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First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic
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ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic ´ Ivo Rudolf a,b,∗ , Kristyna Venclíková a,b,c , Hana Blaˇzejová a , Lenka Betáˇsová a , Jan Mendel a , a,b Zdenˇek Hubálek , Philippe Parola d a
Institute of Vertebrate Biology, v.v.i., Academy of Sciences, Kvetna 8, 603 65, Brno, Czech Republic Masaryk University, Department of Experimental Biology, Kotlarska 2, 611 37 Brno, Czech Republic c Institute of Macromolecular Chemistry, v.v.i., Academy of Sciences, 162 06 Prague, Czech Republic d Aix Marseille University, CNRS 7278, URMITE, INSERM 1095, UM63,IRD Dakar 198, 27 Bd Jean Moulin, F-13385 Marseille 5, France b
a r t i c l e
i n f o
Article history: Received 22 March 2016 Received in revised form 19 July 2016 Accepted 19 July 2016 Available online xxx Keywords: Rickettsia spp. Dermacentor spp. DEBONEL SENLAT
a b s t r a c t Tick-borne rickettsioses nowadays present a significant health threat among emerging tick-borne infections. In the study, we screened a total of 997 questing adult Dermacentor reticulatus ticks (576 females and 421 males) collected in Southern Moravia (Czech Republic) from April to September of 2010 and 2011 for the presence of human pathogenic rickettsiae. Here we provide the first evidence on the occurrence of human pathogenic Rickettsia raoultii in the local D. reticulatus population indicating possible future emergence of Dermacentor-borne necrotic erythema and lymphadenopathy (DEBONEL) in the region. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction Dermacentor reticulatus is the second most reported tick species after Ixodes ricinus in Central Europe. It has a broad geographical overlap with D. marginatus, but generally extends more to the north (Rubel et al., 2016). Preferred habitats constitute alluvial forests (typically flood plain forest-meadow ecosystem) and swamps where it can survive flooding for certain periods (Nosek, 1972; Hubálek et al., 1996, 1998). It also colonizes drier habitats like fallow and heath land as well as grassland interspersed with bushes and trees. Larvae and nymphs feed mainly on rodent species, whereas adults feed on larger mammals like cattle, deer and dog, and occasionally bite humans (Hubálek and Rudolf, 2011; Hubálek et al., 1996, 1998). In Central Europe, D. reticulatus serves as a facultative vector of Francisella tularensis, a causative agent of tularaemia and Babesia canis which causes canine babesiosis (Hubálek et al., 1998; Hubálek and Rudolf, 2011). However, the research on pathogenic Rickettsia raoultii and R. slovaca in Dermacentor spp. has turned the attention of public health experts also to this group of emerging diseases
∗ Corresponding author at: Institute of Vertebrate Biology, v.v.i., Academy of Sciences of the Czech Republic, Kvetna 8, CZ-603 65 Brno, Czech Republic. E-mail address: [email protected] (I. Rudolf).
(Oteo and Portillo, 2012). Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group rickettsiae. In the last decades, the importance of tick-associated rickettsial pathogens have increased dramatically and several species that were considered nonpathogenic for decades are now associated with human infections (Parola et al., 2013). D. reticulatus and D. marginatus are implicated as vectors of emerging rickettsioses in Europe. Rickettsia raoultii and R. slovaca are associated with a syndrome characterized by scalp eschars and neck lymphadenopathy following tick bites, and the term ‘SENLAT’ (scalp eschar and neck lymphadenopathy after tick bite) was proposed recently by the Marseille group (National Reference Center for Rickettsial Diseases) for this clinical entity (Angelakis et al., 2010; Parola et al., 2013). Initially, this syndrome was named TIBOLA (tick-borne lymphadenopathy) or DEBONEL (Dermacentor-borne necrotic erythema and lymphadenopathy). Recently, R. rioja was detected in a tick removed from a patient with DEBONEL/TIBOLA (Pérez-Pérez et al., 2010). In the Czech Republic, data on emerging Rickettsia spp. pathogenic to humans, especially their prevalence in host-seeking ticks, possible reservoirs as well as its relevance for public and animal health are insufficient. To date, no Rickettsia spp. have been detected in D. reticulatus ticks in the Czech Republic, despite the fact that R. raoultii and R. slovaca were detected in particular species in several neighbouring countries including Poland
http://dx.doi.org/10.1016/j.ttbdis.2016.07.011 1877-959X/© 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Rudolf, I., et al., First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.07.011
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I. Rudolf et al. / Ticks and Tick-borne Diseases xxx (2016) xxx–xxx
Table 1 Prevalence for Rickettsia spp., R. raoultii and R. helvetica in field-collected D. reticulatus ticks, Czech Republic, 2010 − 2011.
