G Model TTBDIS-444; No. of Pages 4
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia Marketa Novakova a,b,∗ , Alexandra Bulkova a , Francisco B. Costa c , Anton Kristin d , Milos Krist e , Frantisek Krause f , Eva Liznarova g , Marcelo B. Labruna c , Ivan Literak a,b a Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic b CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic c Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine, University of São Paulo, São Paulo, Brazil d Institute of Forest Ecology, Slovak Academy of Sciences, Zvolen, Slovakia e Department of Zoology and Laboratory of Ornithology, Faculty of Science, Palacky University, Olomouc, Czech Republic f Breclav, Czech Republic g Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic
a r t i c l e
i n f o
Article history: Received 18 May 2014 Received in revised form 30 January 2015 Accepted 13 February 2015 Available online xxx Keywords: Ticks Birds Ixodes arboricola ‘Candidatus Rickettsia vini’ Rickettsia Central Europe
a b s t r a c t The aim of this study was to analyze the prevalence of rickettsiae in the tree-hole tick Ixodes arboricola in the Czech Republic and Slovakia. During May to September of 2009 and 2013, bird boxes belonging to three different areas were screened for ticks. In total, 454 nestlings and 109 nests of 10 hole-breeding bird species were examined. Ticks were found on Ficedula albicollis, Parus major, Cyanistes caeruleus and Sitta europaea and/or in their nests. In total, 166 ticks (17 nymphs, 10 males and 139 females) were found at 3 areas (arithmetic mean ± standard error: 55.3 ± 45.9). All ticks were tested for the presence of Rickettsia species by polymerase chain reaction targeting the rickettsial genes gltA, ompA, ompB and htrA and amplicon sequencing. All individuals except 3 nymphs were infected with ‘Candidatus Rickettsia vini’. Multilocus sequence typing showed closest proximity to Rickettsia japonica and Rickettsia heilongjiangensis cluster. The presence of ‘Ca. R. vini’ is reported for the first time in Slovakia. © 2015 Elsevier GmbH. All rights reserved.
Introduction Ixodes arboricola Schulze and Schlottke, also known as the treehole tick, is a nidicolous ectoparasite that preferably feeds on hole-breeding birds. The whole life cycle of the tick is restricted to different types of cavities, such as tree holes or nest boxes. Birds play a significant role as reservoirs of various tick-borne pathogens (Hubálek, 2004; Palomar et al., 2012a). Tick-borne rickettsioses belong to emerging and reemerging infections. These zoonoses are among the longest-known vector-borne diseases and several rickettsial species have been associated with human infections (Weinert et al., 2009; Parola et al., 2013). Still, new Rickettsia species are discovered, including the recently described ‘Candidatus Rickettsia vini’ found in I. arboricola individuals from Spain, the Czech
∗ Corresponding author at: Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho trida 1/3, 612 42 Brno, Czech Republic. Tel.: +420 732 846 020. E-mail address: [email protected] (M. Novakova).
Republic and Turkey (Keskin et al., 2014; Palomar et al., 2012a; Spitalska et al., 2011). In this study, we screened I. arboricola ticks for Rickettsia spp., and compared the genetic sequences from positive samples with those from known species in GenBank. Ticks were collected from nestlings and nest-boxes from the Czech Republic and Slovakia. Material and methods During the nestling period (May–June), nest boxes installed in the forests were surveyed at 3 locations: Breclav, 48◦ 43 N, 16◦ 54 E, 150 m above sea level (a.s.l.), an oak-ash flood-plain forest, 2009 and 2013; Velky Kosir, 49◦ 32 N, 17◦ 2 E, 350 m a.s.l., an oak hill forest, the nestling period and September (the after-breeding period) 2013 – both in the Czech Republic; and Ziar nad Hronom, Slovakia, 48◦ 34 N, 18◦ 52 E, 450 m a.s.l., a beech-oak mountain forest, 2013. Individual bird nestlings were identified using Svensson et al. (2010), ringed, examined for the presence of ticks and put back into their nests. Ticks were collected by tweezers and stored in 96% ethyl alcohol. If there were no nestlings because fledged or predated, nest
http://dx.doi.org/10.1016/j.ttbdis.2015.02.006 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
G Model
ARTICLE IN PRESS
TTBDIS-444; No. of Pages 4
M. Novakova et al. / Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
2
Table 1 Nest boxes and nestlings surveyed for the presence of Ixodes arboricola. Birds
Study area/year
Family
Species
Certhiidae Muscicapidae Paridae
Certhia sp. Ficedula albicollis Cyanistes caeruleus Parus major Parus palustris Periparus ater Passer montanus Sitta europaea Sturnus vulgaris Jynx torquilla
Passeridae Sittidae Sturnidae Picidae
1 (0) 87 (147) 10 (17) 27 (47) 1 (0)
12 (51)
Velky Kosir 2013
Ziar nad Hronom 2013
55 (102)
17 (27)
1 (10)
1 (0) 171 (327) 10 (17) 32 (68) 1 (0) 1 (11) 3 (5) 3 (4) 1 (6) 5 (16)
4 (11) 1 (11)
3 (5) 1 (0) 1 (6) 5 (16) 136 (238)
Total
Total
Breclav 2009 Breclav 2013 No. of nest boxes (no. of nestlings)
2 (4)
13 (61)
was placed into zip-lock plastic bag. In the laboratory, nests were examined for the presence of ticks and identified to species according to characteristic appearance of the nest. Ticks were classified to species, stage and blood meal volume according to Nosek and Sixl (1972) and sequencing of the tick mitochondrial 16S rDNA gene (Mangold et al., 1998). DNA was extracted from all individuals. Success of DNA extraction was confirmed by polymerase chain reaction (PCR) using primers 16S+1 and 16S–1 amplifying a ≈460-bp fragment of the tick mitochondrial 16S rDNA gene. PCR with primers CS-78 and CS323, CS-239 and CS-1069 was used to amplify a ≈1090-bp fragment of the gltA gene that occurs in all Rickettsia species (Labruna et al., 2004). Detection of Rickettsia belonging to the spotted fever group (SFG) was performed by PCR using primers Rr190.70 and Rr190.602 targeting a 532-bp fragment, a part of the ompA gene that occurs in the majority of SFG rickettsiae (Regnery et al., 1991). This gene is usually highly polymorphic and is considered the most valuable ˜ et al., 2013). for the identification of Rickettsia species (Santibánez To enhance the identification, nested PCR with primers rompB OF, rompB OR, rompB SFG IF and rompB SFG/TG was used to amplify a 425-bp fragment of the ompB gene and primers 17kD1 and 17kD2 to amplify a 433-bp fragment of htrA gene (Choi et al., 2005; Webb et al., 1990). PCR products were purified by ExoSAP-IT® and DNA-sequenced by Sanger dideoxy sequencing. Sequences were subjected to National Center for Biotechnology Information nucleotide BLAST analysis to compare to published sequences of species. Alignment of sequences was conducted using MEGA 6 (Tamura et al., 2013).
