Prevalence of Tick-Borne Pathogens in Questing Ixodes ricinus and Dermacentor reticulatus Ticks Collected from Recreational Areas in Northeastern Poland with Analysis of Environmental Factors

Ticks, such as Ixodes ricinus and Dermacentor reticulatus, act as vectors for multiple pathogens posing a threat to both human and animal health. As the process of urbanization is progressing, those arachnids are being more commonly encountered in urban surroundings. In total, 1112 I. ricinus (n = 842) and D. reticulatus (n = 270) ticks were collected from several sites, including recreational urban parks, located in Augustów and Białystok, Poland. Afterwards, the specimens were examined for the presence of Borrelia spp., Babesia spp., Anaplasma phagocytophilum, Rickettsia spp., Bartonella spp., and Coxiella burnetii using the PCR method. Overall obtained infection rate reached 22.4% (249/1112). In total, 26.7% (225/842) of I. ricinus was infected, namely with Borrelia spp. (25.2%; 212/842), Babesia spp. (2.0%; 17/842), and A. phagocytophilum (1.2%; 10/842). Among D. reticulatus ticks, 8.9% (24/270) were infected, specifically with Babesia spp. (7.0%; 19/270), A. phagocytophilum (1.1%; 3/270), and Borrelia burgdorferi s.l. (0.7%; 2/270). No specimen tested positively for Rickettsia spp., Bartonella spp., or Coxiella burnetii. Co-infections were detected in 14 specimens. Results obtained in this study confirm that I. ricinus and D. reticulatus ticks found within the study sites of northeastern Poland are infected with at least three pathogens. Evaluation of the prevalence of pathogens in ticks collected from urban environments provides valuable information, especially in light of the growing number of tick-borne infections in humans and domesticated animals.


Introduction
Over the past few decades, the phenomenon of urbanization has increased significantly worldwide. Currently, more than 50% of the human population lives in urban areas, and by 2050, this number is expected to rise to 75% [1]. The transformation of wild landscapes into cities and recreational areas causes major changes in the distribution of wildlife. Ticks are an example of arthropods that adapted to the new conditions, increasing the risk of human exposure to tick-borne pathogens. Those arachnids are typically associated with forests, meadows, and other rural landscapes. However, in recent decades, reports of their presence in urban surroundings are becoming increasingly frequent [1][2][3]. The presence of well-known tick-borne pathogens, such as Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, or Babesia spp., among others, has been detected in ticks collected from recreational areas in multiple studies across Europe [4][5][6][7][8][9][10][11].
positive, components were tested again, separately, in order to obtain the exact number of infected specimens.
Further PCR and electrophoresis, as well as sequencing analysis for the detection of chosen pathogens, were performed according to the methods previously described by Grochowska et al. [15].
For identification of Borrelia spp., a 120-bp fragment of the 16S rRNA gene encoding small ribosomal subunit was amplified. PCR was performed with the Borrelia burgdorferi PCR kit (GeneProof, Brno, Czech Republic) for in vitro diagnostics. The reaction program was designed in compatibility with GeneProof instruction with its own modifications and consisted of the following steps: UDG decontamination at 37 • C for 2 min, initial denaturation at 95 • C for 10 min, amplification for 45 cycles (denaturation at 95 • C for 5 s, annealing at 60 • C for 40 s, extension at 72 • C for 20 s), and final extension at 72 • C for 2 min.
For A. phagocytophilum DNA detection, a nested PCR, targeting a fragment of 16S rDNA gene encoding small ribosomal 16S RNA subunit, was used. Reactions were performed with the Anaplasma PCR kit (Blirt-DNA Gdańsk, Gdańsk, Poland), according to the manufacturer's instructions.
For Rickettsia spp., Bartonella spp., and C. burnetii identification, the Vet PCR RICK-ETTSIA, The Hum PCR BARTONELLA, and The Hum PCR Coxiella burnetii detection kits (BioIngenTech, Concepción, Chile) were used, respectively. All reactions were performed in accordance with manufacturer's instructions.
Samples positive for Borrelia spp. and Babesia spp. were sequenced by Macrogen (Amsterdam, The Netherlands). In total, 5 µL of obtained amplification products were mixed with specific primers: BIG BOR-F1 (5 µL, 50 mM) and BIG BOR-R1 (5 µL, 50 mM) for Borrelia spp. and those used previously for PCR for Babesia spp. Prepared samples were sent to Macrogen, where they were sequenced from both sides. All positive A. phagocytophilum amplicons were purified with the Wizard ® SV Gel and PCR Clean-Up System (Promega, Madison, WIS, USA) and subjected to Sanger sequencing at a commercial facility (Macrogen Europe, Maastricht, The Netherlands).
Afterwards, the results were compared with sequences deposited in the GenBank using the BLAST program. Sequences with the highest compatibility were recorded.

