Tick-Borne Encephalitis (TBE)
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ORIGINAL ARTICLE 10.1111/j.1469-0691.2009.02764.x Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk, non-risk and former risk areas of TBE, and in ticks removed from humans in Germany C. Klaus1, B. Hoffmann2, U. Hering3, B. Mielke4, K. Sachse1, M. Beer2 and J. Su¨ss1 1) Institute of Bacterial Infections and Zoonoses, Friedrich Loeffler Institute, Jena, 2) Institute of Diagnostic Virology, Friedrich Loeffler Institute, Greifswald-Insel Riems, 3) Medical Laboratory Rosenheim (Medizinisches Labor Rosenheim), Rosenheim and 4) Health and Veterinary Office (Gesundheits- und Veterina¨ramt), Magdeburg, Germany Abstract Tick-borne encephalitis (TBE) is recognized as the most important viral tick-borne zoonosis in 27 countries in Europe. In this study, ticks were collected in Germany from two non-risk areas in the states of Saxony-Anhalt and Mecklenburg-Western Pomerania, where several single human TBE cases have occurred in recent years. Ticks were also collected from a region in Thuringia, known to be a for- mer risk area for TBE virus (TBEV), where numerous human cases were reported between 1960 and 1975. Detection of TBEV RNA was conducted by real-time RT-PCR. No TBEV was detected in any field-collected ticks. However, ticks were also collected from vol- unteers living in Bavaria. Three of 239 ticks from this collection were positive for TBEV genome and two genetically distinct TBEV strains were detected and characterized. Keywords: Ixodes ricinus, Neudoerfl strain, TBE virus (TBEV), TBEV strain 263, tick-borne encephalitis (TBE), ticks, virus genome characterization Original Submission: 19 August 2008: Revised Submission: 21 November 2008; Accepted: 21 November 2008 Editor: E. Gould Article published online: 10 November 2009 Clin Microbiol Infect 2010; 16: 238–244 single human TBE cases have been registered in areas that Corresponding author and reprint requests: C. Klaus, Friedrich have been classified as non-risk areas in the past. Loeffler Institute, Naumburger Strasse 96a, 07743 Jena, Germany E-mail: christine.klaus@fli.bund.de Systematic examination of ticks will help to develop further understanding of the prevailing epidemiological situa- tion. The prevalence of TBEV in ticks is a suitable marker Introduction for TBE risk analysis in natural foci [3,4]. In the 1960s, Su¨ss et al. found many human TBE cases in the area of the present Hainich National Park (51°03–05¢ N, Tick-borne encephalitis (TBE) is the most important viral tick- 10°23–28¢ E), Thuringia, as well as isolated TBEV strains in borne disease in Europe, the Far East and other parts of Asia. mice, humans and ticks [5]. This region was subsequently Based on molecular biological data, three subtypes of tick- used for military purposes. After 1990, military usage ended borne encephalitis virus (TBEV) have been classified: the Cen- and the area was established as a national park, which is tral European, the Far Eastern and the Siberian subtypes [1]. now visited by many people. Current TBEV prevalence in Tick-borne encephalitis is a notifiable disease in 16 Euro- the area is therefore of the utmost importance. pean countries, although case definitions and definitions of Some single human TBE cases have been recorded in two risk area differ among individual countries. In Germany, TBE other regions in Saxony-Anhalt and Mecklenburg-Western risk areas have been monitored for TBE incidence since Pomerania [6]. However, despite previous limited epidemio- 2007 [2]. The number of TBE risk areas has increased in the logical analyses of TBEV prevalence [4,7,8], little is known last 10 years, from 63 to 132, and the range of risk has about the recent distribution and characteristics of TBEV spread from south to north [3]. Furthermore, a number of strains in Germany. ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases CMI Klaus et al. Tick-borne encephalitis virus prevalence in ticks 239 Recent studies have shown that the prevalence of TBEV in The 239 engorged ticks collected in 2006 from volunteers engorged ticks removed from humans was higher than in at doctors’ surgeries in districts around Rosenheim, Bavaria field-collected ticks [7,8]. Therefore, we also examined (Table 2) were cooled and stored at ) 24 °C, and finally engorged ticks collected from volunteers at doctors’ surger- examined as individual ticks. The city of Munich has been ies. Figs 1 and 2 show the geographical locations of tick classified as a non-risk area until now, but all the other collection. districts are considered to be TBE risk areas. Detection of TBEV-RNA and sequencing Materials and Methods Collected ticks were ground up in a mixer mill with