D. reticulatus males D. reticulatus females Total
Rickettsia spp.
R. raoultii
R. helvetica
15.30 (10.71–21.10)* 20.76 (15.40–27.43) 18.08 (14.44–22.33)
13.46 (9.36–18.63) 17.50 (13.00–23.00) 15.59 (12.44–19.26)
0.49 (0.00–1.49) 0.53 (0.13–1.38) 0.51 (0.18–1.10)
Explanations: ∗ EP, estimated prevalence for “Variable Pool Size and Perfect Test”, per cent individuals (EpiTools: “Pooled Prevalence Calculator”); 95% confidence intervals in parentheses
(Chmielewski et al., 2009), Hungary (Földvari et al., 2013), Sloˇ ˇ cek, 1984; Spitalská vakia (Rehᡠet al., 2012) and Austria (Duscher et al., 2016). Molecular based survey on the prevalence of rickettsial pathogens in adult host-seeking D. reticulatus ticks was therefore performed in order to fill in the knowledge gap on their distribution in the Czech Republic and to evaluate the public health risk for the local human population. 2. Materials and methods
according to the manufacturer’s instructions, and purified with EtOH/EDTA precipitation. The sequencing was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA). PCR amplicons were bidirectionally sequenced once to ensure high quality reads. The DNA sequences were edited and aligned using the Seqman module within Lasergene v. 6.0 (DNASTAR Inc., USA) and also checked manually. The BLAST program (http://www. ncbi.nlm.nih.gov/blast) of the National Center for Biotechnology Information (Bethesda, MD, USA) were used for database searches.
2.1. Study site and sample collection Questing D. reticulatus ticks were collected in South Moravia at Soutok study site (48◦ 40 N 16◦ 56 E) during the seasons of 2010 and 2011. This habitat is in long-term known as a natural focus of tularaemia (Hubálek et al., 1996). The focus is situated on the left bank of the Dyje (Thaya) river, at 151 m above sea level, close to the Austrian village of Rabensburg (lying on the opposite river bank). The climate of the region is relatively warm and dry: the mean annual temperature is 9 ◦ C, and the mean annual precipitation is 525 mm. The area is dominated by a deciduous floodplain forest ecosystem. As for typical flora, the biotope is characterized by Quercus robur L., Fraxinus angustifolia Vahl, F. excelsior L., Populus nigra L., and others. The fauna consists of small (rodents, insectivores) and medium-sized (hares, foxes) mammals along with large game animals (roe deers and red deers) and occasionally wild boar (for more detailed description of the focus see Hubálek et al., 1996). Ticks were collected by flagging low vegetation (with white flannel cloths 90 × 60 cm during afternoon) in April and September (February to April and September to November represent peak activity of questing D. reticulatus in Central Europe) (Szell et al., 2006; Duscher et al., 2013). 2.2. Sample processing, DNA extraction, PCR analysis The sampled ticks were divided into test tubes according to sex and developmental stage and pooled (maximum 10 adults per tube) before being frozen at −60 ◦ C. All D. reticulatus ticks were rinsed with 70% ethanol (PCR quality) and mechanically disrupted using the TissueLyser apparatus (Qiagen, Hilden, Germany) in 245 l of PBS (Oxoid, England). The total genomic DNA was extracted from 100 l of the tick homogenate with a QIAamp DNA Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Single-step PCR was used for Rickettsia spp. detection using primers Rp877p (5 -GGGGGCCTGCTCACGGCGG-3 ) and Rp1258n (5 -ATTGCAAAAAGTACAGTGAACA-3 ) designed for the part of citratsynthase gene (gltA), which corresponds with a PCR product length of 382 bp (Regneri et al., 1991). 2.3. Sequence analysis The PCR product was purified by precipitation with PEG/Mg/NaAc (26% polyethylene glycol, 6.5 mM MgCl2 ·6H2 O, 0.6 M NaAc.3H2 O). Direct sequencing of the purified PCR product was performed with the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit version 1.1 (Applied Biosystems, U.S.A)
