Introduction

Nineteen species of ticks are known to occur in Poland comprising the established tick fauna of the region (Nowak-Chmura and Siuda 2012). The most common species of hard ticks (Ixodidae) are Ixodes ricinus and Dermacentor reticulatus. These two species are subjects of numerous field and laboratory studies, covering the majority of the territory of the country (Asman et al. 2017; Kiewra et al. 2014, 2016; Mierzejewska et al. 2015, 2016; Stańczak et al. 2015, 2018; Wodecka et al. 2014; Wójcik-Fatla et al. 2016; Zając et al. 2017). However, the relict tick Haemaphysalis concinna has never been the subject of such studies because it has been so rare in the Poland. A recent overview of the Polish tick fauna has reported only one previous finding of this tick species in Poland (Nowak-Chmura and Siuda 2012). A single female H. concinna was found in close proximity to the German-Polish border and Baltic Sea shore, in Troszyn, N. West Pomerania in 1953, 65 years ago (Lachmajer et al. 1956).

Interestingly, although the number of field studies on ticks (involving intensive dragging in different habitats and studies on the tick fauna of a wide range of hosts, including wildlife) has increased substantially over the last 20 years, no new foci of H. concinna have been reported in Poland. In contrast, the rapid expansion of D. reticulatus has been well documented in Europe (Rubel et al. 2016; Földvári et al. 2016) and also in Central and Western Poland (Mierzejewska et al. 2016, 2017). In a recent review of the distribution of H. concinna in Central Europe, Poland was still designated as a ‘white spot’ region with minimal records, while neighboring countries were recorded as ‘red spot’ foci reflecting the well-documented presence of the tick: for example in Germany, South Czech Republic, Slovakia, Ukraine and Belarus, South and Eastern Russia (Rubel et al. 2018). Thus, with more vigilant monitoring in the future it will not be surprising to find that more foci of H. concinna are recorded in Poland. Moreover, given the well documented climate change and the evidence that it has promoted tick expansion and tick population growth in Europe (Karbowiak 2014; Medlock et al. 2013; Sréter et al. 2005), substantial changes in the distribution of H. concinna are also likely to have taken place since its first and only report in the country 65 years ago.

Following our earlier study on the expansion of the distribution of D. reticulatus in the period 2012–2014, we also monitored this process subsequently between 2016 and 2018, in order to document further the continued colonization of Western and Central Poland with this important vector of Babesia canis. This study was performed by dragging of suitable open habitats in Spring (March–May) and Autumn (September–November) of each year (Dwużnik unpublished). Additionally, the study was broadened by targeted monitoring of the instars of D. reticulatus. For this reason we collected ticks from several rodent communities in Central and Western Poland in the period June–August, when juvenile D. reticulatus ticks can be found on rodent hosts (Karbowiak 2009; Paziewska et al. 2010). It was during this work that we encountered also various developmental stages of H. concinna in our sampling of both vegetation and on the rodents. In the present study we report a new location for the confirmed presence of H. concinna in Western Poland, identified both in the samples of collected questing ticks encountered on vegetation and also feeding ticks removed from rodent hosts.

Materials and methods

Field study

The study was completed in 2018 in Western Poland (Fig. 1). Juvenile ticks were collected from rodents from three sites: Słonin (52.119201N; 16.747274E); Nowy Młyn 1 (52.152127N; 16.119467E) and Nowy Młyn 2 (52.148505N; 16.114098E). Trapping was performed at all sites in June–July (the season for occurrence of D. reticulatus larvae) and in August in Nowy Młyn 2 and Słonin (D. reticulatus nymph season). The distance between Słonin and Nowy Młyn is about 50 km, and that between Nowy Młyn 1 and Nowy Młyn 2 is about 1 km. Additionally, Nowy Młyn 1 and Nowy Młyn 2 sites were separated by a road (Fig. 1).

Fig. 1
figure 1

Map of Haemaphysalis concinna occurrence in Poland

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The habitats at the three locations were similar, comprising submerged meadows and fallow lands in close proximity to water bodies (water ditch in Słonin, Dojca River in Nowy Młyn 1, Dojca River and large breeding pond in Nowy Młyn 2). Each of the trapping sites was located in the vicinity of forests. All locations were selected on the basis of habitat qualities typical of the preferred habitats for Microtus spp. voles which are the main hosts of D. reticulatus instars, this tick species being the main focus of our field work (Paziewska et al. 2010).

Small rodents were live-trapped and live-processed at the site of trapping, as described in our previous papers (Bajer et al. 2014; Grzybek et al. 2015; Tołkacz et al. 2017), including the removal of ticks and other ectoparasites from anesthetized animals. Rodents were then released near to their trapping points.

Additionally, during the inspection of traps in Nowy Młyn 2 site in August 2018 questing adult ticks and nymphs were collected along a 300 m transect from two researchers and their accompanying dog.

