Ticks and Tick-borne Diseases 5 (2014) 139–144
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Detection of Candidatus Neoehrlichia mikurensis, Borrelia burgdorferi sensu lato genospecies and Anaplasma phagocytophilum in a tick population from Austria Martin Glatz a,b,∗,1 , Robert R. Müllegger a,c,1 , Florian Maurer d , Volker Fingerle e , Yvonne Achermann f , Bettina Wilske g , Guido V. Bloemberg d a
Department of Dermatology, Medical University of Graz, Graz, Austria Department of Dermatology, University Hospital Zurich, Zurich, Switzerland c Department of Dermatology, State Hospital Wiener Neustadt, Wiener Neustadt, Austria d Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland e German National Reference Centre for Borrelia, Bavarian Health and Food Safety Authority, Oberschleißheim, Germany f Division of Infectious Diseases and Hospital Epidemiology, University and University Hospital Zurich, Zurich, Switzerland g Max von Pettenkofer Institute, Ludwig Maximilian University of Munich, Munich, Germany b
a r t i c l e
i n f o
Article history: Received 25 July 2013 Received in revised form 23 September 2013 Accepted 2 October 2013 Available online 15 December 2013 Keywords: Tick-borne disease Borrelia burgdorferi Anaplasma phagocytophilum Candidatus Neoehrlichia mikurensis Ixodes ricinus
a b s t r a c t Candidatus Neoehrlichia mikurensis DNA was discovered in Ixodes ricinus ticks in 1999 and is referred to as an emerging human pathogen since its first detection in patients with febrile illness reported in 2010. In recent years, Ca. Neoehrlichia mikurensis has been detected in ticks from several European, Asian, and African countries. However, no epidemiological data exist for Austria, which is a highly endemic region for tick-transmitted diseases. To assess the geographic spread and prevalence of Ca. Neoehrlichia mikurensis sympatric with other tick-transmitted pathogens, we analysed 518 I. ricinus ticks collected in 2002 and 2003 in Graz, Austria. The prevalence of Ca. Neoehrlichia mikurensis was 4.2%, that of Borrelia burgdorferi sensu lato 25.7%, and that of Anaplasma phagocytophilum 1%. Coinfections with Ca. Neoehrlichia mikurensis and B. burgdorferi sensu lato were found in 2.3% of all ticks. Thus, the results show a relatively high prevalence of Ca. Neoehrlichia mikurensis in Austrian ticks suggesting a high probability for the occurrence of undiagnosed human infections in Austria. © 2013 Elsevier GmbH. All rights reserved.
Introduction Ticks are second to mosquitoes as vectors of human vectorborne pathogens worldwide (de la Fuente et al., 2008). In western and central Europe, the hard tick Ixodes ricinus is the primary tick vector for many human pathogens (Heyman et al., 2010). The incidence of tick-borne diseases has increased over the past decades due to global warming with subsequent altitudinal and latitudinal migration and extended activity of the tick vector. This has been exemplarily shown for Lyme disease/borreliosis, which is the most prevalent tick-borne disease in temperate regions in the Northern hemisphere (Medlock et al., 2013, and references therein). It is caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) group, which includes human pathogenic genospecies such
∗ Corresponding author at: Dermatology Branch, Center for Cancer Research, National Cancer Institute, NIH, Building 10/12N260, 10 Center Drive, Bethesda, MD 20892, USA. Tel.: +1 301 496 9002; fax: +1 301 496 5370. E-mail address: [email protected] (M. Glatz). 1 These authors contributed equally to this research. 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.10.006
as B. afzelii, B. garinii, and B. burgdorferi sensu stricto (Hengge et al., 2003). Lyme borreliosis commonly begins with an erythema migrans at the site of an infectious tick bite and with flu-like symptoms. Untreated patients may develop a multisystem disorder with affection of the nervous- or musculoskeletal system, or the heart (Hengge et al., 2003). Because of the high prevalence of Lyme borreliosis, physicians are aware of a possible infection with B. burgdorferi s.l. after a tick bite. Hence, they routinely perform easy-accessible diagnostic procedures and are familiar with therapy strategies. Besides B. burgdorferi s.l., I. ricinus ticks less often transmit a variety of other pathogens that can cause harmful diseases such as Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis. Both species belong to the family of Anaplasmataceae (Dumler et al., 2007) and were recently described to cause infectious diseases with potentially lifethreatening outcome. Anaplasma phagocytophilum causes human granulocytic anaplasmosis, which is a moderate, self-limited flulike illness in most cases, but fatal courses have been observed in immunocompromised individuals (Dumler et al., 2007). The first European case of human granulocytic anaplasmosis was reported from Slovenia in 1997 (Petrovec et al., 1997) and probably less than