55(102)
24(53)
228 (454)
Phylogenetic analysis was performed by PAUP* 4.0 (Swofford, 2003). Statistical analyses were performed within the R environment (R Development Core Team, 2011). Infestation prevalence in two infested bird species was compared by Two-sample test for equality of proportions. The difference in infestation rate on studied localities was tested using Pearson’s Chi-squared test for count data. We compared localities only for year 2013, because in 2009 the only locality studied was Breclav. Results In total, 228 nest boxes were surveyed, in which 454 pulli were found (Table 1). One hundred nine nests were taken for observation in the laboratory. In total, 166 (17 nymphs, 10 males and 139 females) I. arboricola ticks were collected: 119 (13 nymphs and 106 females) nestling-derived ticks, 47 (4 nymphs, 10 males and 33 females) individuals found in nests; arithmetic mean ± standard error: 55.3 ± 45.9 (Table 2). Ticks were derived from nests and/or nestlings of four out of ten hole-breeding songbird species (Table 2). By BLAST analysis, the 16S rDNA partial sequence was 100% (410/410) identical to the corresponding sequence of I. arboricola from Spain (JF791812). GenBank nucleotide sequence accession numbers are KP713675 (a nymph from the Czech Republic) and KP713676 (a nymph from Slovakia). Ticks feeding on nestlings were observed on F. albicollis (mean infestation prevalence 11.5%) and P. major (mean infestation prevalence 7%). The difference in the infestation prevalence was not
Table 2 Ticks Ixodes arboricola collected on nestlings or from nests in Breclav (BR), Velky Kosir (VK) and Ziar nad Hronom (ZH) in 2009 and 2013. Nymph (N), male (M), female (F). Bird species
Study area/year
Certhia sp. Ficedula albicollis
BR 13 BR 09 BR 13 VK 13 ZH 13 BR 13 BR 09 BR 13 ZH 13 BR 13 ZH 13 BR 13 BR 13 ZH 13 BR 13 BR 13
Cyanistes caeruleus Parus major
Parus palustris Periparus ater Passer montanus Sitta europaea Sturnus vulgaris Jynx torquilla Total
No. infested/no. captured pulli (% prevalence)
Mean infestation intensity
No. of ticks on pulli (N/M/F)
16/51 (31) 21/147 (14) 1/102 (1) 0/27 (0) 0/17 (0) 1/47 (2) 1/10 (10) 1/11 (9)
2.2 3.9 1.0
1/0/34 10/0/70 0/0/1
1.0 1.0 1.0
0/0/1 1/0/0 1/0/0
No. of nests
No. of ticks in nests (N/M/F)
1
0/11 (0) 0/5 (0)
3 1 2 1 1
0/4 (0) 0/6 (0) 0/16 (0) 41/454 (9)
43 21 9 6 21
13/0/106
109
0/6/21 3/1/9 0/0/1 0/1/1
1/1/1
0/1/0
4/10/33
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
G Model TTBDIS-444; No. of Pages 4
ARTICLE IN PRESS M. Novakova et al. / Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
3
and aligned. All of them were 100% identical to each other considering each of the four rickettsial genes across all three locations. By BLAST analysis, the gltA partial sequence was 100% (350/350) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF803266); the ompA partial sequence was 100% (491/491) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF758828) and Turkey (KF192804); the ompB partial sequence was 100% (373/373) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF758826); the htrA partial sequence was 100% (394/394) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF758827). GenBank nucleotide sequence accession numbers for the partial sequences (one sequence per one location) are: KJ626330, KJ626332, KJ626334 for gltA, KJ626331, KJ626333, KJ626335 for ompA, KM590518, KM590519, KM590520 for ompB and KP713672, KP713673, KP713674 for htrA. The phylogenetic tree based on concatenated partial sequences of gltA-ompA-ompB-htrA genes showed the nearest proximity of ‘Ca. R. vini’ to R. japonica and R. heilongjiangensis cluster (Fig. 1).
Discussion
Fig. 1. Molecular phylogenetic analysis of ‘Candidatus Rickettsia vini’ isolates from the Czech Republic, Slovakia (CS) and Spain (SP). Czech and Slovakian strains were unified as they were 100% identical. Four genes were concatenated (gltA-ompAompB-htrA), a total of 2325 positions were tested. The evolutionary relationships were computed using the maximum likelihood method. The bootstrap values obtained by 1000 replicates are shown next to the branches. The analyses were made by PAUP* 4.0 program (Swofford, 2003). GenBank accession numbers of the sequences of the Rickettsia species used to make the concatenated analysis are available as Supplement Data.