Tick identification

Ticks were fixed in 70% EtOH, transported to the laboratory of the Department of Parasitology, Faculty of Biology, University of Warsaw and identified to species and stage levels using a stereoscopic microscope Zeiss Stemi 508 equipped with a camera. All ticks were assigned to species and stages according to the key of Estrada-Peña et al. (2004). Photographical documentation was performed. Ticks were counted and two infestation parameters were calculated: prevalence (% infested rodents) and abundance (mean number of tick/individual). For questing ticks we calculated the mean density/100 m2 of territory.

Genotyping of ticks

Genomic DNA was extracted from four ticks (4 nymphs) collected from rodents at the Nowy Młyn 2 site and identified as H. concinna, using the A&A Biotechnology DNA extraction kit (Gdańsk, Poland). Variable mitochondrial (mt) 16S rRNA gene fragment (440 bp) was amplified following Kulakova et al. (2014).

PCR products were sequenced by a private company (Genomed, Warsaw, Poland). DNA sequence alignments and analyses were conducted using MEGA 7. Consensus sequences were compared with sequences deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/).

For the phylogenetic analysis, the Akaike information criterion was used in jModel Test to identify the most appropriate model of nucleotide substitution. A representative tree for mt16S rDNA was constructed using MEGA 7 by the Maximum Likelihood method and the General Time Reversible model with Gamma distribution (Kumar et al. 2016).

Ethics approval

All of the procedures were conducted with the approval of the First Warsaw Local Ethics Committee for Animal Experimentation in Poland (ethical license number: 706/2015) according to the principles governing experimental conditions and care of laboratory animals required by the European Union and the Polish Law on Animal Protection.

Results

Ticks from rodents

A total of 106 rodents were examined for ectoparasites, including 22 Apodemus agrarius, 14 A. flavicollis, six A. sylvaticus, 51 Microtus oeconomus, six M. agrestis, one M. arvalis, three Mus musculus and three juvenile Microtus voles (Microtus spp.), which could not be identified to species level due to their very small body size (juvenile M. agrestis and M. oeconomus are morphologically almost identical).

Altogether 1482 feeding ticks were collected from these rodents at our three study sites, including 13 D. reticulatus (1 larva and 12 nymphs), 1042 I. ricinus (972 larvae and 70 nymphs) and 427 H. concinna (405 larvae and 22 nymphs). The H. concinna ticks were collected only at Nowy Młyn 2 site (Figs. 1 and 2). A representative larva and nymph, identified as H. concinna, are illustrated in Fig. 3.

Fig. 2
figure 2

Prevalence of three tick species (Dermacentor reticulatus, Ixodes ricinus and Haemaphysalis concinna) by site and host species (Apodemus agrarius, A. flavicollis, A. sylvaticus, Microtus oeconomus, M. agrestis and Mus musculus)

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Fig. 3
figure 3

Pictures of Haemaphysalis concinna larva (A), nymph (B), female (C), male (D) and hypostom (E)

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Ixodes ricinus was the most common species of tick on rodents from the three sites (Table 1). Total prevalence of this species ranged only between 82 and 95%, mainly due to high infestation with I. ricinus larvae. Mean abundance was also similar between sites (NS). Juvenile D. reticulatus ticks were found only in two rodent communities in low numbers, resulting in an overall low prevalence and abundance (Table 1). Ticks identified as H. concinna were found at the Nowy Młyn 2 site and at this site constituted the second most numerous tick species: 427 H. concinna were collected from 39 rodents in comparison to 443 I. ricinus and 10 D. reticulatus. Prevalence of infestation with H. concinna was above 50% at this site, and mean abundance exceeded 4 ticks/rodent, mainly due to the high number of H. concinna larvae.

Table 1 Comparison of the prevalence and mean (± SE) abundance of tick infestation (Dermacentor reticulatus, Ixodes ricinus and Haemaphysalis concinna) between trapping areas
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Comparisons of the prevalence and abundance of the three tick species on the different species of rodents at individual sites are presented in Supplementary file 1 (Supplementary Tables 1–3). Prevalence of tick species, by host species and site, is presented in Fig. 2. Juvenile I. ricinus ticks were found on all inspected rodent species. Juvenile D. reticulatus ticks were found on A. agrarius and M. oeconomus, and H. concinna ticks were also found on A. agrarius and two vole species: M. oeconomus and M. agrestis (and on three unidentified juvenile Microtus) (Fig. 2). Both high prevalence (65–80%) and high mean abundance (4–16 ticks/host) of H. concinna infestation were noted on Microtus voles from Nowy Młyn 2 (Supplementary File 1: Supplementary Table 3). Interestingly, no H. concinna ticks were found on rodents in early summer (June–July) but both larvae and nymphs appeared on rodents in August (Table 2). At that time both prevalence and abundance of H. concinna were highest on M. agrestis and M. oeconomus, exceeding 83% and 21 ticks/host for the latter species (Table 2).