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M. Glatz et al. / Ticks and Tick-borne Diseases 5 (2014) 139–144
100 laboratory-confirmed European cases are currently known (Edouard et al., 2012; Jin et al., 2012). Candidatus Neoehrlichia mikurensis was first detected in I. ricinus ticks from the Netherlands in 1999 and referred to as an Ehrlichia-like species (‘Schotti variant’). Since then, it was found in questing ticks from several European, Asian, and African countries (Alekseev et al., 2001; Andersson et al., 2013; Brouqui et al., 2003; Capelli et al., 2012; Fertner et al., 2012; Jahfari et al., 2012; Kamani et al., 2013; Li et al., 2012; Lommano et al., 2012; Maurer et al., 2013; Movila et al., 2013; Nijhof et al., 2007; Rar et al., 2010; Richter and Matuschka, 2012; Schouls et al., 1999; Shpynov et al., 2006; van Overbeek et al., 2008; Wielinga et al., 2006). Therefore, ticks are generally considered as the main vectors for this pathogen to humans. The first human infections with Ca. Neoehrlichia mikurensis were reported in 2010, and only 15 cases of human neoehrlichiosis have been published in Europe and Asia until September 2013. They were characterized by fever, malaise, weight loss, and septicaemia in previously healthy individuals as well as in immunocompromised patients (Fehr et al., 2010; Li et al., 2012; Maurer et al., 2013; Pekova et al., 2011; von Loewenich et al., 2010; Welinder-Olsson et al., 2010). Due to its recent detection in ticks and the small but growing number of diagnosed clinical cases, human infection with Ca. Neoehrlichia mikurensis is referred to as an emerging infectious disease (Lommano et al., 2012; Maurer et al., 2013). Austria with its location in central Europe and its temperate climate is predestined to be a highly endemic region for ticktransmitted diseases, which is particularly proved for B. burgdorferi s.l. The spirochaete has been found in 16% of ticks (Leschnik et al., 2012), which explains the high rate of seropositive individuals in high-risk groups for tick bites (e.g., 54% among healthy hunters) (Cetin et al., 2006) and one of the highest Lyme borreliosis incidences in central Europe with 130 cases per 100,000 individuals (Hengge et al., 2003). Anaplasma phagocytophilum has been identified only occasionally in ticks from Austria (Leschnik et al., 2012; Polin et al., 2004; Sixl et al., 2003), and only 8 cases of human infection have been diagnosed in this area so far (Haschke-Becher et al., 2010; Vogl et al., 2010; Walder et al., 2006). However, we have shown that 20% of patients with erythema migrans from southeastern Austria are seropositive for A. phagocytophilum (unpublished data). Candidatus Neoehrlichia mikurensis has not been detected in Austrian ticks so far, and no case of human disease has been reported in Austria by September 2013. In 2002 and 2003, we collected questing I. ricinus ticks in southeastern Austria with the aim to determine the prevalence of B. burgdorferi s.l. and A. phagocytophilum. Recent reports of a high prevalence of Ca. Neoehrlichia mikurensis in ticks from neighbouring countries such as Switzerland, Germany, and Italy (Capelli et al., 2012; Lommano et al., 2012; Richter and Matuschka, 2012) prompted us to investigate these ticks collected in 2002/2003 also for the presence of this pathogen. This helps to assess the chronological and geographical spread of Ca. Neoehrlichia mikurensis in Europe.
Materials and methods Study area and tick sampling This study was carried out in a mixed woodland recreational area in the city of Graz in southeastern Austria (47◦ 4 0 N, 15◦ 26 0 E, altitude 353 m). The climate is generally temperate with average annual temperatures around 10 ◦ C and annual rainfalls of 800 mm. The sampling area is popular for walkers and hobby joggers and is well known for a high tick activity. Ticks were collected by the flagging method in June and September 2002 and 2003. Briefly, we dragged a 1.5-m2 white flannel cloth over the low vegetation for a
distance of 2–5 m for each drag. Then, the cloth was turned around and the attached I. ricinus ticks were gently removed with plastic tweezers. Groups of 10 ticks were put into a 50-ml conical tube with humidified sterile gaze and stored at 4 ◦ C until DNA extraction. Directly before DNA extraction, determination of I. ricinus was reassured, and the life stages were identified based on morphological characters.