3. Results and discussion In the study we screened a total of 997 D. reticulatus ticks (576 females and 421 males) for the presence of spotted fever group rickettsiae. Of the 107 tick pools analysed, 89 contained rickettsial DNA. PCR positive samples were subjected to sequence analysis which revealed 84 amplicons with 100% sequence identity to R. raoultii (GenBank accession no. KJ663737) and 5 amplicons with 100% sequence identity to R. helvetica (GenBank accession no. JX040636), respectively. Estimated prevalence for Rickettsia spp. and particular rickettsial species was calculated (Table 1). R. raoultii was only proven indirectly by serology or by molecular identification in ticks collected from humans, never in the blood or in an eschar biopsy of a patient. As for the incidence of R. raoultii infections in Europe, human cases were so far reported from France (Parola et al., 2009), Slovakia (Sekeyova et al., 2012), Hungary (Földvari et al., 2013) and Poland (Switaj et al., 2012). However, especially in early spring (March to May) and autumn (September to November), which corresponds with peak activity of adult Dermacentor spp., clinicians should be aware of its typical clinical symptoms mainly in places, where D. reticulatus or D. marginatus are present, on the grounds of high pathogen prevalence in field collected D. reticulatus in Central Europe, e.g. 22% in Slovakia ˇ (Spitalská et al., 2012), 56% in Poland (Chmielewski et al., 2009), 15% in Austria (Duscher et al., 2016) and 58% in Hungary (Szekeres et al., 2016). Disease caused by R. helvetica, the second agent reported in our study, is a self-limited illness associated with headache, myalgia and less frequently with rash and/or eschar, which was documented by serology and molecular tools in Austria, France, Italy, Denmark, Switzerland and Slovakia. However, no definitive, convincing cases have been reported since 2005 (Parola et al., 2013). It is worth mentioning that R. helvetica is usually associated with I. ricinus and sporadic detection in D. reticulatus does not mean that these ticks are vectors of R. helvetica, as ticks may have been infected on a R. helvetica rickettsiemic animal. Our study provides the first evidence of the occurrence of emerging R. raoultii in Czech D. reticulatus ticks and warrants further investigation in this area despite the fact that these ticks bite humans very rarely. Since all spotted fever group Rickettsia spp. have to be considered as potential pathogens, a special surveillance should be carried out (including intensive searching for human cases which are mostly underrecognized and consequently, from an epidemiological point of view, underreported). Summing up, monitoring tick vectors and the pathogens they transmit, within the scope of epidemiological surveillance, is an
Please cite this article in press as: Rudolf, I., et al., First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.07.011
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important tool for better prevention and control of tick-borne diseases. Acknowledgement Authors would like to thank projects (MUNI/A/1437/2014) and (MUNI/A/1013/2015) for support. References Angelakis, E., Pulcini, C., Waton, J., Imbert, P., Socolovschi, C., Edouard, S., Dellamonica, P., Raoult, D., 2010. Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after tick bite. Clin. Infect. Dis. 50, 549–551. Chmielewski, T., Podsiadly, E., Karbowiak, G., Tylewska-Wierzbanowska, S., 2009. Rickettsia spp. in ticks, Poland. Emerg. Infect. Dis. 15, 486–488. Duscher, G.G., Feiler, A., Leschnik, M., Joachim, A., 2013. Seasonal and spatial distribution of ixodid tick species feeding on naturally infested dogs from Eastern Austria and the influence of acaricides/repellents on these parameters. Parasites Vectors 6, 76. ´ A., Weiler, M., Vaux, A.G.C., Rudolf, I., Sixl, W., Medlock, J.M., Duscher, G., Hodˇzic, Versteirt, V., Hubalek, Z., 2016. First report of Rickettsia raoultii in field collected Dermacentor reticulatus ticks from Austria. Ticks Tick-Borne Dis. 7, 720–722. Földvari, G., Rigo, K., Lakos, A., 2013. Transmission of Rickettsia slovaca and Rickettsia raoultii by male Dermacentor marginatus and Dermacentor reticulatus ticks to humans. Diagn. Microbiol. Infect. Dis. 76, 387–389. Hubálek, Z., Rudolf, I., 2011. Microbial Zoonoses and Sapronoses. Springer, Dordrecht-Heidelberg-London-New York, 457 pp. ˇ Hubálek, Z., Treml, F., Halouzka, J., Juˇricová, Z., Hunády, M., Janík, V., 1996. Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia. Med. Vet. Entomol. 10, 241–246. Hubálek, Z., Sixl, W., Halouzka, J., 1998. Francisella tularensis in Dermacentor reticulatus ticks from the Czech Republic and Austria. Wien. Klin. Wochenschr. 110, 909–910. Nosek, J., 1972. The ecology and public health importance of Dermacentor marginatus and D. reticulatus in Central Europe. Folia Parasitol. 19, 93–102.
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Oteo, J.A., Portillo, A., 2012. Tick-borne rickettsioses in europe. Ticks Tick-Borne Dis. 3, 270–277. Pérez-Pérez, L., Portillo, A., Allegue, F., Zulaica, A., Oteo, J.A., Caeiro, J.L., Fabeiro, C.M., 2010. Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL): a case associated with Rickettsia rioja. Acta Dermato Venereol. 90, 214–215. Parola, P., Rovery, C., Rolain, J.M., Brouqui, P., Davoust, B., Raoult, D., 2009. Rickettsia slovaca and R. raoultii in tick-borne rickettsioses. Emerg. Infect. Dis. 15, 1105–1108. Parola, P., Paddock, C.D., Socholovschi, 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 rickettsioses around the world: a geographic approach. Clin. Microbiol. Rev. 26, 657–702. ˇ Rehᡠcek, J., 1984. Rickettsia Slovaca – the Organism and Its Ecology, Acta Sci. Natur. Acad. Sci. Bohemoslovaca (Brno), 54 pp. Regneri, R.L., Spruill, C.L., Plikaytis, B.D., 1991. Genotypic identification of rickettsiae and estimation of intraspecies seqence divergence for portion of 2 rickettsial genes. J. Bacteriol. 173, 1576–1589. Rubel, F., Brugger, K., Pfeffer, M., Dobler, L.C., Didyk, Y.M., Leverenz, S., Dautel, H., Kahl, O., 2016. Geographical distribution of Dermacentor marginatus and Dermacentor reticulatus in Europe. Tick Tick-Borne Dis. 7, 224–233. Sekeyova, Z., Subramanian, G., Mediannikov, O., Diaz, M.Q., Nyitray, A., Blaskovicova, H., Raoult, D., 2012. Evaluation of clinical specimens for Rickettsia, Bartonella, Borrelia, Coxiella, Anaplasma, Francisella and Diplorickettsia positivity using serological and molecular biology methods. FEMS Immunol. Med. Microbiol. 64, 82–91. ˇ ˇ Spitalská, E., Stefanidesová, K., Kocianová, E., Boldiˇs, V., 2012. Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic. Exp. Appl. Acarol. 57, 189–197. Switaj, K., Chmielewski, T., Borkowski, P., Tylewska-Wierzbanowska, S., Olszynska-Krowicka, M., 2012. Spotted fever rickettsiosis caused by Rickettsia raoultii – case report. Przegl. Epidemiol. 66, 347–350. Szekeres, S., van Leeuven, A.D., Rigo, K., Jablonszky, M., Majoros, G., Sprong, H., Földvari, G., 2016. Prevalence and diversity of human pathogenic rickettsiae in urban versus rural habitats, Hungary. Exp. Appl. Acarol. 68, 223–226. Szell, Z., Sreter-Lancz, Z., Marialigeti, K., Sreter, T., 2006. Temporal distribution of Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary. Vet. Parasitol. 141, 377–379.