significant among these two bird species (Two-sample test for equality of proportions, X21 = 28.10, p = 0.12). The significantly highest abundance of ticks was observed in a lowland oak-ash flood-plain forest near Breclav in 2013 (Pearson’s Chi-squared test, X22 = 13.02, p = 0.0015, infestation prevalence 14%, mean infestation intensity 3.9). The highest number of ticks observed per bird box was 48 (5 nymphs and 43 females), including 16 females on one pullus of F. albicollis. All ticks were tested individually for the presence of Rickettsia. All males, all females and 14 of 17 nymphs yielded positive results by the gltA, ompA, ompB and htrA PCR assays. The three Rickettsia spp. non-infected nymphs were collected from F. albicollis in Breclav in 2013. There were found one larva and one nymph of Ixodes ricinus on 2 pulli of F. albicollis at Velky Kosir in June (prevalence 2%). These ticks did not occur along with I. arboricola ticks. None of them contained rickettsial DNA. All PCR products from Velky Kosir and Ziar nad Hronom and randomly selected PCR products from Breclav (all males, all nymphs and randomly selected 28 of 127 females) were DNA-sequenced
I. arboricola is commonly found in tree cavities and nest boxes in Europe (Heylen et al., 2014; Jaenson et al., 1994; Petney et al., 2012; Siuda, 1986). In this study, we found it on birds belonging to Passeriformes, which is in compliance with previous studies (Heylen and Matthysen, 2011; Palomar et al., 2012a). We collected nymphal and female ticks attached to F. albicollis and P. major. I. arboricola ticks have been previously recorded on 11 bird species (including the 4 species mentioned in Table 2) in the Czech Republic and Slovakia (Cerny and Balat, 1960; Literak et al., 2007). The difference in the infestation prevalence was not significant between F. albicollis and P. major as well as it has been observed before between captured P. major and C. caeruleus (Heylen et al., 2014). Larvae were found neither on nestlings nor in nests. Although larvae occur during the whole year, they could have escaped our notice due to their small size (Heylen et al., 2014). The significantly higher abundance of ticks in Breclav could be caused by a higher density of hole-breeding birds in the area in the long term due to the plentiful presence of insect. For this reason, a stable population of I. arboricola ticks could have been established there. Higher humidity could also be another supporting factor. The two other study areas are more arid so the abundance of invertebrates is lower. The analysis of the partial sequences of the four rickettsial genes (gltA, ompA, ompB and htrA) showed that all infected ticks contained DNA of ‘Ca. R. vini’. By phylogenetic analysis based on these four genes concatenated, ‘Ca. R. vini’ is closely related to R. heilongjiangensis and R. japonica (Ando et al., 2010; Mahara, 2006; Palomar et al., 2012b). These two latter species are human pathogens and have not been reported in Europe (Oteo and Portillo, 2012). Other rickettsial genospecies that are closely related to R. heilongjiangensis have been detected in Ixodes spp. collected from birds in Sweden and United Kingdom (Elfving et al., 2010; Graham et al., 2010). This high diversity of rickettsial genospecies suggests that birds could play an important role in dispersal of various rickettsiae. In a previous study, Rickettsia sp. was detected in 44% larvae and in 24.5% nymphs of I. arboricola from the Czech Republic (Spitalska et al., 2011). That identification was not conclusive because it was based only on the gltA partial sequences. Those sequences (GenBank accession numbers JF303663–JF303666) were 98–100% identical with the gltA partial sequences obtained in this study. Here, we identified a recently described ‘Ca. R. vini’ in I. arboricola ticks from
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
G Model TTBDIS-444; No. of Pages 4
ARTICLE IN PRESS M. Novakova et al. / Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
4
the Czech Republic and Slovakia. Previously, this rickettsia has been confirmed in I. arboricola and I. ricinus ticks from Spain and in I. arboricola ticks from Turkey (Keskin et al., 2014; Palomar et al., 2012a). Since cofeeding of I. ricinus and I. arboricola ticks has previously been observed due to the overlapping of hosts and seasonal feeding activity of these two tick species, ‘Ca. R. vini’ could be bridged to other hosts that stay outside of the enzootic cycle (Heylen et al., 2014). In the present study, all adults and 82% nymphs contained DNA of ‘Ca. R. vini’. These high infection rates can be explained by an exclusive endosymbiotic relation of this rickettsia to I. arboricola ticks. Similarly, DNA of Rickettsia phylotype G021 was found with 100% prevalence in Ixodes pacificus ticks (Cheng et al., 2013). Rickettsiae have been described in the ovaries of Ixodes scapularis and I. ricinus ticks (Noda et al., 1997; Zhu et al., 1992). Regarding the presence of ‘Ca. R. vini’ DNA in males, we suggest that this rickettsia could occur in other tissues, such as Malpighian tubules, as various bacterial symbionts have been observed there (Noda et al., 1997). At present, we cannot discard ‘Ca. R. vini’ as a potential human pathogen, since several examples in rickettsiology have shown that previously ‘non-pathogenic’ rickettsiae were further demonstrated to be agents of human diseases (Parola et al., 2013). Further studies could elucidate, if this rickettsia is fully adapted to its tick host or is able to infect and propagate in vertebrate host. Acknowledgements ˚ zek, Michaela We thank Peter Adamík, Libor Schröpfer, Pavel Ruˇ Galová, Miluˇse Krauseová and Martin Janˇca for their cooperation ˇ in the field. We appreciate Eva Spitalska’s help in the early period of study. We are grateful to Linda Grillová and Álvaro Adolfo Faccini M. for providing skilled advice. The study was supported by the project CEITEC – Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) of the European Regional Development Fund., FAPESP – Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, Brazil 2010/52395-0 and Project for Consolidation of Student’s Mobilities in non-EU Countries (CSM15) of the Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.02.006. References Ando, S., Kurosawa, M., Sakata, A., Fujita, H., Sakai, K., Sekine, M., Katsumi, M., Saitou, W., Yano, Y., Takada, N., Takano, A., Kawabata, H., Hanaoka, N., Watanabe, H., Kurane, I., Kishimoto, T., 2010. Human Rickettsia heilongjiangensis infection, Japan. Emerg. Infect. Dis. 16, 1306–1308. Cerny, V., Balat, F., 1960. A contribution to the bionomics of the tick Ixodes arboricola P. Schulze. Zoologicke Listy 9, 217–226 (in Czech). Cheng, D., Vigil, K., Schanes, P., Brown, R.N., Zhong, J., 2013. Prevalence and burden of two rickettsial phylotypes (G021 and G022) in Ixodes pacificus from California by real-time quantitative PCR. Ticks Tick Borne Dis. 4, 280–287. Choi, Y.J., Jang, W.J., Kim, J.H., Ryu, J.S., Lee, S.H., Park, K.H., Paik, H.S., Koh, Y.S., Choi, M.S., Kim, I.S., 2005. Spotted fever group and typhus group rickettsioses in humans, South Korea. Emerg. Infect. Dis. 11, 237–244. Elfving, K., Olsen, B., Bergström, S., Waldenström, J., Lundkvist, A., Sjöstedt, A., Mejlon, H., Nilsson, K., 2010. Dissemination of spotted fever Rickettsia agents in Europe by migrating birds. PLoS ONE 5, e8572.
