The diversity of tick-borne bacteria and parasites in ticks collected from the Strandja Nature Park in south-eastern Bulgaria

Ticks are important carriers of many different zoonotic pathogens. To date, there are many studies about ticks and tick-borne pathogens (TBP), but only a few were carried out in Bulgaria. The present study intends to detect the prevalence of tick-borne bacteria and parasites occurring at the Black Sea in Bulgaria to evaluate the zoonotic potential of the tick-borne pathogens transmitted by ticks in this area. In total, cDNA from 1541 ticks (Dermacentor spp., Haemaphysalis spp., Hyalomma spp., Ixodes spp. and Rhipicephalus spp.) collected in Bulgaria by flagging method or from hosts was tested in pools of ten individuals each for Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi (s.l.), Rickettsia spp. and “Candidatus Neoehrlichia mikurensis” via conventional and quantitative real-time PCR. Subsequently, samples from positive pools were tested individually and a randomized selection of positive PCR samples was purified, sequenced, and analyzed. Altogether, 23.2% of ticks were infected with at least one of the tested pathogens. The highest infection levels were noted in nymphs (32.3%) and females (27.5%). Very high prevalence was detected for Rickettsia spp. (48.3%), followed by A. phagocytophilum (6.2%), Borrelia burgdorferi (s.l.) (1.7%), Babesia spp. (0.4%) and “Ca. Neoehrlichia mikurensis” (0.1%). Co-infections were found in 2.5% of the tested ticks (mainly Ixodes spp.). Sequencing revealed the presence of Rickettsia monacensis, R. helvetica, and R. aeschlimannii, Babesia microti and B. caballi, and Theileria buffeli and Borrelia afzelli. This study shows very high prevalence of zoonotic Rickettsia spp. in ticks from Bulgaria and moderate to low prevalence for all other pathogens tested. One should take into account that tick bites from this area could lead to Rickettsia infection in humans and mammals.

and abdominal pain. The Mediterranean spotted fever (MSF), which is mainly caused by R. conorii, may have a far more severe outcome. MSF is endemic in some regions in Bulgaria, and severe cases have been reported [2,3]. Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus spp. are mainly involved in the circulation of Rickettsia species in Europe.
"Candidatus Neoehrlichia mikurensis" (CNM) is also a gram-negative, obligate intracellular bacterium transmitted by ticks that are of considerable risk for human and animal health [8][9][10]. To our knowledge, the occurrence of CNM has not been reported in Bulgaria thus far.
Anaplasma phagocytophilum is a gram-negative obligate intracellular bacterium belonging to the family Anaplasmataceae. In Europe, A. phagocytophilum is mainly transmitted by I. ricinus. To our knowledge, only one study from Bulgaria examined A. phagocytophilum in I. ricinus ticks, with a surprisingly high prevalence (35%) [7].
Babesia spp. are single-celled Apicomplexa which parasitize erythrocytes and may cause babesiosis in humans, horses, dogs and cattle. Ticks such as Rhipicephalus sanguineus, I. ricinus and D. reticulatus are the most important vectors for several different Babesia species in Bulgaria [11].
To our knowledge, until now, most of the studies examining ticks and tick-borne pathogens in Bulgaria were conducted on small sample sizes mainly from central Bulgaria [4,5,12]. The current study is focused on ticks from the largest protected area in Bulgaria, Strandja Nature Park, which is located in the south-eastern part of the country at the Black Sea [13]. It is often frequented by visitors for leisure activities in the natural surroundings and thus of public health relevance.
As knowledge is lacking on the distribution of ticks and tick-borne bacteria and parasites in this area, the aim of this study was to examine the prevalence of tickborne pathogens in ticks occurring in this region.