Please cite this article in press as: Rudolf, I., et al., First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.07.011
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic ´ Ivo Rudolf a,b,∗ , Kristyna Venclíková a,b,c , Hana Blaˇzejová a , Lenka Betáˇsová a , Jan Mendel a , a,b Zdenˇek Hubálek , Philippe Parola d a
Institute of Vertebrate Biology, v.v.i., Academy of Sciences, Kvetna 8, 603 65, Brno, Czech Republic Masaryk University, Department of Experimental Biology, Kotlarska 2, 611 37 Brno, Czech Republic c Institute of Macromolecular Chemistry, v.v.i., Academy of Sciences, 162 06 Prague, Czech Republic d Aix Marseille University, CNRS 7278, URMITE, INSERM 1095, UM63,IRD Dakar 198, 27 Bd Jean Moulin, F-13385 Marseille 5, France b
a r t i c l e
i n f o
Article history: Received 22 March 2016 Received in revised form 19 July 2016 Accepted 19 July 2016 Available online xxx Keywords: Rickettsia spp. Dermacentor spp. DEBONEL SENLAT
a b s t r a c t Tick-borne rickettsioses nowadays present a significant health threat among emerging tick-borne infections. In the study, we screened a total of 997 questing adult Dermacentor reticulatus ticks (576 females and 421 males) collected in Southern Moravia (Czech Republic) from April to September of 2010 and 2011 for the presence of human pathogenic rickettsiae. Here we provide the first evidence on the occurrence of human pathogenic Rickettsia raoultii in the local D. reticulatus population indicating possible future emergence of Dermacentor-borne necrotic erythema and lymphadenopathy (DEBONEL) in the region. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction Dermacentor reticulatus is the second most reported tick species after Ixodes ricinus in Central Europe. It has a broad geographical overlap with D. marginatus, but generally extends more to the north (Rubel et al., 2016). Preferred habitats constitute alluvial forests (typically flood plain forest-meadow ecosystem) and swamps where it can survive flooding for certain periods (Nosek, 1972; Hubálek et al., 1996, 1998). It also colonizes drier habitats like fallow and heath land as well as grassland interspersed with bushes and trees. Larvae and nymphs feed mainly on rodent species, whereas adults feed on larger mammals like cattle, deer and dog, and occasionally bite humans (Hubálek and Rudolf, 2011; Hubálek et al., 1996, 1998). In Central Europe, D. reticulatus serves as a facultative vector of Francisella tularensis, a causative agent of tularaemia and Babesia canis which causes canine babesiosis (Hubálek et al., 1998; Hubálek and Rudolf, 2011). However, the research on pathogenic Rickettsia raoultii and R. slovaca in Dermacentor spp. has turned the attention of public health experts also to this group of emerging diseases
∗ Corresponding author at: Institute of Vertebrate Biology, v.v.i., Academy of Sciences of the Czech Republic, Kvetna 8, CZ-603 65 Brno, Czech Republic. E-mail address: [email protected] (I. Rudolf).