Graham, R.I., Mainwaring, M.C., Du Feu, R., 2010. Detection of spotted fever group Rickettsia spp. from bird ticks in the U.K. Med. Vet. Entomol. 24, 340–343. Heylen, D.J.A., Matthysen, E., 2011. Experimental evidence for host preference in a tick parasitizing songbird nestlings. Oikos 120, 1209–1216. Heylen, D.J.A., Van Oosten, A.R., Devriendt, N., Elst, J., De Bruyn, L., Matthysen, E., 2014. Seasonal feeding activity of the tree-hole tick, Ixodes arboricola. Parasitology 141, 1044–1051. Hubálek, Z., 2004. An annotated checklist of pathogenic microorganisms associated with migratory birds. J. Wildl. Dis. 40, 639–659. Jaenson, T.G.T., Talleklint, L., Lundqvist, L., Olsen, B., Chirico, J., Mejlon, H., 1994. Geographical distribution, host associations, and vector roles of ticks (Acari: Ixodidae, Argasidae) in Sweden. J. Med. Entomol. 31, 240–256. Keskin, A., Koprulu, T.K., Bursali, A., Ozsemir, A.C., Yavuz, K.E., Tekin, S., 2014. First record of Ixodes arboricola (Ixodida: Ixodidae) from Turkey with presence of Candidatus Rickettsia vini (Rickettsiales: Rickettsiaceae). J. Med. Entomol. 51, 864–867. Labruna, M.B., Whitworth, T., Horta, M.C., Bouyer, D.H., McBride, J.W., Pinter, A., Popov, V., Gennari, S.M., Walker, D.H., 2004. Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where Brazilian spotted fever is endemic. J. Clin. Microbiol. 42, 90–98. Literak, I., Kocianova, E., Dusbabek, F., Martinu, J., Podzemny, P., Sychra, O., 2007. Winter infestation of wild birds by ticks and chiggers (Acari: Ixodidae, Trombiculidae) in the Czech Republic. Parasitol. Res. 101, 1709–1711. Mahara, F., 2006. Rickettsioses in Japan and the Far East. Ann. N. Y. Acad. Sci. 1078, 60–73. Mangold, A.J., Bargues, M.D., Mas-Coma, S., 1998. Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari, Ixodidae). Parasitol. Res. 84, 478–484. Noda, H., Munderloh, U.G., Kurtti, T.J., 1997. Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals. Appl. Environ. Microbiol. 63, 3926–3932. Nosek, J., Sixl, W., 1972. Central-European ticks (Ixodoidea). Mitt. Abt. Zool. Landesmus. Joanneum 1, 61–92. Oteo, J.A., Portillo, A., 2012. Tick-borne rickettsioses in Europe. Ticks Tick Borne Dis. 3, 271–278. ˜ ˜ Palomar, A.M., Santibánez, P., Mazuelas, D., Roncero, L., Santibánez, S., Portillo, A., Oteo, J.A., 2012a. Role of birds in dispersal of etiologic agents of tick-borne zoonoses, Spain, 2009. Emerg. Infect. Dis. 18, 1188–1191. ˜ ˜ Palomar, A.M., Portillo, A., Santibánez, P., Santibánez, S., García-Álvarez, L., Oteo, J.A., 2012b. Genetic characterization of Candidatus Rickettsia vini, a new rickettsia amplified in ticks from La Rioja, Spain. Ticks Tick Borne Dis. 3, 318–320. Parola, P., Paddock, C.D., Socolovschi, C., Labruna, M.B., Mediannikov, O., Kernif, T., Abdad, M.Y., Stenos, J., Bitam, I., Fournier, P.E., Raoult, D., 2013. Update on tick-borne rickettioses around the world: a geographic approach. Clin. Microbiol. Rev. 26, 657–702. Petney, T.N., Pfäffle, M.P., Skuballa, J.D., 2012. An annotated checklist of the ticks (Acari: Ixodida) of Germany. Syst. Appl. Acarol. 17, 115–170. R Core Team, 2011. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Regnery, R.L., Spruill, C.L., Plikaytis, B.D., 1991. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J. Bacteriol. 173, 1576–1589. ˜ ˜ Santibánez, S., Portillo, A., Santibánez, P., Palomar, A.M., Oteo, J.A., 2013. Usefulness of rickettsial PCR assays for the molecular diagnosis of human rickettsioses. Enferm. Infecc. Microbiol. Clin. 31, 283–288. Siuda, K., 1986. Ixodidae (Ixodida, Acari) pasozyty ptakow Polski. Bezwzglednie ornitofilne. Wiad. Parazytol. 4, 479–482 (in Polish). Spitalska, E., Literak, I., Kocianova, E., Taragelova, V., 2011. The importance of Ixodes arboricola in transmission of Rickettsia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato in the Czech Republic, Central Europe. Vector Borne Zoonotic Dis. 11, 1235–1241. Svensson, L., Mullarney, K., Zetterström, D., 2010. Birds of Europe, second edn. Princeton University Press, New Jersey. Swofford, D.L., 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, MA. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725– 2729. Webb, L., Carl, M., Malloy, D.C., Dasch, G.A., Azad, A.F., 1990. Detection of murine typhus infection in fleas by using the polymerase chain reaction. J. Clin. Microbiol. 28, 530–534. Weinert, L.A., Werren, J.H., Aebi, A., Stone, G.N., Jiggins, F.M., 2009. Evolution and diversity of Rickettsia bacteria. BMC Biol. 7, 6. Zhu, Z., Aeschlimann, A., Gern, L., 1992. Rickettsia-like microorganisms in the ovarian primordia of molting Ixodes ricinus (Acari: Ixodidae) larvae and nymphs. Ann. Parasitol. Hum. Comp. 67, 99–110.