DNA extraction Ticks were removed from the 50-ml conical tubes and separately washed twice in 70% ethanol. After mechanical crushing with a sterile steel probe, DNA was extracted from each tick individually using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), protocol ‘DNA purification from tissues’. Briefly, crushed ticks were incubated with 180 l buffer ATL and 20 l proteinase K on a shaking water bath at 56 ◦ C overnight to ensure tissue lysis. The next steps were performed according to the manufacturer’s protocol. DNA was eluted in a total volume of 200 l of elution buffer. Quantity and purity of DNA was measured at 260 nm and 280 nm in a Beckman DU 600 spectrometer (Beckman Coulter, Vienna, Austria). DNA was stored at −80 ◦ C until analysis was done. PCR for Ixodes ricinus housekeeping gene To confirm successful extraction of tick DNA, 5 randomly chosen samples of each extraction batch consisting of 20 ticks were subjected to a PCR targeting the tick 16S mitochondrial ribosomal DNA gene as described previously (Norris et al., 1996). Briefly, the 50-l reaction mixture contained 1× PCR, 2.5 mM MgCl2 , 200 mM dNTPs, 1.25 U Taq polymerase, 0.5 mM of forward primer 16S+1 (5 -CCGGTCTGAACTCAGATCAAGT-3 ) and reverse primer 16S−1 (5 -CTGCTCAATGATTTTTTAAATTGCTGTGG-3 ). The cycling protocol included a polymerase activation step at 95 ◦ C for 5 min, followed by 35 cycles at 94 ◦ C for 30 s, 48 ◦ C for 30 s, 72 ◦ C for 45 s, and a final extension step at 72 ◦ C for 7 min and was performed on a GeneAmp 2700 (Applied Biosystems, Vienna, Austria). PCR products were visualized on a 2% agarose gel (Sea Kem LE Agarose, Biozym, Hessisch Oldendorf, Germany) stained with 1 mg/ml ethidium bromide (Bio-Rad, Vienna, Austria). For each sample, a PCR amplicon was detected.
PCR for Borrelia burgdorferi sensu lato Extracted DNA was subjected to a semi-nested conventional PCR that was previously developed as a reliable detection method for all European B. burgdorferi s.l. genospecies (Michel et al., 2004). This PCR amplifies an 818-bp fragment of the ospA gene. Briefly, 5 l of DNA extract was tested in a 50-l reaction volume containing 5 l of 10× PCR buffer containing 1.5 mM MgCl2 , 200 mM dNTPs, and 0.5 U Taq polymerase. Primers used in the first PCR were V1a forward (5 -GGGAATAGGTCTAATATTAGC3 ), V1b forward (5 -GGGGATAGGTCTAATATTAGC-3 ), and R2 reverse (5 -CATAAATTCTCCTTATTTTAAAGC-3 ) at a concentration of 10 pmol each. For the semi-nested PCR, 5 l of the reaction mixture of the first PCR was used with 100 pmol of primers V3a forward (5 -GCCTTAATAGCATGTAAGC-3 ), V3b forward (5 GCCTTAATAGCATGCAAGC-3 ), and R2. All PCR reagents and primers were obtained from Applied Biosystems. Cycling conditions for both PCR runs were 95 ◦ C for 5 min, followed by 30 cycles at 94 ◦ C for 45 s, 48 ◦ C for 45 s, 72 ◦ C for 1 min, and a final extension step at 72 ◦ C for 7 min. The B. garinii strain Pfri (In-house strain, Max von Pettenkofer Institute, Munich, Germany) was used as a positive control in each PCR experiment. PCR amplicons were visualized on a
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2% agarose gel (Sea Kem LE Agarose, Biozym) stained with 1 mg/ml ethidium bromide (Bio-Rad).
respectively. Nymphs were the most frequently collected life stage, followed by female and male adult ticks in a comparable frequency.
Restriction fragment length polymorphism (RFLP) analysis for Borrelia burgdorferi genospecies
Prevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum
The semi-nested PCR product from each B. burgdorferi-positive tick was digested separately with 5 different restriction enzymes, Kpn21 (MBI Fermentas, St. Leon-Rot, Germany), BglII, SspI, SfuI (Roche, Vienna, Austria), and HindIII (Invitrogen, Vienna, Austria). Digestion was performed overnight, and the digested products were visualized on a 2% agarose gel as described earlier (Michel et al., 2004).