Table 2 Prevalence and mean (± SE) abundance of tick infestation (Dermacentor reticulatus, Ixodes ricinus and Haemaphysalis concinna) by host species, site and season
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Questing ticks

In August 2018, 20 questing ticks were collected in Nowy Młyn 2 from two researchers and their accompanying dog, to whom they had attached while checking traps over a 300 m long transect. All ticks were morphologically identified as H. concinna, including 3 H. concinna females, one male and 16 nymphs. Representative female and male of H. concinna are presented in Fig. 3. The mean density of H. concinna in this location was estimated at 3.33 ticks/100 m2.

Genotyping and phylogenetic analysis

Four mt 16S rDNA sequences were obtained from 4 nymphs morphologically identified as H. concinna. Three consensus sequences showed the highest similarity (404/405 = 99.75%) to a H. concinna sequence from Japan (GenBank: AB819173) and 99.51% (404/406) to a H. concinna sequence from Hungary (GenBank: KU170525). This first identified genotype has been deposited in the GenBank database under accession number MK418863. Fourth sequence (deposited with accession number MK463620) differed by five positions (one insertion, four A–T substitutions) from the three former sequences, representing second genotype, and showed the highest similarity—98.76% (399/404) to a sequence from Japan (Gen Bank: AB819171).

A phylogenetic tree incorporating sequences obtained in this study is presented in Fig. 4. All sequences grouped with H. concinna from Europe and Japan.

Fig. 4
figure 4

Molecular phylogenetic analysis of Ixodidae by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model (Nei and Kumar 2000). The tree with the highest log likelihood (− 2079.4170) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 0.3748)). The rate variation model allowed for some sites to be evolutionarily invariable ([+ I], 20.7364% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 56 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 286 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016)

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Discussion

A new well-established focus of H. concinna has been identified in Western Poland both through the collection of questing ticks from vegetation and feeding ticks from several rodent hosts. The identity of this tick species was confirmed by morphological criteria and by amplification and sequencing of mt 16S rDNA.

In comparison to the dynamic changes in distribution of many arthropod vectors, including ticks, the finding of one new focus of H. concinna 65 years after the first identification of this species in Western Poland may suggest a low overall ability of this tick to expand locally and to establish in a new territory. Looking at the distances involved, the new site is located about 100 km from the German-Polish border, where at least one known focus is present (Hagedorn 2013; Rubel et al. 2018). It is also located 200 km to the south of a previously described focus in Troszyn in NW Poland. There is no evidence that these sites could have served as the source of this newly recognized H. concinna population. Interestingly, the distribution of this tick species is very focal and rather restricted: no ticks of this species were found on rodents trapped 1 km south from the new focus at Nowy Młyn 1, nor 50 km east in Słonin. Accordingly, no questing H. concinna ticks were found during our previous monitoring surveys of D. reticulatus expansion in Central and Western Poland in 2012–2014 (Mierzejewska et al. 2016), nor during the present monitoring in 2016–2018 (Dwużnik, unpublished), when in this period a high number of sites (> 100) were regularly visited and sampled. Interestingly, in support of the rare occurrence of H. concinna in Poland, no ticks of this species were found on dogs from Central (Zygner and Wedrychowicz 2006; Mierzejewska et al. 2015), South (Kilar 2011) or Western Poland (Król et al. 2016a, b). Also in our long-term studies on rodent parasites in the Mazury Lake District (Bajer 2008; Bajer et al. 2001, 2002, 2005, 2014; Behnke et al. 2001, 2008a, b; Grzybek et al. 2015; Tołkacz et al. 2017), where the number of studied rodents is currently in excess of 4000, yet to-date no H. concinna have been found (Pawełczyk and Siński 2004; Welc-Falęciak et al. 2008; Paziewska et al. 2010).

Such a limited distribution of H. concinna in Poland is surprising, given that this tick species is known to be capable of inhabiting different habitats and climatic zones (Rubel et al. 2018) and is widespread in Germany (Hagedorn 2013) and Austria (where it is the dominant tick species on dogs in summer months; Duscher et al. 2013).

As H. concinna is a proven or suggested vector of a wide range of pathogens, including tick-borne encephalitis, Anaplasma phagocytophilum and Rickettsia spp. (reviewed in Rubel et al. 2018), the finding of this new focus in Poland is of considerable local concern. This tick species may feed on both human and domestic animal hosts, as found also in our study although ticks had been removed before they started feeding. Our results are therefore of public health relevance, contributing to the understanding of the risk of tick-borne diseases in regions where this tick species is endemic, and we emphasize the need for increased awareness of its presence and continued monitoring for potential expansion of its range.

Conclusions

In the present study we report a new focus of H. concinna in Western Poland, identified both by the collection of questing ticks from vegetation and feeding ticks from several rodent hosts. Moreover, we confirm that this is still a rare and uncommon species in other regions of Poland.