PCR-screening for tick-borne bacteria and parasites
cDNA from 1541 ticks collected from the vegetation by flagging method (n = 1140), from humans by humanlanding catch (n = 74) and from hosts (n = 327): dogs (n = 56), cattle (n = 83), tortoises (n = 22), goats (n = 20), rodents (n = 60), shrews (n = 1) and hedgehogs (n = 85) in the Burgas Province (south-east Bulgaria) was provided by Ohlendorf et al. (unpublished) ( Table 1). A description of sampling sites and sample processing will be published elsewhere. Pooled cDNA samples were screened by quantitative real-time PCR (qPCR) for the presence of Rickettsia spp. targeting the gltA gene (70 bp) [14], B. burgdorferi (s.l.) complex targeting the p41 gene (96 bp) [15], A. phagocytophilum targeting the msp2 gene (77 bp) [16], and CNM targeting the groEL gene (99 bp) [10,17]. All qPCR reactions were carried out using Mx3000P Real-Time Cycler (Stratagene, Agilent Technologies Deutschland GmbH, Waldbronn,  [18] was carried out. This PCR also amplifies DNA of Theileria spp. but is only referred to Babesia spp. in the following text. All positive pools were further analyzed separately, to identify positive samples, except for Rickettsia spp. due to high prevalence. To determine infection levels of Rickettsia spp. in ticks, 563 samples were selected (based on established criteria such as collecting method and location, tick species, development stage and sex) for qPCR. Then randomly selected Rickettsia-positive samples yielding a cycle threshold (C t ) value below 35 were further investigated by a conventional PCR targeting 811 bp of the ompB (the outer membrane protein B) gene [19]. Samples positive for B. burgdorferi (s.l.) by qPCR (C t < 33) were further examined by single-locus sequence typing targeting the recG gene (722 bp) [20,21]. Conventional PCRs were carried out in the Eppendorf MasterCycler Gradient Thermal Cycler (Eppendorf AG, Hamburg, Germany) and the products were visualized by gel-electrophoresis on 1.5% agarose gel stained with Midori Green (NIPPON, Genetics, Düren, Germany). Positive conventional PCR products, all for Babesia spp. and a randomized selection for Rickettsia spp. (n = 31) and Borrelia spp. (n = 2), were purified using the NucleoSpin® and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. Purified PCR products were sequenced commercially (Interdisziplinäres Zentrum für Klinische Forschung, Leipzig, Germany) with forward and reverse primers used for PCR. Obtained sequences were assembled and analyzed with Bionumerics (Version 7.6) and compared to GenBank entries in NCBI BLAST.

Statistical analysis
Confidence intervals (95%CI) for the prevalence in questing and engorged ticks were determined by the Clopper and Pearson method using the GraphPad Software (GraphPad Software Inc., San Diego, Ca., USA). The Fisher's exact was applied to test the independence of compared prevalence values.

Discussion
Until today, studies in Bulgaria were mostly focused on Lyme disease in humans, sheep, cows and dogs [4,22,23]. Most studies from Bulgaria on tick-borne pathogens are serological surveys in humans, cattle and dogs [2,[22][23][24] and there are only a few studies investigating ticks for tick-borne pathogens [5,22,25]. Further, these studies examined only a small sample size of ticks (n = 94-299) [4,6,7,12]. The current study reports tick-borne bacteria and parasites on a larger scale in a nature park at the Black Sea in Bulgaria with a high frequency of visitors.
Ixodes ricinus was the predominant tick species in this study which is not surprising as it is the most common tick species in the Northern Hemisphere [26]. The infection rate for tick-borne pathogens was also significantly higher in I. ricinus compared to all other tick species, which is not unusual as I. ricinus is known to be the most important vector of tick-borne pathogens in Europe [27].
Rickettsia spp. were found in every tick genus examined. However, a higher diversity of tick species infected by Rickettsia spp. were collected from hosts (ticks belonging to Ixodes, Hyalomma, Dermacentor and Rhipicephalus) than from vegetation (only Ixodes). In general, the prevalence in questing ticks was higher compared to the one obtained from ticks collected from animals. The infection levels in almost all tick genera (Ixodes -both from vegetation and hosts, Hyalomma and Dermacentor from hosts) were very high, i.e. at least 50%, except for Rhipicephalus ticks from hosts which were infected only in few percentage. Interestingly, most Rickettsia-positive ticks collected from small mammals, were parasitizing southern white-breasted hedgehogs, Erinaceus concolor.