Evolutionary Relationships of Taxa
The evolutionary history of the various Borrelia and Babesia genospecies was inferred by using the Neighbor-Joining method [21]. The evolutionary distances were computed using the Tamura-Nei method [22] were are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA X [23] with subsequent phylogenetic tree visualization using iTOL v61 [24]. This analysis involved

Statistical Analysis of Previous and Present Research
This study is the expansion of the previous study, focusing on D. reticulatus ticks collected in Białystok in 2018 [15]. Since the specimens were analyzed for the presence of the same six pathogens and were obtained in the same area, it was decided to combine the results and perform statistical analysis on a larger study group, including all collection years, in order to obtain more accurate results.
Aforementioned research included 368 D. reticulatus ticks collected in the Zwierzyniecki Forest Nature Reserve in Białystok, Poland, from April to October 2018. Among those, 9.2% were infected with Babesia spp., 0.8% with A. phagocytophilum, and 0.3% with B. burgdorferi s.l.
Statistical analysis was performed using the Statistica 12.0 program (StatSoft, Tulsa, OK, USA).
The Mann-Whitney test was used to assess the prevalence of pathogens in relation to temperature (above and below 20 • C) and humidity (above and below 80%), both with division to the sampling season (April-July, August-October). Overall infection rate, as well as the prevalence of individual pathogens between the two tick species, and developmental stages were also compared using the same test.
Additionally, logistic regression analysis was performed in order to compare the influence of multiple factors.
Statistical significance was established as p < 0.05.

Phylogenetic Analysis
The results of the phylogenetic analysis are presented in a graphical form in Figures 4-7.

Statistical Analysis
Statistical analysis revealed significant differences in several categories. Data from previous and present research used in the evaluation is presented in Figure 8.

Mann-Whitney Test
Statistically significant results were obtained in the following categories (Table 3).

Overall Infection Rate
Comparative analysis revealed a statistically significant difference between Borrelia spp. infection rate in I. ricinus and D. reticulatus, with higher prevalence in I. ricinus ticks. Moreover, significantly more D. reticulatus ticks were infected with Babesia spp.

Air Temperature
For April-July, a statistically significant difference was confirmed in Borrelia spp. infection rates in ticks collected in over 20 • C temperature. Opposite results were obtained for Babesia spp. The same relations in both pathogens were observed in August-October.

Relative Air Humidity
The comparative analysis revealed a higher amount of Babesia spp. infections in ticks collected during periods of air relative humidity below 80% humidity in April-July. In contradiction, Borrelia spp. was found more frequently in ticks sampled in over 80% humidity in August-October.

Sampling Season
Comparative analysis of infection rates in individual developmental stages in relation to the sampling season revealed statistically significant differences for Borrelia spp. It was established that more adults were positive for this pathogen if collected in April-July, while higher prevalence was noticed for nymphs sampled in August-October.

Statistical Analysis
Statistical analysis revealed significant differences in several categories. Data from previous and present research used in the evaluation is presented in Figure 8.

Mann-Whitney Test
Statistically significant results were obtained in the following categories (Table 3).