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia Marketa Novakova a,b,∗ , Alexandra Bulkova a , Francisco B. Costa c , Anton Kristin d , Milos Krist e , Frantisek Krause f , Eva Liznarova g , Marcelo B. Labruna c , Ivan Literak a,b a Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic b CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic c Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine, University of São Paulo, São Paulo, Brazil d Institute of Forest Ecology, Slovak Academy of Sciences, Zvolen, Slovakia e Department of Zoology and Laboratory of Ornithology, Faculty of Science, Palacky University, Olomouc, Czech Republic f Breclav, Czech Republic g Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic
a r t i c l e
i n f o
Article history: Received 18 May 2014 Received in revised form 30 January 2015 Accepted 13 February 2015 Available online xxx Keywords: Ticks Birds Ixodes arboricola ‘Candidatus Rickettsia vini’ Rickettsia Central Europe
a b s t r a c t The aim of this study was to analyze the prevalence of rickettsiae in the tree-hole tick Ixodes arboricola in the Czech Republic and Slovakia. During May to September of 2009 and 2013, bird boxes belonging to three different areas were screened for ticks. In total, 454 nestlings and 109 nests of 10 hole-breeding bird species were examined. Ticks were found on Ficedula albicollis, Parus major, Cyanistes caeruleus and Sitta europaea and/or in their nests. In total, 166 ticks (17 nymphs, 10 males and 139 females) were found at 3 areas (arithmetic mean ± standard error: 55.3 ± 45.9). All ticks were tested for the presence of Rickettsia species by polymerase chain reaction targeting the rickettsial genes gltA, ompA, ompB and htrA and amplicon sequencing. All individuals except 3 nymphs were infected with ‘Candidatus Rickettsia vini’. Multilocus sequence typing showed closest proximity to Rickettsia japonica and Rickettsia heilongjiangensis cluster. The presence of ‘Ca. R. vini’ is reported for the first time in Slovakia. © 2015 Elsevier GmbH. All rights reserved.
Introduction Ixodes arboricola Schulze and Schlottke, also known as the treehole tick, is a nidicolous ectoparasite that preferably feeds on hole-breeding birds. The whole life cycle of the tick is restricted to different types of cavities, such as tree holes or nest boxes. Birds play a significant role as reservoirs of various tick-borne pathogens (Hubálek, 2004; Palomar et al., 2012a). Tick-borne rickettsioses belong to emerging and reemerging infections. These zoonoses are among the longest-known vector-borne diseases and several rickettsial species have been associated with human infections (Weinert et al., 2009; Parola et al., 2013). Still, new Rickettsia species are discovered, including the recently described ‘Candidatus Rickettsia vini’ found in I. arboricola individuals from Spain, the Czech
∗ Corresponding author at: Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho trida 1/3, 612 42 Brno, Czech Republic. Tel.: +420 732 846 020. E-mail address: [email protected] (M. Novakova).
Republic and Turkey (Keskin et al., 2014; Palomar et al., 2012a; Spitalska et al., 2011). In this study, we screened I. arboricola ticks for Rickettsia spp., and compared the genetic sequences from positive samples with those from known species in GenBank. Ticks were collected from nestlings and nest-boxes from the Czech Republic and Slovakia. Material and methods During the nestling period (May–June), nest boxes installed in the forests were surveyed at 3 locations: Breclav, 48◦ 43 N, 16◦ 54 E, 150 m above sea level (a.s.l.), an oak-ash flood-plain forest, 2009 and 2013; Velky Kosir, 49◦ 32 N, 17◦ 2 E, 350 m a.s.l., an oak hill forest, the nestling period and September (the after-breeding period) 2013 – both in the Czech Republic; and Ziar nad Hronom, Slovakia, 48◦ 34 N, 18◦ 52 E, 450 m a.s.l., a beech-oak mountain forest, 2013. Individual bird nestlings were identified using Svensson et al. (2010), ringed, examined for the presence of ticks and put back into their nests. Ticks were collected by tweezers and stored in 96% ethyl alcohol. If there were no nestlings because fledged or predated, nest
http://dx.doi.org/10.1016/j.ttbdis.2015.02.006 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
G Model
ARTICLE IN PRESS
TTBDIS-444; No. of Pages 4
M. Novakova et al. / Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
2
Table 1 Nest boxes and nestlings surveyed for the presence of Ixodes arboricola. Birds
Study area/year
Family
Species
Certhiidae Muscicapidae Paridae
Certhia sp. Ficedula albicollis Cyanistes caeruleus Parus major Parus palustris Periparus ater Passer montanus Sitta europaea Sturnus vulgaris Jynx torquilla
Passeridae Sittidae Sturnidae Picidae
1 (0) 87 (147) 10 (17) 27 (47) 1 (0)
12 (51)
Velky Kosir 2013
Ziar nad Hronom 2013
55 (102)
17 (27)
1 (10)
1 (0) 171 (327) 10 (17) 32 (68) 1 (0) 1 (11) 3 (5) 3 (4) 1 (6) 5 (16)
4 (11) 1 (11)
3 (5) 1 (0) 1 (6) 5 (16) 136 (238)
Total
Total
Breclav 2009 Breclav 2013 No. of nest boxes (no. of nestlings)
2 (4)
13 (61)
was placed into zip-lock plastic bag. In the laboratory, nests were examined for the presence of ticks and identified to species according to characteristic appearance of the nest. Ticks were classified to species, stage and blood meal volume according to Nosek and Sixl (1972) and sequencing of the tick mitochondrial 16S rDNA gene (Mangold et al., 1998). DNA was extracted from all individuals. Success of DNA extraction was confirmed by polymerase chain reaction (PCR) using primers 16S+1 and 16S–1 amplifying a ≈460-bp fragment of the tick mitochondrial 16S rDNA gene. PCR with primers CS-78 and CS323, CS-239 and CS-1069 was used to amplify a ≈1090-bp fragment of the gltA gene that occurs in all Rickettsia species (Labruna et al., 2004). Detection of Rickettsia belonging to the spotted fever group (SFG) was performed by PCR using primers Rr190.70 and Rr190.602 targeting a 532-bp fragment, a part of the ompA gene that occurs in the majority of SFG rickettsiae (Regnery et al., 1991). This gene is usually highly polymorphic and is considered the most valuable ˜ et al., 2013). for the identification of Rickettsia species (Santibánez To enhance the identification, nested PCR with primers rompB OF, rompB OR, rompB SFG IF and rompB SFG/TG was used to amplify a 425-bp fragment of the ompB gene and primers 17kD1 and 17kD2 to amplify a 433-bp fragment of htrA gene (Choi et al., 2005; Webb et al., 1990). PCR products were purified by ExoSAP-IT® and DNA-sequenced by Sanger dideoxy sequencing. Sequences were subjected to National Center for Biotechnology Information nucleotide BLAST analysis to compare to published sequences of species. Alignment of sequences was conducted using MEGA 6 (Tamura et al., 2013).