All ticks were analysed for the presence of A. phagocytophilum DNA by amplifying a 546-bp fragment of the 16S rRNA gene as described earlier (Massung et al., 1998). Briefly, 5 l of DNA extract was tested in a 50-l reaction volume containing 5 l of 10× PCR buffer, 1.5 mM MgCl2 , 200 M dNTPs, and 2.5 U Taq polymerase. Primers for the first PCR run were ge3a forward (5 -CACATGCAAGTCGAACGGATTATTC-3 ), and ge10 reverse (5 -TTCCGTTAAGAAGGATCTAATCTCC-3 ) at a concentration of 0.5 M each. For the nested PCR run, 1 l of the primary PCR reaction product was used as a template with primers ge9 forward (5 -AACGGATTATTCTTTATAGCTTGCT-3 ) and ge2 reverse (5 -GGCAGTATTAAAAGCAGCTCCAGG-3 ) at a concentration of 0.2 M each. All PCR reagents and primers were obtained from Applied Biosystems.
All ticks were individually analysed for the presence of B. burgdorferi s.l. and A. phagocytophilum by conventional 2-step PCR (Tables 1 and 2). B. burgdorferi s.l. was found in about a quarter of all ticks and was therefore significantly more abundant than A. phagocytophilum, which was detected in only 1% of ticks. For B. burgdorferi s.l., the proportion of positive ticks was the same in both years of collection, whereas 4 of the 5 ticks positive for A. phagocytophilum were found in the much smaller 2002 tick group. The analysis of the different life stages showed that none of the larvae was infected and that adult ticks were more frequently infected with B. burgdorferi s.l. than nymphs. No difference between female and male ticks was observed. The prevalence of A. phagocytophilum in nymphs and adults is comparable, although the number of infected ticks was too low for a reliable statistical analysis. A coinfection with B. burgdorferi s.l. (genospecies B. afzelii) and A. phagocytophilum was identified in only 1 female tick. Genotyping of B. burgdorferi s.l. by RFLP showed that B. afzelii was the most abundant genospecies. It represented together with B. burgdorferi s.s. the majority of identified genospecies. The numbers of B. garinii and B. valaisiana were low. A co-infection with 2 genospecies was found in 6 adult ticks. In these ticks, B. afzelii was associated with either B. burgdorferi s.s. (5 ticks) or B. valaisiana (1 tick). We did not observe a significant prevalence of a particular genospecies in particular instars.
Real-time PCR for Candidatus Neoehrlichia mikurensis
Prevalence of Candidatus Neoehrlichia mikurensis
Before ticks that had been collected in 2002 and 2003 were analysed for the presence of Ca. Neoehrlichia mikurensis in 2012, tick DNA was tested for integrity after 9 and 10 years of storage at −80 ◦ C. For this purpose, 10 samples of tick DNA that were tested positive for B. burgdorferi in 2002 or 2003 were randomly selected. These samples were again tested by the originally applied B. burgdorferi-specific PCR (Michel et al., 2004). Again, all samples gave a positive result confirming the integrity of DNA after storage under ideal conditions. For Ca. Neoehrlichia mikurensis, all ticks were analysed using a quantitative real-time PCR with TaqMan probes as described recently (Maurer et al., 2013). In a first step, equivalent volumes of DNA extracts from 10 ticks were pooled, and 5 l of each pool was subjected to the real-time PCR. In a second step, ticks in positive pools were individually tested with the same PCR.
The prevalence of Ca. Neoehrlichia mikurensis was tested in DNA pooled from 10 individual ticks using a recently published Taqman real-time PCR assay (Maurer et al., 2013). One third of the pools gave corresponding positive signals for the Ca. Neoehrlichia mikurensis probe. Subsequently, the prevalence of Ca. Neoehrlichia mikurensis was determined by testing each individual tick of each positive tested pool. Ca. Neoehrlichia mikurensis was detected in 22 ticks (4.2%) (Table 1). In 2002, 7/131 ticks (5.3%) and in 2003 15/387 ticks (3.9%) were tested positive for Ca. Neoehrlichia mikurensis (p = 0.7). No significant difference according to life stage of positive ticks (p = 1) or to coinfection (p = 1) were found between both collection periods (data not shown). Twelve ticks (55%), almost exclusively nymphs, were coinfected with B. burgdorferi s.l., all of them belonging to the B. afzelii genospecies, but no tick was found coinfected with A. phagocytophilum.