There are no data about Rickettsia infection in ticks collected from E. concolor but other hedgehog species such as E. europaeus, are known to serve as potential reservoirs for certain Rickettsia spp. from urban and suburban areas [28][29][30]. Sequence analysis revealed a variety of different Rickettsia species such as R. helvetica, R. aeschlimannii and R. monacensis in the current study. All of them are considered as agents of human diseases and occur in Europe [1,31]. Rickettsia species were detected only in their respective vectors: R. helvetica and R. monacensis were exclusively in I. ricinus, and R. aeschlimannii was found only Hy. marginatum  in Hyalomma spp. [1,32]. All R. aeschlimannii samples were very closely associated with the Crimean isolate obtained from Hy. marginatum (KU961544, unpublished). Migrating birds from Africa are considered as reservoirs for R. aeschlimannii in Europe and Hyalomma spp. are remarkably contributing to its transmission in southern Europe [32,33]. The R. helvetica sequences detected in the current study were almost identical with the one previously detected in I. persulcatus from Novosibirsk Region, Russia (KU310591, unpublished). The ubiquitously occurring R. helvetica is mostly transmitted by I. ricinus ticks which are considered as its main vector and reservoir, but it was previously detected also in tissues of many vertebrates, e.g. rodents, hedgehogs, dogs, deer, birds and dogs [1,[34][35][36]. Rickettsia monacensis sequences obtained in this study had a high similarity to (i) a Crimean isolate acquired from Ha. punctata (KU961543, unpublished), (ii) a variant isolated from I. ricinus ticks from Germany (EU330640, unpublished), and (iii) a strain detected in I. ricinus from an urban park in Munich, Germany (JN036418.1; [37]). Widely distributed in Europe, R. monacensis was detected previously not only in I. ricinus ticks but on hosts, mainly migratory birds and lizards [38][39][40][41]. In the current study, R. monacensis was detected in Ixodes ticks collected from the southern white-breasted hedgehogs, Erinaceus concolor for the first time.
Borrelia burgdorferi (s.l.) was found with a low prevalence (1.7%) compared to other studies (32-37.3%) from Bulgaria [4,12]. All positive ticks from this study belonged to the genus Ixodes, which is in line with previous studies from Bulgaria. However, there is also a study reporting Borrelia-positive D. marginatus and Ha. punctata which were collected from humans with Lyme disease in Bulgaria [6]. In this study, most Borrelia-positive ticks were collected from small mammals, especially from E. concolor. Sequencing unveiled presence of pathogenic B. afzelii with a 100% identity with a sequence obtained from human skin in Austria (CP009058; [42]). Again, there is no information about Borrelia-infected ticks collected from E. concolor; however, many studies report the prevalence of Borrelia species, including B. afzelii, in ticks collected from other hedgehog species in the neighbouring country Romania [30,43,44].
In this study, CNM was found in a single specimen of I. ricinus from vegetation only. To our knowledge, this is the first detection of CNM in Bulgaria. Nevertheless, the prevalence (0.1%) for CNM in this study was lower compared to other studies from central Europe (2.2-45%) [10,17,45]. However, results from south-eastern Europe show a similar low prevalence (0-1.3%) leading to the assumption that CNM in ticks is occurring more often in central Europe, where also clinical cases of neoehrlichiosis were reported than in south-eastern Europe where clinical cases are thus far absent [46,47].
