First Molecular Evidence of Zoonotic Bacteria in Ticks in Bosnia and Herzegovina A

Transboundary and Emerging Diseases

SHORT COMMUNICATION First Molecular Evidence of Zoonotic Bacteria in Ticks in Bosnia and Herzegovina A. Hodzi c, H.-P. Fuehrer and G. G. Duscher

Department of Pathobiology, Institute of Parasitology, University of Veterinary Medicine Vienna, Vienna, Austria

Keywords: Summary spotted fever group rickettsiae; Anaplasma phagocytophilum; Francisella tularensis; In Bosnia and Herzegovina, the tick fauna is very diverse, but data on the occur- zoonoses; ticks; Bosnia and Herzegovina rence of zoonotic tick-borne bacteria are lacking. Thus, the aim of this study was to investigate the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocy- Correspondence: tophilum, ‘Candidatus Neoehrlichia mikurensis’, spotted fever group (SFG) rick- G. G. Duscher. Department of Pathobiology, ettsiae and Francisella tularensis in questing ticks. In 19 (21.8%) of 87 ticks Institute of Parasitology, University of (Ixodes ricinus, n = 30; Dermacentor reticulatus, n = 54; D. marginatus, n = 3) Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Tel.: +43 1 25077- collected by flagging the vegetation at the collection site in the Glamoc Munici- 2211; Fax: +43 1 25077-2290; pality (south-western Bosnia and Herzegovina), Rickettsia monacensis (1.1%), E-mail: [email protected] R. helvetica (5.7%), R. raoultii (5.7%), R. slovaca (8.0%), A. phagocytophilum (1.1%) and F. tularensis subsp. holartica (1.1%) were detected and identified by Received for publication November 3, 2015 molecular methods. None of the tested ticks were positive for B. burgdorferi s.l. and ‘Candidatus N. mikurensis’, and co-infection of R. slovaca and F. tularensis doi:10.1111/tbed.12473 subsp. holarctica was detected in only one D. marginatus (1.1%). This study reports the occurrence of emerging zoonotic bacteria in ticks from Bosnia and Herzegovina for the first time, indicating a public health threat to humans. Therefore, physicians and practitioners should be aware of the presence of these tick-borne bacteria, especially when they are faced with acute febrile illnesses after tick exposure.

tick-borne bacteria, A. phagocytophilum,‘Candidatus Introduction Neoehrlichia mikurensis’ and F. tularensis are also zoonotic In Bosnia and Herzegovina, Ixodes ricinus and Dermacentor pathogens, causing human granulocytic anaplasmosis, marginatus are the most common and widely distributed neoehrlichiosis and tularaemia, respectively (Parola and tick species, whereas D. reticulatus has more focal occur- Raoult, 2001; Lommano et al., 2012; Reye et al., 2013). All rence (Omeragic, 2011). These tick species are principal these are considered to be emerging pathogens (Parola and vectors of many viral, bacterial and protozoan pathogens of Raoult, 2001; Lommano et al., 2012). To our knowledge, medical and veterinary importance (Reye et al., 2013). In only a few clinical cases of Lyme borreliosis and tularaemia the past three decades, the interest of the scientiﬁc commu- (Dautovic-Krkic et al., 2008; Hukic et al., 2010; Arapovic nity for tick-borne diseases has increased with the emer- et al., 2014) have been reported in the country, but there gence and identiﬁcation of B. burgdorferi sensu lato (s.l.) as are no any data available on human infections caused by a causative agent of Lyme disease in humans in 1982. Spot- other tick-borne bacteria. The main reason may be an ted fever group (SFG) rickettsiae are mainly transmitted by underdiagnosing due to the lack of knowledge and aware- ticks, which act both as vectors and as reservoirs, and they ness among physicians and practitioners. Furthermore, are the most common pathogens detected in I. ricinus and information on the occurrence of zoonotic bacteria in ticks D. reticulatus in Europe (Reye et al., 2013). These obligate in Bosnia and Herzegovina is lacking, and thus, this study intracellular bacteria are able to cause mild to severe dis- aimed to investigate the presence of abovementioned eases in humans (Parola and Raoult, 2001). Other pathogens in questing ticks by PCR and sequencing.