Multivariate Logistic Regression Model
Multivariate logistic regression analysis showed that, for Borrelia spp. infections, D. reticulatus ticks were 97.16 times less likely to be infected with this pathogen as compared to I. ricinus. Moreover, the chance of detecting Borrelia spp. increased by 1.35 times in the successive sampling years and decreased by 1.59 times in males and nymphs, as compared to females (Table 4).

Discussion
Up until the 1980s, reports on infections in ticks in urban landscapes were incidental. Ever since then, the number of publications on this subject rose, presumably due to rapid development of recreational areas and green tourism, as well as progressing global urbanization [2].
Sequencing analysis of Borrelia-positive I. ricinus ticks identified the majority as B. afzelii and B. garinii, both in ticks collected in Białystok and Augustów. Those two Borrelia species are predominant in Europe [25], which was confirmed in other studies, including those from urban areas [6,7,[26][27][28]. In this study, B. lusitaniae was confirmed in four I. ricinus ticks. Other than the current study, its presence was detected in two studies focusing on urban surroundings [7,27].
Interestingly, 6.4-8.4% of I. ricinus ticks were positive for B. miyamotoi, the causative agent of relapsing fever. Literature data regarding this spirochete presence in urban surrounding is scarce. However, B. miyamotoi was detected in such studies in Poland (4.7%) [26] and Switzerland (4.2%) [7]. Krause et al. suggests that B. miyamotoi may be prevalent in endemic borreliosis areas [29]. Human cases of B. miyamotoi infection were first reported in Russia in 2011 [30] and were since then described in multiple studies across Europe, the USA, and Japan [29,31,32].
It is worth emphasizing once again that Lyme disease incidence in the study region (107.7 per 100,000 people) is twice as high as the average in Poland (53.7 per 100,000 people) [14]. It is also worth noting that overall B. burgdorferi s.l. infection rates (23.5% and 23.0% for Białystok and Augustów, respectively) obtained in this study were also higher than the mean prevalence of B. burgdorferi s.l. in I. ricinus ticks in Europe (12.3%). Strnad et al. highlight that infection rates appear to increase significantly from western to eastern Europe [33].
B. afzelii was detected in only two D. reticulatus ticks (0.7%). This spirochete was also identified in other studies from Poland, although only in those from rural areas (0.09-1.6%) [34][35][36]. Low prevalence of B. burgdorferi s.l. in D. reticulatus ticks was confirmed in multiple studies in Europe [37][38][39][40], which may suggest that D. reticulatus ticks are ineffective vectors for this pathogen. In their study, Rudolf et al. examined the effect of D. reticulatus salivary glands and midgut extract on the growth, motility, and morphology of B. garinii in vitro. It was revealed that the extracts inhibited the growth of the spirochete [41].
The statistical analysis revealed higher median Borrelia spp. infection rate in ticks collected in temperatures above 20 • C in both seasons. It is known that the questing activity of I. ricinus nymphs and adults ranges from March to October [42], with a peak in AprilMay [43]. In a previous study that collected data on I. ricinus ticks from urban areas in Europe, it was revealed that temperature over 20 • C was connected to greater B. burgdorferi s.l. prevalence [44]. A relationship between higher mean temperatures and an increase in Lyme disease incidence was also observed by other studies [45,46]. As Keith et al. note, this may be further connected to the increase of human recreational activity in the warmer weather, thus higher tick exposure [45]. Babesia spp. infections were detected more frequently in temperatures below 20 • C and <80% humidity. In this study, D. reticulatus ticks were found to be primarily infected with Babesia spp. Additionally, all of the specimens were adults, who are most active during early spring (March-April) and autumn (September-October) [47], which, in Poland, are associated with lower temperatures. In comparison to I. ricinus ticks, D. reticulatus show higher resilience to colder environmental conditions [47,48].