Statistics
Discussion
Statistical calculations were performed with GraphPad Prism (Version 5.0, 2010). Nominal variables (positive ticks) were calculated with Fisher’s exact test and Chi-square test. A 2-tiered p value of
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Detection of Candidatus Neoehrlichia mikurensis, Borrelia burgdorferi sensu lato genospecies and Anaplasma phagocytophilum in a tick population from Austria Martin Glatz a,b,∗,1 , Robert R. Müllegger a,c,1 , Florian Maurer d , Volker Fingerle e , Yvonne Achermann f , Bettina Wilske g , Guido V. Bloemberg d a
Department of Dermatology, Medical University of Graz, Graz, Austria Department of Dermatology, University Hospital Zurich, Zurich, Switzerland c Department of Dermatology, State Hospital Wiener Neustadt, Wiener Neustadt, Austria d Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland e German National Reference Centre for Borrelia, Bavarian Health and Food Safety Authority, Oberschleißheim, Germany f Division of Infectious Diseases and Hospital Epidemiology, University and University Hospital Zurich, Zurich, Switzerland g Max von Pettenkofer Institute, Ludwig Maximilian University of Munich, Munich, Germany b
a r t i c l e
i n f o
Article history: Received 25 July 2013 Received in revised form 23 September 2013 Accepted 2 October 2013 Available online 15 December 2013 Keywords: Tick-borne disease Borrelia burgdorferi Anaplasma phagocytophilum Candidatus Neoehrlichia mikurensis Ixodes ricinus
a b s t r a c t Candidatus Neoehrlichia mikurensis DNA was discovered in Ixodes ricinus ticks in 1999 and is referred to as an emerging human pathogen since its first detection in patients with febrile illness reported in 2010. In recent years, Ca. Neoehrlichia mikurensis has been detected in ticks from several European, Asian, and African countries. However, no epidemiological data exist for Austria, which is a highly endemic region for tick-transmitted diseases. To assess the geographic spread and prevalence of Ca. Neoehrlichia mikurensis sympatric with other tick-transmitted pathogens, we analysed 518 I. ricinus ticks collected in 2002 and 2003 in Graz, Austria. The prevalence of Ca. Neoehrlichia mikurensis was 4.2%, that of Borrelia burgdorferi sensu lato 25.7%, and that of Anaplasma phagocytophilum 1%. Coinfections with Ca. Neoehrlichia mikurensis and B. burgdorferi sensu lato were found in 2.3% of all ticks. Thus, the results show a relatively high prevalence of Ca. Neoehrlichia mikurensis in Austrian ticks suggesting a high probability for the occurrence of undiagnosed human infections in Austria. © 2013 Elsevier GmbH. All rights reserved.
Introduction Ticks are second to mosquitoes as vectors of human vectorborne pathogens worldwide (de la Fuente et al., 2008). In western and central Europe, the hard tick Ixodes ricinus is the primary tick vector for many human pathogens (Heyman et al., 2010). The incidence of tick-borne diseases has increased over the past decades due to global warming with subsequent altitudinal and latitudinal migration and extended activity of the tick vector. This has been exemplarily shown for Lyme disease/borreliosis, which is the most prevalent tick-borne disease in temperate regions in the Northern hemisphere (Medlock et al., 2013, and references therein). It is caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) group, which includes human pathogenic genospecies such
∗ Corresponding author at: Dermatology Branch, Center for Cancer Research, National Cancer Institute, NIH, Building 10/12N260, 10 Center Drive, Bethesda, MD 20892, USA. Tel.: +1 301 496 9002; fax: +1 301 496 5370. E-mail address: [email protected] (M. Glatz). 1 These authors contributed equally to this research. 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.10.006
as B. afzelii, B. garinii, and B. burgdorferi sensu stricto (Hengge et al., 2003). Lyme borreliosis commonly begins with an erythema migrans at the site of an infectious tick bite and with flu-like symptoms. Untreated patients may develop a multisystem disorder with affection of the nervous- or musculoskeletal system, or the heart (Hengge et al., 2003). Because of the high prevalence of Lyme borreliosis, physicians are aware of a possible infection with B. burgdorferi s.l. after a tick bite. Hence, they routinely perform easy-accessible diagnostic procedures and are familiar with therapy strategies. Besides B. burgdorferi s.l., I. ricinus ticks less often transmit a variety of other pathogens that can cause harmful diseases such as Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis. Both species belong to the family of Anaplasmataceae (Dumler et al., 2007) and were recently described to cause infectious diseases with potentially lifethreatening outcome. Anaplasma phagocytophilum causes human granulocytic anaplasmosis, which is a moderate, self-limited flulike illness in most cases, but fatal courses have been observed in immunocompromised individuals (Dumler et al., 2007). The first European case of human granulocytic anaplasmosis was reported from Slovenia in 1997 (Petrovec et al., 1997) and probably less than