© 2016 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH 1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made. Zoonotic Bacteria in Questing Ticks A. Hodzic et al.

published elsewhere (Regnery et al., 1991; Roux et al., Methods 1996; Sjostedt€ et al., 1997; Liebisch et al., 1998; Brown In June 2015, questing ticks were collected from pastures et al., 2001). Amplified PCR products were visualized by and wooded habitats by flagging the vegetation, near to agarose gel electrophoresis, purified and directly sequenced human settlements and military training camp, in the at a commercial company (LGC Genomics, Germany). Glamoc Municipality (44°02044″N, 16°50054″E; elevation Confidence interval (95% CI) for the prevalence rate was 936 m), south-western Bosnia and Herzegovina (Fig. 1). computed with SPSS 20.0 statistical software. Two 200-m2-sized transects were selected within the collection site. The sampled area is characterized by crops, pas- Results tures for sheep flocks and cattle herds and coniferous forest with small lawns and shrubs, and it was previously known A total of 87 adult ticks (75 females and 12 males) collected for high tick abundance and relatively high diversity of tick on the vegetation were identified as I. ricinus (n = 30), species (Omeragic, 2011). All collected ticks were morpho- D. reticulatus (n = 54) and D. marginatus (n = 3). At least logically identified to species level and sexed by using stan- one tested pathogen was detected by molecular methods in dard taxonomic keys (Estrada-Pena~ et al., 2004) and stored 21.8% (19/87) of all sampled ticks. PCR for gltA gene of at 20°C until further procedure. Prior to DNA extraction, Rickettsia spp. delivered 18 (20.7%) positive samples in the ticks were washed in 3% H2O2 and 70% ethanol, rinsed gel electrophoresis, whereas rickettsial ompA gene frag- with distilled water and dried on sterile filter paper. Each ments were detected in only 13 (15%) specimens. The tick was then crushed and homogenized individually in sequences obtained from I. ricinus (n = 6) showed 100% 100 ll of DEPC water and 3 mm steel beads, using a Tis- similarity to gltA and ompA sequences of R. monacensis â sueLyser II (Qiagen, Hilden, Germany). After centrifuga- (n = 1) [e.g. GenBank accession nos. KP215387.1 and tion (3000 g/10 s), the supernatant was collected and DNA FJ919640.1] and gltA sequences of R. helvetica (n = 5) [e.g. was extracted with the peqGOLD TriFast Isolation System KP283018.1]. Sequences analyses of five PCR-positive (Peqlab, Germany) following the manufacturer’s instruc- D. reticulatus ticks revealed 100% similarity to gltA [e.g. tions. The presence of B. burgdorferi s.l., A. phagocy- KP742992.1] and 99–100% similarity to ompA sequences tophilum, ‘Candidatus N. mikurensis’, SFG rickettsiae and [e.g. HM161792.1] of R. raoultii. The sequences of F. tularensis DNA in ticks was tested using PCR protocols R. slovaca from D. reticulatus (n = 5) and D. marginatus

Fig. 1. Map of Bosnia and Herzegovina showing location (Glamoc) where ticks were collected.

(n = 2) ticks were 99–100% identical to gltA and ompA 32.9)

sequences available in GenBank [e.g. KJ410267.1 and – 6.2) 6.2) 15.7) 12.8) 12.8) 6.2) – – – – – –

KF791242.1]. a Of 87 ticks analysed, two sequences obtained from PCR- positive D. reticulatus and D. marginatus showed 100% % (95% CI) similarity to 16S rRNA and TUL4 sequences Total infections attributed to A. phagocytophilum [e.g. KM215233.1] and n F. tularensis subsp. holarctica [e.g. KF607099.1], respectively. A co-infection of R. slovaca and F. tularensis subsp. 100.0 20 23.0 (15.4 79.2 1 1.1 (0.2 56.1 1 1.1 (0.2 93.9 7 8.0 (4.0 56.1 5 5.7 (2.5 56.1 5 5.7 (2.5 holarctica was detected in one D. marginatus tick (1.1%). 56.1 1 1.1 (0.2 – – – – – – – None of the tested ticks were positive for B. burgdorferi s.l. and ‘Candidatus N. mikurensis’. Sequences are deposited in â GenBank and are available under accession numbers KT805283-KT805303. Infection rates with detected pathogens in examined ticks are shown in Table 1.