Overall, 1.1% of D. reticulatus ticks were infected with A. phagocytophilum in the current study, which is consistent with previous findings [15]. Similar results were obtained in Kyiv, Ukraine (0-1%) [5,53], while in the outskirts of Berlin, Germany, none of the collected D. reticulatus ticks were positive for this pathogen [39]. Comparable values were reported in studies conducted in rural areas of Poland and Serbia (0-1.1% and 1.9%, respectively) [36,40,54]. Results obtained in the current study most likely reflect the availability and population density of A. phagocytophilum hosts, such as rodents, hedgehogs, ungulates, foxes, and birds [5,11,51], which are necessary for the completion of the A. phagocytophilum life cycle, since this bacterium is not transmitted transovarially [55].The majority of Babesia in I. ricinus ticks were identified as B. microti in this study. Similar results were obtained by Wójcik-Fatla et al. in their research on recreational sites of eastern Poland [56]. It is worth noting that other studies focused on urban areas identified B. venatorum as the most prevalent [7,49,57]. Interestingly, one of the identified B. microti sequences (KP055650.1) is 100% identical to the pathogenic Jena/Germany strain. However, as stressed by Obiegala et al., it does not mean that the newly detected sequence is also pathogenic [58].
B. canis was the predominant pathogen identified in D. reticulatus ticks in this study (6.7% of all specimens), similar to the previous study (6.8%) [15]. Comparable results (4.18%) were obtained by Mierzejewska et al., who studied D. reticulatus ticks collected from multiple localities in eastern, central, and western Poland. In that study, B. canis was found only in ticks collected in the Eastern part of the country [34]. Noteworthy, Eastern Poland belongs to the European macro-region for D. reticulatus presence [59]. It is reflected by reported canine babesiosis cases. In a study conducted by Dwużnik et al., the authors collected data from 42 veterinary clinics from Eastern and Western Poland and reported 1558 cases of canine babesiosis. Interestingly, the majority (1532) of them came from clinics in the Eastern part of the country [60]. B. canis was also detected in a number of different studies on D. reticulatus ticks from Ukraine, Latvia, Lithuania, Slovakia, and Poland (0.63-3.4%) [53,56,[61][62][63]. Notably, two studies from Poland and Serbia reported exceptionally high B. canis prevalence (21.3% and 20.8%, respectively) [35,40].
In this study, one D. reticulatus was infected with B. microti (0.4%), which is consistent with previous findings (0.8%) [15]. Other studies from Poland report 0.04-4.5% infection rate [34,36,54,64]. It is worth noting that the detected sequence was the same potentially pathogenic sequence as described in I. ricinus. Although it is known that D. reticulatus ticks rarely feed on humans [65], they significantly contribute to the circulation of pathogens, including those potentially harmful to humans, in the environment.
Bartonella spp. was not detected in any I. ricinus ticks investigated in this study. Similar values were reported in other European studies, both in urban Germany [77,78] and rural areas [38,40,79]. This pathogen has been reported in other research conducted in Poland with 1.7-4.8% prevalence, although all infected ticks were collected either from vegetation in rural areas or from animals [63,80,81]. In the current study, no D. reticulatus ticks were infected with Bartonella spp. Comparable results were obtained in research focused on urban areas: 0.5% in Warsaw, Poland [80] and 1.0% in Kyiv, Ukraine [53], as well as in rural surroundings: 0.6% in Belarus [38] and 0% in Serbia [40].
Interestingly, also no D. reticulatus ticks were found to be infected with Rickettsia spp. in the current study. This pathogen has been reported with a high prevalence rate (40.7-56.7%) in natural sites in Poland [34,36,66,[85][86][87]. Studies from urban areas in Kyiv, Ukraine, revealed 10.1-35.7% infection rate [5,53]. In other European countries, reported infection rates in natural sites were similar (14-21.4%) [65,88]. Given such discrepancies between Rickettsia spp. prevalence obtained in this and other studies, further research in the study area, focusing on this pathogen, is needed.

Conclusions
In conclusion, the molecular investigation carried out in this study confirms that I. ricinus and D. reticulatus ticks present within urban areas of the northeastern Poland are infected with at least three pathogens: Borrelia spp., A. phagocytophilum, and Babesia spp. Moreover, results reveal that the prevalence of B. burgdorferi s.l. is equal or even higher than in natural ecosystems. As this is the first study on ticks in cities of northeastern Poland, it provides valuable information for tick-borne pathogen surveillance.