based on partial sequences of amoebae and plants [2, 3]. A number of Rickettsia species four rickettsial genes (gltA, ompA, ompB, sca4)showedthat can propagate in vertebrates, some of them cause diseases ‘Ca. R. vini’ segregated closest to Rickettsia heilongjiangensis in humans and animals, to which they are transmitted by and Rickettsia japonica, supported by high bootstrap values arthropod vectors such as fleas, lice, mites or ticks. Some [14]. The latter two species are causative agents of the Far species are considered non-pathogenic, and novel Rickettsia East spotted fever (R. heilongjiangensis) and the Japanese species reveal to be nearly cosmopolitan [4]. spotted fever (R. japonica), and both have been reported in Originally, pathogenic rickettsiae used to be divided Asia [4]. into two groups, the typhus group that included Rickett- In order to describe ‘Ca. R. vini’ as a new species, we sia prowazekii and Rickettsia typhi, and the spotted fever isolated the bacterium in cell culture for the first time, group (SFG) composed by about 20 species [5]. The and performed both molecular and phenotypical taxonomic position of other rickettsial species has characterization of the isolates. remained unclear because of their genetic anomalies. Due to findings of intriguing variety of rickettsiae in ar- Methods thropods and molecular analysis of rickettsial plasmids, Field study in Breclav, Czech Republic the genus Rickettsia has been reclassified into SFG rick- Free-living I. arboricola ticks were collected manually ettsiae, typhus group rickettsiae, the transitional group, from nest boxes during after-breeding season in Breclav, the Rickettsia bellii group, the Rickettsia canadensis Czech Republic (48°43'N, 16°54'E, 150 m above sea level, group, and several basal groups [3, 6]. However, some an oak-ash flood-plain forest), an area attractive to tour- authors do not support the creation of the transitional ists. Nesting bird species had been previously identified group claiming that it is not monophyletic and is un- during the breeding season using a bird guide book [15] helpful as it does not take into account epidemiological and confirmed according to characteristic appearance of criteria [1]. the nest during tick collecting. Ticks were identified to Tick-borne rickettsioses are caused by rickettsiae be- species according to Nosek & Sixl [16]. Collected ticks longing to the SFG [4]. Rapid development of molecular were brought alive to the laboratory and incubated at methods brought reversed approach to tick-borne patho- 12 °C (relative humidity of 80 %) for 3 months and then gen research, when disease cases are detected years after at 24 °C (relative humidity of 80 %) for 7 days before be- the tick-borne microorganism was first discovered [7]. ing subjected to the hemolymph test. There have been species of rickettisae detected in ticks years or decades before they became associated with hu- Hemolymph test and isolation of rickettsiae man illness cases, e.g. Rickettsia monacensis, Rickettsia Selected individuals were tested for the presence of Rick- parkeri, Rickettsia massiliae and Rickettsia slovaca [4, 8]. ettsia-like structures using the hemolymph test [17]. It is not clear if these novel tick-borne diseases were not Shortly, the distal part of a tick leg was cut, then a drop noticed by physicians or whether they were absent. of hemolymph was dried on a microscope slide and While it has been suggested that any novel described stained using Gimenez method [18]. The whole-body rickettsia from ticks should be considered a potential remnants were immediately stored at -80 °C to preserve pathogen [5], many tick species just do not bite humans living rickettsial organisms. under natural conditions, or some rickettsial agents are Isolation of rickettsiae from the tick samples was per- just tick endosymbionts. formed according to previous protocols [19] with some Recently, a novel SFG rickettsia has been found by mo- modifications. Briefly, ticks were surface-sterilized by lecular methods in bird-associated ticks. It was named immersion in iodine-alcohol for 10 min, washed in sterile ‘Candidatus Rickettsia vini’ and until now it has been de- water, macerated, and resuspended in 600 μl of brain heart tected in Spain, the Czech Republic, Slovakia and Turkey infusion (BHI). For each tick sample, two shell vials with a [9–11]. This bacterium has been molecularly detected confluent monolayer of Vero cells were each inoculated mainly in Ixodes arboricola ticks, in which the prevalence is with 300 μl of the BHI suspension and then centrifuged high (reaching 90–100 %) [11, 12]. It has rarely been found for 1 h at 700× g and 22 °C. The monolayers were washed Novakova et al. Parasites & Vectors (2016) 9:469 Page 3 of 8 and fed with 1 ml of minimal essential medium supple- Molecular characterization mented
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