55(102)
24(53)
228 (454)
Phylogenetic analysis was performed by PAUP* 4.0 (Swofford, 2003). Statistical analyses were performed within the R environment (R Development Core Team, 2011). Infestation prevalence in two infested bird species was compared by Two-sample test for equality of proportions. The difference in infestation rate on studied localities was tested using Pearson’s Chi-squared test for count data. We compared localities only for year 2013, because in 2009 the only locality studied was Breclav. Results In total, 228 nest boxes were surveyed, in which 454 pulli were found (Table 1). One hundred nine nests were taken for observation in the laboratory. In total, 166 (17 nymphs, 10 males and 139 females) I. arboricola ticks were collected: 119 (13 nymphs and 106 females) nestling-derived ticks, 47 (4 nymphs, 10 males and 33 females) individuals found in nests; arithmetic mean ± standard error: 55.3 ± 45.9 (Table 2). Ticks were derived from nests and/or nestlings of four out of ten hole-breeding songbird species (Table 2). By BLAST analysis, the 16S rDNA partial sequence was 100% (410/410) identical to the corresponding sequence of I. arboricola from Spain (JF791812). GenBank nucleotide sequence accession numbers are KP713675 (a nymph from the Czech Republic) and KP713676 (a nymph from Slovakia). Ticks feeding on nestlings were observed on F. albicollis (mean infestation prevalence 11.5%) and P. major (mean infestation prevalence 7%). The difference in the infestation prevalence was not
Table 2 Ticks Ixodes arboricola collected on nestlings or from nests in Breclav (BR), Velky Kosir (VK) and Ziar nad Hronom (ZH) in 2009 and 2013. Nymph (N), male (M), female (F). Bird species
Study area/year
Certhia sp. Ficedula albicollis
BR 13 BR 09 BR 13 VK 13 ZH 13 BR 13 BR 09 BR 13 ZH 13 BR 13 ZH 13 BR 13 BR 13 ZH 13 BR 13 BR 13
Cyanistes caeruleus Parus major
Parus palustris Periparus ater Passer montanus Sitta europaea Sturnus vulgaris Jynx torquilla Total
No. infested/no. captured pulli (% prevalence)
Mean infestation intensity
No. of ticks on pulli (N/M/F)
16/51 (31) 21/147 (14) 1/102 (1) 0/27 (0) 0/17 (0) 1/47 (2) 1/10 (10) 1/11 (9)
2.2 3.9 1.0
1/0/34 10/0/70 0/0/1
1.0 1.0 1.0
0/0/1 1/0/0 1/0/0
No. of nests
No. of ticks in nests (N/M/F)
1
0/11 (0) 0/5 (0)
3 1 2 1 1
0/4 (0) 0/6 (0) 0/16 (0) 41/454 (9)
43 21 9 6 21
13/0/106
109
0/6/21 3/1/9 0/0/1 0/1/1
1/1/1
0/1/0
4/10/33
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
G Model TTBDIS-444; No. of Pages 4
ARTICLE IN PRESS M. Novakova et al. / Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
3
and aligned. All of them were 100% identical to each other considering each of the four rickettsial genes across all three locations. By BLAST analysis, the gltA partial sequence was 100% (350/350) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF803266); the ompA partial sequence was 100% (491/491) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF758828) and Turkey (KF192804); the ompB partial sequence was 100% (373/373) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF758826); the htrA partial sequence was 100% (394/394) identical to the corresponding sequence of ‘Ca. R. vini’ detected in I. arboricola from Spain (JF758827). GenBank nucleotide sequence accession numbers for the partial sequences (one sequence per one location) are: KJ626330, KJ626332, KJ626334 for gltA, KJ626331, KJ626333, KJ626335 for ompA, KM590518, KM590519, KM590520 for ompB and KP713672, KP713673, KP713674 for htrA. The phylogenetic tree based on concatenated partial sequences of gltA-ompA-ompB-htrA genes showed the nearest proximity of ‘Ca. R. vini’ to R. japonica and R. heilongjiangensis cluster (Fig. 1).
Discussion
Fig. 1. Molecular phylogenetic analysis of ‘Candidatus Rickettsia vini’ isolates from the Czech Republic, Slovakia (CS) and Spain (SP). Czech and Slovakian strains were unified as they were 100% identical. Four genes were concatenated (gltA-ompAompB-htrA), a total of 2325 positions were tested. The evolutionary relationships were computed using the maximum likelihood method. The bootstrap values obtained by 1000 replicates are shown next to the branches. The analyses were made by PAUP* 4.0 program (Swofford, 2003). GenBank accession numbers of the sequences of the Rickettsia species used to make the concatenated analysis are available as Supplement Data.