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100 laboratory-confirmed European cases are currently known (Edouard et al., 2012; Jin et al., 2012). Candidatus Neoehrlichia mikurensis was first detected in I. ricinus ticks from the Netherlands in 1999 and referred to as an Ehrlichia-like species (‘Schotti variant’). Since then, it was found in questing ticks from several European, Asian, and African countries (Alekseev et al., 2001; Andersson et al., 2013; Brouqui et al., 2003; Capelli et al., 2012; Fertner et al., 2012; Jahfari et al., 2012; Kamani et al., 2013; Li et al., 2012; Lommano et al., 2012; Maurer et al., 2013; Movila et al., 2013; Nijhof et al., 2007; Rar et al., 2010; Richter and Matuschka, 2012; Schouls et al., 1999; Shpynov et al., 2006; van Overbeek et al., 2008; Wielinga et al., 2006). Therefore, ticks are generally considered as the main vectors for this pathogen to humans. The first human infections with Ca. Neoehrlichia mikurensis were reported in 2010, and only 15 cases of human neoehrlichiosis have been published in Europe and Asia until September 2013. They were characterized by fever, malaise, weight loss, and septicaemia in previously healthy individuals as well as in immunocompromised patients (Fehr et al., 2010; Li et al., 2012; Maurer et al., 2013; Pekova et al., 2011; von Loewenich et al., 2010; Welinder-Olsson et al., 2010). Due to its recent detection in ticks and the small but growing number of diagnosed clinical cases, human infection with Ca. Neoehrlichia mikurensis is referred to as an emerging infectious disease (Lommano et al., 2012; Maurer et al., 2013). Austria with its location in central Europe and its temperate climate is predestined to be a highly endemic region for ticktransmitted diseases, which is particularly proved for B. burgdorferi s.l. The spirochaete has been found in 16% of ticks (Leschnik et al., 2012), which explains the high rate of seropositive individuals in high-risk groups for tick bites (e.g., 54% among healthy hunters) (Cetin et al., 2006) and one of the highest Lyme borreliosis incidences in central Europe with 130 cases per 100,000 individuals (Hengge et al., 2003). Anaplasma phagocytophilum has been identified only occasionally in ticks from Austria (Leschnik et al., 2012; Polin et al., 2004; Sixl et al., 2003), and only 8 cases of human infection have been diagnosed in this area so far (Haschke-Becher et al., 2010; Vogl et al., 2010; Walder et al., 2006). However, we have shown that 20% of patients with erythema migrans from southeastern Austria are seropositive for A. phagocytophilum (unpublished data). Candidatus Neoehrlichia mikurensis has not been detected in Austrian ticks so far, and no case of human disease has been reported in Austria by September 2013. In 2002 and 2003, we collected questing I. ricinus ticks in southeastern Austria with the aim to determine the prevalence of B. burgdorferi s.l. and A. phagocytophilum. Recent reports of a high prevalence of Ca. Neoehrlichia mikurensis in ticks from neighbouring countries such as Switzerland, Germany, and Italy (Capelli et al., 2012; Lommano et al., 2012; Richter and Matuschka, 2012) prompted us to investigate these ticks collected in 2002/2003 also for the presence of this pathogen. This helps to assess the chronological and geographical spread of Ca. Neoehrlichia mikurensis in Europe.
Materials and methods Study area and tick sampling This study was carried out in a mixed woodland recreational area in the city of Graz in southeastern Austria (47◦ 4 0 N, 15◦ 26 0 E, altitude 353 m). The climate is generally temperate with average annual temperatures around 10 ◦ C and annual rainfalls of 800 mm. The sampling area is popular for walkers and hobby joggers and is well known for a high tick activity. Ticks were collected by the flagging method in June and September 2002 and 2003. Briefly, we dragged a 1.5-m2 white flannel cloth over the low vegetation for a
distance of 2–5 m for each drag. Then, the cloth was turned around and the attached I. ricinus ticks were gently removed with plastic tweezers. Groups of 10 ticks were put into a 50-ml conical tube with humidified sterile gaze and stored at 4 ◦ C until DNA extraction. Directly before DNA extraction, determination of I. ricinus was reassured, and the life stages were identified based on morphological characters.