The majority of Anaplasma phagocytophilum-positive ticks in this study belonged to I. ricinus (over 90%), which is in line with other studies from Europe suggesting I. ricinus as the main vector [48,49]. The current study reports a high prevalence of A. phagocytophilum in ticks collected from small mammals compared to questing ticks and ticks collected from any other animal species. This finding is in contrast to other European studies reporting low or even zero prevalence in ticks collected from small mammal species such as Apodemus spp. and Myodes spp. [45,50]. However, one should take into account that infected ticks obtained from small mammals in this study were collected mainly from southern whitebreasted hedgehogs, E. concolor. There are no available data on Anaplasma infections in ticks from E. concolor but in general, the hedgehog E. europaeus is a suspected reservoir host for A. phagocytophilum [30,43,51,52]. In Romania which is a neighbouring country to Bulgaria, A. phagocytophilum was detected in ticks collected from another hedgehog species, Erinaceus roumanicus with a prevalence of 12% [44].
Babesia spp. and Theileria spp. were found with a remarkable low prevalence in ticks in this study (less than 1%) in comparison to the prevalence in blood samples of dogs and ticks collected from humans and the environment from Bulgaria in previous studies (3.6-31.4%) [11,24]. Babesia spp. and Theileria spp. were detected only in ticks collected from hosts and were belonging to three genera: Hyalomma, Rhipicephalus and Ixodes, which is not surprising as these tick species are known to be vectors for these protozoans especially in neighbouring countries such as Turkey [53][54][55]. Sequence analysis revealed the presence of three species. Babesia microti detected in I. ricinus from the yellow-necked mouse A. flavicollis,, which is known to serve as a reservoir, was most closely related with an isolate obtained from questing I. ricinus in Kyiv Botanical Garden, Ukraine (KX591647; [56]). Babesia microti is responsible for human babesiosis cases mostly in the USA, but it was also detected in I. ricinus ticks in Europe [57,58]. However, European strains of B. microti are known to be less pathogenic. Only the 'Jena' strain is considered as pathogenic for humans in Europe [57]. The sequences for B. caballi detected in a female Hy. marginatum tick feeding on cattle in the current study, showed the closest similarity to a sequence found as well in a female Hy. marginatum tick collected from vegetation in Italy (KX375824, unpublished). Babesia caballi is known as the etiological agent of equine piroplasmosis, and ticks from following genera have been identified as significant vectors of this protozoon: Boophilus, Dermacentor, Haemaphysalis, Hyalomma and Rhipicephalus [59]. Theileria buffeli detected in R. bursa, and Hyalomma spp. from cattle in the current study was identical with two sequences obtained from R. annulatus nymphs parasitizing cattle in Italy which were most likely misnamed as T. sergenti (KX375822, KX375823; [60]). According to Uilenberg [61], there is confusion in the nomenclature, and T. sergenti should be named as T. buffeli which is responsible for bovine theileriosis worldwide, since the name 'T. sergenti' has been used earlier to describe a Theileria species infesting sheep [62,63].
Altogether, the prevalence in ticks from hosts was higher for most pathogens. Moreover, more tick genera collected from hosts were found to be positive in general in comparison to ticks which were collected from vegetation. These facts point out that the uptake of pathogens during a blood meal on potential reservoir hosts is more probable than the vertical transmission of the pathogen in ticks. Co-infections in ticks were detected in combination with almost all pathogens besides CNM and the combination of infection of Borrelia spp. and Babesia spp. Co-infections have been described for Rickettsia spp., Borrelia spp., Babesia spp. and A. phagocytophilum [45,64]. As co-infection levels in this study were rather low, no significant combination of pathogens could be found.

Conclusions
In conclusion, this study presents the prevalence of tickborne bacteria and parasites in ticks on a large scale for the first time in a natural reserve in Bulgaria. To our knowledge, this study reports the first detection of "Candidatus Neoehrlichia mikurensis" and R. aeschlimannii in ticks from Bulgaria. A high diversity of tick-borne pathogens (R. monacensis, A. phagocytophilum and B. afzelii) was detected in ticks collected from the southern white-breasted hedgehog, E. concolor, for the first time suggesting it as a host maintaining circulation of tick-borne pathogens. Although most tick-borne pathogens studied were only found with a low prevalence, the prevalence of Rickettsia spp. was very high and diverse species were found. This may be of health impact as humans may suffer from spotted fever after having a tick bite from this region in Bulgaria.