(Oteo and Portillo, 2012). Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group rickettsiae. In the last decades, the importance of tick-associated rickettsial pathogens have increased dramatically and several species that were considered nonpathogenic for decades are now associated with human infections (Parola et al., 2013). D. reticulatus and D. marginatus are implicated as vectors of emerging rickettsioses in Europe. Rickettsia raoultii and R. slovaca are associated with a syndrome characterized by scalp eschars and neck lymphadenopathy following tick bites, and the term ‘SENLAT’ (scalp eschar and neck lymphadenopathy after tick bite) was proposed recently by the Marseille group (National Reference Center for Rickettsial Diseases) for this clinical entity (Angelakis et al., 2010; Parola et al., 2013). Initially, this syndrome was named TIBOLA (tick-borne lymphadenopathy) or DEBONEL (Dermacentor-borne necrotic erythema and lymphadenopathy). Recently, R. rioja was detected in a tick removed from a patient with DEBONEL/TIBOLA (Pérez-Pérez et al., 2010). In the Czech Republic, data on emerging Rickettsia spp. pathogenic to humans, especially their prevalence in host-seeking ticks, possible reservoirs as well as its relevance for public and animal health are insufficient. To date, no Rickettsia spp. have been detected in D. reticulatus ticks in the Czech Republic, despite the fact that R. raoultii and R. slovaca were detected in particular species in several neighbouring countries including Poland
http://dx.doi.org/10.1016/j.ttbdis.2016.07.011 1877-959X/© 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Rudolf, I., et al., First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.07.011
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ARTICLE IN PRESS
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I. Rudolf et al. / Ticks and Tick-borne Diseases xxx (2016) xxx–xxx
Table 1 Prevalence for Rickettsia spp., R. raoultii and R. helvetica in field-collected D. reticulatus ticks, Czech Republic, 2010 − 2011.
D. reticulatus males D. reticulatus females Total
Rickettsia spp.
R. raoultii
R. helvetica
15.30 (10.71–21.10)* 20.76 (15.40–27.43) 18.08 (14.44–22.33)
13.46 (9.36–18.63) 17.50 (13.00–23.00) 15.59 (12.44–19.26)
0.49 (0.00–1.49) 0.53 (0.13–1.38) 0.51 (0.18–1.10)
Explanations: ∗ EP, estimated prevalence for “Variable Pool Size and Perfect Test”, per cent individuals (EpiTools: “Pooled Prevalence Calculator”); 95% confidence intervals in parentheses
(Chmielewski et al., 2009), Hungary (Földvari et al., 2013), Sloˇ ˇ cek, 1984; Spitalská vakia (Rehᡠet al., 2012) and Austria (Duscher et al., 2016). Molecular based survey on the prevalence of rickettsial pathogens in adult host-seeking D. reticulatus ticks was therefore performed in order to fill in the knowledge gap on their distribution in the Czech Republic and to evaluate the public health risk for the local human population. 2. Materials and methods
according to the manufacturer’s instructions, and purified with EtOH/EDTA precipitation. The sequencing was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA). PCR amplicons were bidirectionally sequenced once to ensure high quality reads. The DNA sequences were edited and aligned using the Seqman module within Lasergene v. 6.0 (DNASTAR Inc., USA) and also checked manually. The BLAST program (http://www. ncbi.nlm.nih.gov/blast) of the National Center for Biotechnology Information (Bethesda, MD, USA) were used for database searches.