DNA extraction Ticks were removed from the 50-ml conical tubes and separately washed twice in 70% ethanol. After mechanical crushing with a sterile steel probe, DNA was extracted from each tick individually using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), protocol ‘DNA purification from tissues’. Briefly, crushed ticks were incubated with 180 l buffer ATL and 20 l proteinase K on a shaking water bath at 56 ◦ C overnight to ensure tissue lysis. The next steps were performed according to the manufacturer’s protocol. DNA was eluted in a total volume of 200 l of elution buffer. Quantity and purity of DNA was measured at 260 nm and 280 nm in a Beckman DU 600 spectrometer (Beckman Coulter, Vienna, Austria). DNA was stored at −80 ◦ C until analysis was done. PCR for Ixodes ricinus housekeeping gene To confirm successful extraction of tick DNA, 5 randomly chosen samples of each extraction batch consisting of 20 ticks were subjected to a PCR targeting the tick 16S mitochondrial ribosomal DNA gene as described previously (Norris et al., 1996). Briefly, the 50-l reaction mixture contained 1× PCR, 2.5 mM MgCl2 , 200 mM dNTPs, 1.25 U Taq polymerase, 0.5 mM of forward primer 16S+1 (5 -CCGGTCTGAACTCAGATCAAGT-3 ) and reverse primer 16S−1 (5 -CTGCTCAATGATTTTTTAAATTGCTGTGG-3 ). The cycling protocol included a polymerase activation step at 95 ◦ C for 5 min, followed by 35 cycles at 94 ◦ C for 30 s, 48 ◦ C for 30 s, 72 ◦ C for 45 s, and a final extension step at 72 ◦ C for 7 min and was performed on a GeneAmp 2700 (Applied Biosystems, Vienna, Austria). PCR products were visualized on a 2% agarose gel (Sea Kem LE Agarose, Biozym, Hessisch Oldendorf, Germany) stained with 1 mg/ml ethidium bromide (Bio-Rad, Vienna, Austria). For each sample, a PCR amplicon was detected.
PCR for Borrelia burgdorferi sensu lato Extracted DNA was subjected to a semi-nested conventional PCR that was previously developed as a reliable detection method for all European B. burgdorferi s.l. genospecies (Michel et al., 2004). This PCR amplifies an 818-bp fragment of the ospA gene. Briefly, 5 l of DNA extract was tested in a 50-l reaction volume containing 5 l of 10× PCR buffer containing 1.5 mM MgCl2 , 200 mM dNTPs, and 0.5 U Taq polymerase. Primers used in the first PCR were V1a forward (5 -GGGAATAGGTCTAATATTAGC3 ), V1b forward (5 -GGGGATAGGTCTAATATTAGC-3 ), and R2 reverse (5 -CATAAATTCTCCTTATTTTAAAGC-3 ) at a concentration of 10 pmol each. For the semi-nested PCR, 5 l of the reaction mixture of the first PCR was used with 100 pmol of primers V3a forward (5 -GCCTTAATAGCATGTAAGC-3 ), V3b forward (5 GCCTTAATAGCATGCAAGC-3 ), and R2. All PCR reagents and primers were obtained from Applied Biosystems. Cycling conditions for both PCR runs were 95 ◦ C for 5 min, followed by 30 cycles at 94 ◦ C for 45 s, 48 ◦ C for 45 s, 72 ◦ C for 1 min, and a final extension step at 72 ◦ C for 7 min. The B. garinii strain Pfri (In-house strain, Max von Pettenkofer Institute, Munich, Germany) was used as a positive control in each PCR experiment. PCR amplicons were visualized on a
M. Glatz et al. / Ticks and Tick-borne Diseases 5 (2014) 139–144
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2% agarose gel (Sea Kem LE Agarose, Biozym) stained with 1 mg/ml ethidium bromide (Bio-Rad).
respectively. Nymphs were the most frequently collected life stage, followed by female and male adult ticks in a comparable frequency.
Restriction fragment length polymorphism (RFLP) analysis for Borrelia burgdorferi genospecies
Prevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum
The semi-nested PCR product from each B. burgdorferi-positive tick was digested separately with 5 different restriction enzymes, Kpn21 (MBI Fermentas, St. Leon-Rot, Germany), BglII, SspI, SfuI (Roche, Vienna, Austria), and HindIII (Invitrogen, Vienna, Austria). Digestion was performed overnight, and the digested products were visualized on a 2% agarose gel as described earlier (Michel et al., 2004).