significant among these two bird species (Two-sample test for equality of proportions, X21 = 28.10, p = 0.12). The significantly highest abundance of ticks was observed in a lowland oak-ash flood-plain forest near Breclav in 2013 (Pearson’s Chi-squared test, X22 = 13.02, p = 0.0015, infestation prevalence 14%, mean infestation intensity 3.9). The highest number of ticks observed per bird box was 48 (5 nymphs and 43 females), including 16 females on one pullus of F. albicollis. All ticks were tested individually for the presence of Rickettsia. All males, all females and 14 of 17 nymphs yielded positive results by the gltA, ompA, ompB and htrA PCR assays. The three Rickettsia spp. non-infected nymphs were collected from F. albicollis in Breclav in 2013. There were found one larva and one nymph of Ixodes ricinus on 2 pulli of F. albicollis at Velky Kosir in June (prevalence 2%). These ticks did not occur along with I. arboricola ticks. None of them contained rickettsial DNA. All PCR products from Velky Kosir and Ziar nad Hronom and randomly selected PCR products from Breclav (all males, all nymphs and randomly selected 28 of 127 females) were DNA-sequenced
I. arboricola is commonly found in tree cavities and nest boxes in Europe (Heylen et al., 2014; Jaenson et al., 1994; Petney et al., 2012; Siuda, 1986). In this study, we found it on birds belonging to Passeriformes, which is in compliance with previous studies (Heylen and Matthysen, 2011; Palomar et al., 2012a). We collected nymphal and female ticks attached to F. albicollis and P. major. I. arboricola ticks have been previously recorded on 11 bird species (including the 4 species mentioned in Table 2) in the Czech Republic and Slovakia (Cerny and Balat, 1960; Literak et al., 2007). The difference in the infestation prevalence was not significant between F. albicollis and P. major as well as it has been observed before between captured P. major and C. caeruleus (Heylen et al., 2014). Larvae were found neither on nestlings nor in nests. Although larvae occur during the whole year, they could have escaped our notice due to their small size (Heylen et al., 2014). The significantly higher abundance of ticks in Breclav could be caused by a higher density of hole-breeding birds in the area in the long term due to the plentiful presence of insect. For this reason, a stable population of I. arboricola ticks could have been established there. Higher humidity could also be another supporting factor. The two other study areas are more arid so the abundance of invertebrates is lower. The analysis of the partial sequences of the four rickettsial genes (gltA, ompA, ompB and htrA) showed that all infected ticks contained DNA of ‘Ca. R. vini’. By phylogenetic analysis based on these four genes concatenated, ‘Ca. R. vini’ is closely related to R. heilongjiangensis and R. japonica (Ando et al., 2010; Mahara, 2006; Palomar et al., 2012b). These two latter species are human pathogens and have not been reported in Europe (Oteo and Portillo, 2012). Other rickettsial genospecies that are closely related to R. heilongjiangensis have been detected in Ixodes spp. collected from birds in Sweden and United Kingdom (Elfving et al., 2010; Graham et al., 2010). This high diversity of rickettsial genospecies suggests that birds could play an important role in dispersal of various rickettsiae. In a previous study, Rickettsia sp. was detected in 44% larvae and in 24.5% nymphs of I. arboricola from the Czech Republic (Spitalska et al., 2011). That identification was not conclusive because it was based only on the gltA partial sequences. Those sequences (GenBank accession numbers JF303663–JF303666) were 98–100% identical with the gltA partial sequences obtained in this study. Here, we identified a recently described ‘Ca. R. vini’ in I. arboricola ticks from
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
G Model TTBDIS-444; No. of Pages 4
ARTICLE IN PRESS M. Novakova et al. / Ticks and Tick-borne Diseases xxx (2015) xxx–xxx
4
the Czech Republic and Slovakia. Previously, this rickettsia has been confirmed in I. arboricola and I. ricinus ticks from Spain and in I. arboricola ticks from Turkey (Keskin et al., 2014; Palomar et al., 2012a). Since cofeeding of I. ricinus and I. arboricola ticks has previously been observed due to the overlapping of hosts and seasonal feeding activity of these two tick species, ‘Ca. R. vini’ could be bridged to other hosts that stay outside of the enzootic cycle (Heylen et al., 2014). In the present study, all adults and 82% nymphs contained DNA of ‘Ca. R. vini’. These high infection rates can be explained by an exclusive endosymbiotic relation of this rickettsia to I. arboricola ticks. Similarly, DNA of Rickettsia phylotype G021 was found with 100% prevalence in Ixodes pacificus ticks (Cheng et al., 2013). Rickettsiae have been described in the ovaries of Ixodes scapularis and I. ricinus ticks (Noda et al., 1997; Zhu et al., 1992). Regarding the presence of ‘Ca. R. vini’ DNA in males, we suggest that this rickettsia could occur in other tissues, such as Malpighian tubules, as various bacterial symbionts have been observed there (Noda et al., 1997). At present, we cannot discard ‘Ca. R. vini’ as a potential human pathogen, since several examples in rickettsiology have shown that previously ‘non-pathogenic’ rickettsiae were further demonstrated to be agents of human diseases (Parola et al., 2013). Further studies could elucidate, if this rickettsia is fully adapted to its tick host or is able to infect and propagate in vertebrate host. Acknowledgements ˚ zek, Michaela We thank Peter Adamík, Libor Schröpfer, Pavel Ruˇ Galová, Miluˇse Krauseová and Martin Janˇca for their cooperation ˇ in the field. We appreciate Eva Spitalska’s help in the early period of study. We are grateful to Linda Grillová and Álvaro Adolfo Faccini M. for providing skilled advice. The study was supported by the project CEITEC – Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) of the European Regional Development Fund., FAPESP – Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, Brazil 2010/52395-0 and Project for Consolidation of Student’s Mobilities in non-EU Countries (CSM15) of the Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.02.006. References Ando, S., Kurosawa, M., Sakata, A., Fujita, H., Sakai, K., Sekine, M., Katsumi, M., Saitou, W., Yano, Y., Takada, N., Takano, A., Kawabata, H., Hanaoka, N., Watanabe, H., Kurane, I., Kishimoto, T., 2010. Human Rickettsia heilongjiangensis infection, Japan. Emerg. Infect. Dis. 16, 1306–1308. Cerny, V., Balat, F., 1960. A contribution to the bionomics of the tick Ixodes arboricola P. Schulze. Zoologicke Listy 9, 217–226 (in Czech). Cheng, D., Vigil, K., Schanes, P., Brown, R.N., Zhong, J., 2013. Prevalence and burden of two rickettsial phylotypes (G021 and G022) in Ixodes pacificus from California by real-time quantitative PCR. Ticks Tick Borne Dis. 4, 280–287. Choi, Y.J., Jang, W.J., Kim, J.H., Ryu, J.S., Lee, S.H., Park, K.H., Paik, H.S., Koh, Y.S., Choi, M.S., Kim, I.S., 2005. Spotted fever group and typhus group rickettsioses in humans, South Korea. Emerg. Infect. Dis. 11, 237–244. Elfving, K., Olsen, B., Bergström, S., Waldenström, J., Lundkvist, A., Sjöstedt, A., Mejlon, H., Nilsson, K., 2010. Dissemination of spotted fever Rickettsia agents in Europe by migrating birds. PLoS ONE 5, e8572.