Discussion c, south-western Bosnia and Herzegovina Ticks play an important role in the transmission of diseases worldwide, mostly due to their great capacity to act as vectors for many pathogens of medical and veterinary impor- ticks in Glamo 32.9 0.0 (0/0) 100.0 (3/3) 43.9 6.6 0 33.3 (1/3) 6.1 9.8 0 0 0.0 19.9 0 66.6 (2/3) 20.8 19.9 0 0 0.0 6.6 0 0 0.0 tance (Otranto et al., 2014). In the present study, four 6.6 0 0 0.0 – – – – – – species of SFG rickettsiae, namely R. monacensis, R. hel- – vetica, R. raoultii and R. slovaca, were identified based on gltA and ompA gene markers. R. monacensis and R. helvetica were considered to be non-pathogenic for humans, D. marginatus but in 1999 R. helvetica was associated with fatal perimy- and ocarditis in two human patients (Nilsson et al., 1999), while R. monacensis has recently been recognized as causative agent of Mediterranean spotted fever (MSF)-like illness in three human patients from Spain and Italy (Jado et al., 2007; Madeddu et al., 2012). The most prevalent pathogen in our study was R. slovaca (8.0%; 7/87), which was first Dermacentor reticulatus identified in D. marginatus in the former Czechoslovakia in , 37.3 42.9 (3/7) 17.0 (8/47) 11.8 11.4 0 0 0.0 11.4 0 2.1 (1/47) 0.3 11.4 28.6 (2/7) 6.4 (3/47) 4.0 11.4 14.3 (1/7) 8.5 (4/47) 4.0 33.6 0 0 0.0 16.7 0 0 0.0 – – – – – – 1968. Since then, it has been found in D. marginatus and – D. reticulatus ticks throughout Europe (Parola et al., 2009). R. slovaca and R. raoultii were described as human Ixodes ricinus pathogens and agents of tick-borne lymphadenopathy (TIBOLA), also known as Dermacentor-borne necrosis ery- thema and lymphadenopathy (DEBONEL). However, R. slovaca is considered to be more pathogenic than R. raoultii, because it has been more frequently associated with human infection (Parola et al., 2009). TIBOLA/ 0 0 0.0 0 0 0.0 0 0 0.0 0 0 0.0 0 20.0 (5/25) 7.3 Ixodes ricinus Dermacentor reticulatus Dermacentor marginatus 0 4.0 (1/25) 0.6 DEBONEL is the most prevalent tick-borne rickettsioses in Male Female 95% CI Male Female 95% CI Male Female 95% CI Europe after MSF (Oteo and Portillo, 2012). The low prevalence of A. phagocytophilum and F. tularensis subsp. holartica in the present study is in line with the very low incidence of diseases they cause in East- ern Europe (Reye et al., 2013). DNA of B. burgdorferi s.l and ‘Candidatus N. mikurensis’ was not detected in sam- The prevalence of tick-borne bacteria in questing pled ticks, most likely due to the small sample size and low prevalence rate of infection in the studied area. All six number of infections. Refers to total number of infections, not total number of infected ticks. n, Prevalence % (no. of infected/tested). Pathogens Table 1. a Total positive/total tested 0.0 (0/5) 24.0 (6/25) 9.5 Francisella tularensis Anaplasma phagocytophilum Rickettsia slovaca Rickettsia raoultii Rickettsia helvetica pathogens detected in this study are zoonotic, and forest Rickettsia monacensis

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