All ticks were analysed for the presence of A. phagocytophilum DNA by amplifying a 546-bp fragment of the 16S rRNA gene as described earlier (Massung et al., 1998). Briefly, 5 l of DNA extract was tested in a 50-l reaction volume containing 5 l of 10× PCR buffer, 1.5 mM MgCl2 , 200 M dNTPs, and 2.5 U Taq polymerase. Primers for the first PCR run were ge3a forward (5 -CACATGCAAGTCGAACGGATTATTC-3 ), and ge10 reverse (5 -TTCCGTTAAGAAGGATCTAATCTCC-3 ) at a concentration of 0.5 M each. For the nested PCR run, 1 l of the primary PCR reaction product was used as a template with primers ge9 forward (5 -AACGGATTATTCTTTATAGCTTGCT-3 ) and ge2 reverse (5 -GGCAGTATTAAAAGCAGCTCCAGG-3 ) at a concentration of 0.2 M each. All PCR reagents and primers were obtained from Applied Biosystems.
All ticks were individually analysed for the presence of B. burgdorferi s.l. and A. phagocytophilum by conventional 2-step PCR (Tables 1 and 2). B. burgdorferi s.l. was found in about a quarter of all ticks and was therefore significantly more abundant than A. phagocytophilum, which was detected in only 1% of ticks. For B. burgdorferi s.l., the proportion of positive ticks was the same in both years of collection, whereas 4 of the 5 ticks positive for A. phagocytophilum were found in the much smaller 2002 tick group. The analysis of the different life stages showed that none of the larvae was infected and that adult ticks were more frequently infected with B. burgdorferi s.l. than nymphs. No difference between female and male ticks was observed. The prevalence of A. phagocytophilum in nymphs and adults is comparable, although the number of infected ticks was too low for a reliable statistical analysis. A coinfection with B. burgdorferi s.l. (genospecies B. afzelii) and A. phagocytophilum was identified in only 1 female tick. Genotyping of B. burgdorferi s.l. by RFLP showed that B. afzelii was the most abundant genospecies. It represented together with B. burgdorferi s.s. the majority of identified genospecies. The numbers of B. garinii and B. valaisiana were low. A co-infection with 2 genospecies was found in 6 adult ticks. In these ticks, B. afzelii was associated with either B. burgdorferi s.s. (5 ticks) or B. valaisiana (1 tick). We did not observe a significant prevalence of a particular genospecies in particular instars.
Real-time PCR for Candidatus Neoehrlichia mikurensis
Prevalence of Candidatus Neoehrlichia mikurensis
Before ticks that had been collected in 2002 and 2003 were analysed for the presence of Ca. Neoehrlichia mikurensis in 2012, tick DNA was tested for integrity after 9 and 10 years of storage at −80 ◦ C. For this purpose, 10 samples of tick DNA that were tested positive for B. burgdorferi in 2002 or 2003 were randomly selected. These samples were again tested by the originally applied B. burgdorferi-specific PCR (Michel et al., 2004). Again, all samples gave a positive result confirming the integrity of DNA after storage under ideal conditions. For Ca. Neoehrlichia mikurensis, all ticks were analysed using a quantitative real-time PCR with TaqMan probes as described recently (Maurer et al., 2013). In a first step, equivalent volumes of DNA extracts from 10 ticks were pooled, and 5 l of each pool was subjected to the real-time PCR. In a second step, ticks in positive pools were individually tested with the same PCR.
The prevalence of Ca. Neoehrlichia mikurensis was tested in DNA pooled from 10 individual ticks using a recently published Taqman real-time PCR assay (Maurer et al., 2013). One third of the pools gave corresponding positive signals for the Ca. Neoehrlichia mikurensis probe. Subsequently, the prevalence of Ca. Neoehrlichia mikurensis was determined by testing each individual tick of each positive tested pool. Ca. Neoehrlichia mikurensis was detected in 22 ticks (4.2%) (Table 1). In 2002, 7/131 ticks (5.3%) and in 2003 15/387 ticks (3.9%) were tested positive for Ca. Neoehrlichia mikurensis (p = 0.7). No significant difference according to life stage of positive ticks (p = 1) or to coinfection (p = 1) were found between both collection periods (data not shown). Twelve ticks (55%), almost exclusively nymphs, were coinfected with B. burgdorferi s.l., all of them belonging to the B. afzelii genospecies, but no tick was found coinfected with A. phagocytophilum.
Statistics
Discussion
Statistical calculations were performed with GraphPad Prism (Version 5.0, 2010). Nominal variables (positive ticks) were calculated with Fisher’s exact test and Chi-square test. A 2-tiered p value of