three stainless steel beads (Retsch GmbH, Haan, Germany) and Tick collecting (Ixodes ricinus) 500 lL RNeasy lysis buffer (RLT) for RNA extraction Ticks were collected in 2006 (n = 888) in Hainich National using the RNeasy Mini Plant Kit (Qiagen GmbH, Hilden, Ger- Park in the state of Thuringia, as well as from bagged roe many) according to the manufacturer’s instructions. In for- and fallow deer in 2005 and 2006 (n = 187) (Table 1). Most mer examinations we also used other RNA isolation kits collection sites consisted of typical beech forest with some with the same success, such as the QIAamp Viral RNA Mini smaller meadows. The former shooting range of Weber- Kit (Qiagen GmbH) or the Instant Virus RNA Kit (Analytic stedt, however, represents a more open landscape, with wet Jena AG Biosolutions, Jena, Germany). In all cases, we rec- meadows, bushes and some clusters of trees. ommend the use of Qiashredder (Qiagen GmbH) in the first Ticks (n = 917) were collected in 2005 and 2006 in the step, which is included in the RNeasy Mini Plant Kit or can Colbitz-Letzlinger Heide (52°19–21¢ N, 11°32–35¢ E), Saxony be ordered separately. Extracted RNA was eluted in a final Anhalt, where a single human case occurred in 2004 volume of 50 lL RNA-safe buffer (RSB: 50 ng/lL carrier- (Table 1) [6]. One collection site was located in a lime forest RNA, 0.05% Tween 20, 0.05% sodium azide in RNase free and two other sites were in mostly coniferous forests with water) for longterm storage of RNA [12]. smaller meadows. Another 1285 ticks were collected from For detection of TBEV-specific RNA, primers and probe of locations around the site connected to the human case in a quantitative real-time RT-PCR protocol according to Schwai- order to analyze the natural boundaries of a suspected TBE ger and Cassinotti [13] were used. TBEV-RNA was amplified area (52°08–09¢ N, 11°39–40¢ E) (Table 1). These sites were in an optimized 25-lL reaction mixture consisting of 12.5 lL characterized by mixed forest and meadows. 2 · reaction mix, 1 lL enzyme mix (SuperScript III One-Step A total of 651 ticks were collected in July 2006 in Meckl- RT-PCR System with Platinum Taq; Invitrogen GmbH, Karls- enburg-Western Pomerania (Table 1). In the town of ruhe, Germany), 2 lL FAM-labelled primer-probe mix Neustrelitz, one human case occurred near Lake Woblitzsee (2.5 pmol/lL TBE-specific primers and 2.0 pmol/lL TBE- (53°18–19¢ N, 13°00–01¢ E). In July 2004, a 61-year-old man probe; TIB Molbiol, Berlin, Germany) and 4.5 lL water as a became ill after tick bite exposure in this area. He was hos- master mix. Finally, 5 lL RNA template was added. Extracted pitalized and infection was clinically and serologically (specific RNA from the low virulent TBE strain Langat was used as a IgM and IgG) confirmed [9]. The lake surroundings consist of positive amplification control. Real-time RT-PCR was carried meadows with some bushes (near the villages of Below and out in a MX3000 real-time PCR cycler (Stratagene Europe, Vosswinkel) or mixed forest (near Gross Quassow). Amsterdam, the Netherlands) with the following temperature Ticks were collected on the Isle of Ru¨gen, near Sassnitz, profile: 30 min at 50 °C (reverse transcription), 2 min at from a mixed but mostly deciduous forest, and near the 94 °C (inactivation reverse transcriptase/activation Taq poly- Ko¨nigsstuhl site from a meadow and a mixed forest merase), followed by 42 cycles of 30 s at 94 °C (denaturation), (54°31–34¢ N, 13°37–39¢ E). Ticks in the field regions were 30 s at 57 °C (annealing) and 30 s at 68 °C (elongation). collected by flagging and immediately cooled to 4–6 °Cina RNAs of the TBEV genome-positive samples were used portable refrigerator. In the laboratory ticks were sorted for a one-step RT-PCR to generate fragments suitable for into larvae, nymphs, females and males using a microscope sequencing, where purified DNA of each PCR amplicon was according to Estrada-Pen˜a et al. [10] and stored at ) 80 °C. used for direct sequencing in both directions using a 3130 The time between collection of live ticks and storage at Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, ) 80 °C was no longer than 24–48 h. Field-collected ticks USA). Based on the generated sequence data, internal for- were pooled into groups of mainly five male or female ward and reverse primers were designed to complete a adults, 10 nymphs or 20 larvae, depending on the estimated 2912-bp sequence region representing the C-gene, M-gene prevalence of TBE in the examined region [11]. and ENV-gene, as well as part of the 5¢end of the NS1-gene ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 238–244 240 Clinical Microbiology and Infection, Volume 16 Number 3, March 2010 CMI FIG.