2.1. Study site and sample collection Questing D. reticulatus ticks were collected in South Moravia at Soutok study site (48◦ 40 N 16◦ 56 E) during the seasons of 2010 and 2011. This habitat is in long-term known as a natural focus of tularaemia (Hubálek et al., 1996). The focus is situated on the left bank of the Dyje (Thaya) river, at 151 m above sea level, close to the Austrian village of Rabensburg (lying on the opposite river bank). The climate of the region is relatively warm and dry: the mean annual temperature is 9 ◦ C, and the mean annual precipitation is 525 mm. The area is dominated by a deciduous floodplain forest ecosystem. As for typical flora, the biotope is characterized by Quercus robur L., Fraxinus angustifolia Vahl, F. excelsior L., Populus nigra L., and others. The fauna consists of small (rodents, insectivores) and medium-sized (hares, foxes) mammals along with large game animals (roe deers and red deers) and occasionally wild boar (for more detailed description of the focus see Hubálek et al., 1996). Ticks were collected by flagging low vegetation (with white flannel cloths 90 × 60 cm during afternoon) in April and September (February to April and September to November represent peak activity of questing D. reticulatus in Central Europe) (Szell et al., 2006; Duscher et al., 2013). 2.2. Sample processing, DNA extraction, PCR analysis The sampled ticks were divided into test tubes according to sex and developmental stage and pooled (maximum 10 adults per tube) before being frozen at −60 ◦ C. All D. reticulatus ticks were rinsed with 70% ethanol (PCR quality) and mechanically disrupted using the TissueLyser apparatus (Qiagen, Hilden, Germany) in 245 l of PBS (Oxoid, England). The total genomic DNA was extracted from 100 l of the tick homogenate with a QIAamp DNA Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Single-step PCR was used for Rickettsia spp. detection using primers Rp877p (5 -GGGGGCCTGCTCACGGCGG-3 ) and Rp1258n (5 -ATTGCAAAAAGTACAGTGAACA-3 ) designed for the part of citratsynthase gene (gltA), which corresponds with a PCR product length of 382 bp (Regneri et al., 1991). 2.3. Sequence analysis The PCR product was purified by precipitation with PEG/Mg/NaAc (26% polyethylene glycol, 6.5 mM MgCl2 ·6H2 O, 0.6 M NaAc.3H2 O). Direct sequencing of the purified PCR product was performed with the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit version 1.1 (Applied Biosystems, U.S.A)
3. Results and discussion In the study we screened a total of 997 D. reticulatus ticks (576 females and 421 males) for the presence of spotted fever group rickettsiae. Of the 107 tick pools analysed, 89 contained rickettsial DNA. PCR positive samples were subjected to sequence analysis which revealed 84 amplicons with 100% sequence identity to R. raoultii (GenBank accession no. KJ663737) and 5 amplicons with 100% sequence identity to R. helvetica (GenBank accession no. JX040636), respectively. Estimated prevalence for Rickettsia spp. and particular rickettsial species was calculated (Table 1). R. raoultii was only proven indirectly by serology or by molecular identification in ticks collected from humans, never in the blood or in an eschar biopsy of a patient. As for the incidence of R. raoultii infections in Europe, human cases were so far reported from France (Parola et al., 2009), Slovakia (Sekeyova et al., 2012), Hungary (Földvari et al., 2013) and Poland (Switaj et al., 2012). However, especially in early spring (March to May) and autumn (September to November), which corresponds with peak activity of adult Dermacentor spp., clinicians should be aware of its typical clinical symptoms mainly in places, where D. reticulatus or D. marginatus are present, on the grounds of high pathogen prevalence in field collected D. reticulatus in Central Europe, e.g. 22% in Slovakia ˇ (Spitalská et al., 2012), 56% in Poland (Chmielewski et al., 2009), 15% in Austria (Duscher et al., 2016) and 58% in Hungary (Szekeres et al., 2016). Disease caused by R. helvetica, the second agent reported in our study, is a self-limited illness associated with headache, myalgia and less frequently with rash and/or eschar, which was documented by serology and molecular tools in Austria, France, Italy, Denmark, Switzerland and Slovakia. However, no definitive, convincing cases have been reported since 2005 (Parola et al., 2013). It is worth mentioning that R. helvetica is usually associated with I. ricinus and sporadic detection in D. reticulatus does not mean that these ticks are vectors of R. helvetica, as ticks may have been infected on a R. helvetica rickettsiemic animal. Our study provides the first evidence of the occurrence of emerging R. raoultii in Czech D. reticulatus ticks and warrants further investigation in this area despite the fact that these ticks bite humans very rarely. Since all spotted fever group Rickettsia spp. have to be considered as potential pathogens, a special surveillance should be carried out (including intensive searching for human cases which are mostly underrecognized and consequently, from an epidemiological point of view, underreported). Summing up, monitoring tick vectors and the pathogens they transmit, within the scope of epidemiological surveillance, is an
Please cite this article in press as: Rudolf, I., et al., First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.07.011
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Please cite this article in press as: Rudolf, I., et al., First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.07.011