Graham, R.I., Mainwaring, M.C., Du Feu, R., 2010. Detection of spotted fever group Rickettsia spp. from bird ticks in the U.K. Med. Vet. Entomol. 24, 340–343. Heylen, D.J.A., Matthysen, E., 2011. Experimental evidence for host preference in a tick parasitizing songbird nestlings. Oikos 120, 1209–1216. Heylen, D.J.A., Van Oosten, A.R., Devriendt, N., Elst, J., De Bruyn, L., Matthysen, E., 2014. Seasonal feeding activity of the tree-hole tick, Ixodes arboricola. Parasitology 141, 1044–1051. Hubálek, Z., 2004. An annotated checklist of pathogenic microorganisms associated with migratory birds. J. Wildl. Dis. 40, 639–659. Jaenson, T.G.T., Talleklint, L., Lundqvist, L., Olsen, B., Chirico, J., Mejlon, H., 1994. Geographical distribution, host associations, and vector roles of ticks (Acari: Ixodidae, Argasidae) in Sweden. J. Med. Entomol. 31, 240–256. Keskin, A., Koprulu, T.K., Bursali, A., Ozsemir, A.C., Yavuz, K.E., Tekin, S., 2014. First record of Ixodes arboricola (Ixodida: Ixodidae) from Turkey with presence of Candidatus Rickettsia vini (Rickettsiales: Rickettsiaceae). J. Med. Entomol. 51, 864–867. Labruna, M.B., Whitworth, T., Horta, M.C., Bouyer, D.H., McBride, J.W., Pinter, A., Popov, V., Gennari, S.M., Walker, D.H., 2004. Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where Brazilian spotted fever is endemic. J. Clin. Microbiol. 42, 90–98. Literak, I., Kocianova, E., Dusbabek, F., Martinu, J., Podzemny, P., Sychra, O., 2007. Winter infestation of wild birds by ticks and chiggers (Acari: Ixodidae, Trombiculidae) in the Czech Republic. Parasitol. Res. 101, 1709–1711. Mahara, F., 2006. Rickettsioses in Japan and the Far East. Ann. N. Y. Acad. Sci. 1078, 60–73. Mangold, A.J., Bargues, M.D., Mas-Coma, S., 1998. Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari, Ixodidae). Parasitol. Res. 84, 478–484. Noda, H., Munderloh, U.G., Kurtti, T.J., 1997. Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals. Appl. Environ. Microbiol. 63, 3926–3932. Nosek, J., Sixl, W., 1972. Central-European ticks (Ixodoidea). Mitt. Abt. Zool. Landesmus. Joanneum 1, 61–92. Oteo, J.A., Portillo, A., 2012. Tick-borne rickettsioses in Europe. Ticks Tick Borne Dis. 3, 271–278. ˜ ˜ Palomar, A.M., Santibánez, P., Mazuelas, D., Roncero, L., Santibánez, S., Portillo, A., Oteo, J.A., 2012a. Role of birds in dispersal of etiologic agents of tick-borne zoonoses, Spain, 2009. Emerg. Infect. Dis. 18, 1188–1191. ˜ ˜ Palomar, A.M., Portillo, A., Santibánez, P., Santibánez, S., García-Álvarez, L., Oteo, J.A., 2012b. Genetic characterization of Candidatus Rickettsia vini, a new rickettsia amplified in ticks from La Rioja, Spain. Ticks Tick Borne Dis. 3, 318–320. Parola, P., Paddock, C.D., Socolovschi, C., Labruna, M.B., Mediannikov, O., Kernif, T., Abdad, M.Y., Stenos, J., Bitam, I., Fournier, P.E., Raoult, D., 2013. Update on tick-borne rickettioses around the world: a geographic approach. Clin. Microbiol. Rev. 26, 657–702. Petney, T.N., Pfäffle, M.P., Skuballa, J.D., 2012. An annotated checklist of the ticks (Acari: Ixodida) of Germany. Syst. Appl. Acarol. 17, 115–170. R Core Team, 2011. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Regnery, R.L., Spruill, C.L., Plikaytis, B.D., 1991. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J. Bacteriol. 173, 1576–1589. ˜ ˜ Santibánez, S., Portillo, A., Santibánez, P., Palomar, A.M., Oteo, J.A., 2013. Usefulness of rickettsial PCR assays for the molecular diagnosis of human rickettsioses. Enferm. Infecc. Microbiol. Clin. 31, 283–288. Siuda, K., 1986. Ixodidae (Ixodida, Acari) pasozyty ptakow Polski. Bezwzglednie ornitofilne. Wiad. Parazytol. 4, 479–482 (in Polish). Spitalska, E., Literak, I., Kocianova, E., Taragelova, V., 2011. The importance of Ixodes arboricola in transmission of Rickettsia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato in the Czech Republic, Central Europe. Vector Borne Zoonotic Dis. 11, 1235–1241. Svensson, L., Mullarney, K., Zetterström, D., 2010. Birds of Europe, second edn. Princeton University Press, New Jersey. Swofford, D.L., 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, MA. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725– 2729. Webb, L., Carl, M., Malloy, D.C., Dasch, G.A., Azad, A.F., 1990. Detection of murine typhus infection in fleas by using the polymerase chain reaction. J. Clin. Microbiol. 28, 530–534. Weinert, L.A., Werren, J.H., Aebi, A., Stone, G.N., Jiggins, F.M., 2009. Evolution and diversity of Rickettsia bacteria. BMC Biol. 7, 6. Zhu, Z., Aeschlimann, A., Gern, L., 1992. Rickettsia-like microorganisms in the ovarian primordia of molting Ixodes ricinus (Acari: Ixodidae) larvae and nymphs. Ann. Parasitol. Hum. Comp. 67, 99–110.
Please cite this article in press as: Novakova, M., et al., Molecular characterization of ‘Candidatus Rickettsia vini’ in Ixodes arboricola from the Czech Republic and Slovakia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.006
