Density of Dermacentor reticulatus Ticks in Eastern Poland.

Dermacentor reticulatus, the ornate cow tick, is second only to Ixodes ricinus as the most important reservoir and vector of infectious diseases in Europe. In recent years, the distribution of D. reticulatus ticks has expanded into new territories, including increased population densities in areas of their previous occurrence. Our investigations around this consisted of two stages. In the first stage, we monitored the seasonal activity of D. reticulatus ticks in Polesie National Park in 2014–2019. The second stage, which was carried out in 2019 at the peak of the spring (March) and autumn (October) activity of this species, included assessment of the density of D. reticulatus ticks in the entire province. To this end, the study area was divided into 101 equal plots that were surveyed for ticks. The seasonal activity of D. reticulatus in Polesie National Park showed peaks of activity in autumn in 2014–2018 and in spring in 2019. A total of 19,559 adult D. reticulatus specimens were collected, with a mean of 96.8 specimens/100 m2 in Lublin Province. The area of Lublin Province is characterized by a high density of the ornate cow tick. An increase in the surface area of meadows and fallow land has contributed to a rise in the number of local populations of D. reticulatus ticks.


Introduction
Dermacentor reticulatus, the ornate cow tick, is one of the most widely distributed tick species throughout Europe. As suggested by some authors, two geographically separated populations of ornate cow ticks, known as the "Eastern European" and "Western European", can be distinguished in Europe. This phenomenon is not found in other widely distributed tick species in Europe, such as Ixodes ricinus or D. marginatus [1][2][3].
In the western part of the continent, the occurrence of D. reticulatus has been confirmed in the northern regions of Spain, France, the southern part of the British Isles, Switzerland, Benelux countries, and Germany [3][4][5]. In Central Europe, numerous localities of this species have been reported in Poland, Hungary, the Slovakian lowlands, Slovenia, and Austria [3,[6][7][8][9]. In the east, the occurrence range of D. reticulatus ticks covers Ukraine, Belarus, Lithuania, Latvia, Estonia, and the European north-west part of Russia. Numerous records have also been reported from the Caucasus and Asia to the Omsk and Novosibirsk regions [2,[10][11][12][13].
Before the beginning of the 21st century, it was thought that the distribution of D. reticulatus in Poland was limited to the territory east of the Vistula River and in the north-eastern part of the country, while western regions were considered non-endemic. Additionally, it was assumed that Poland was the boundary between the eastern and western populations of D. reticulatus [13][14][15].
Similar changes in the dynamics and spread of D. reticulatus into new areas have also been observed in other European countries, including in Germany, where its recorded distribution before 1970 was limited to western areas, with low numbers reported [23]. In the following years, the distribution of D. reticulatus was reported to expand into eastern areas bordering Poland [6]. Many new sites of D. reticulatus occurrence have been discovered in the Czech Republic. In Slovakia, new localities of the ornate cow tick, representing both eastern and western European populations, have been reported [24,25]. More recently, D. reticulatus ticks have been reported in Hungary [26][27][28], where only two sites were documented before the 1970s [9]. Increased abundance and new localities of D. reticulatus have also been observed in Romania [29,30]. Clustered populations of D. reticulatus have been confirmed in the Balkans [31][32][33][34] and Iberian Peninsula [35]. The spread of ornate cow ticks to Northern Italy [36], which was previously regarded as tick-free, has been observed recently.
D. reticulatus populations in Europe were previously separated by a geographical border, which is now breaking down due to new introductions. Although genetic studies of specimens of both populations have shown differences in the structure of their genomes [37], the results of these studies do not indicate equivocally the cause of the occurrence of gaps in D. reticulatus distribution in Europe.
The aim of this study was to conduct concurrent large-scale tick surveillance to determine the relative density and distribution of D. reticulatus in Lublin Province, Eastern Poland.

Materials and Methods
The investigations consisted of two stages. In the first stage (2014-2019), we monitored the seasonal activity of D. reticulatus ticks in a habitat preferred by the species in Polesie National Park.
The results facilitated precise determination of the rhythms of seasonal activity of the species in the spring-autumn period between March and October. The second stage (2019) was focused on assessment of the density of D. reticulatus ticks in the entire Lublin Province.

Polesie National Park
Polesie National Park (51.496° N, 23.102° E) is located in the lowlands of Central-Eastern Poland. The park, covering a surface area of 97.6 km 2 , is mainly occupied by lakes, ponds, marshes, peat bogs, and meadows [38].

Lublin Province
The Province has an area of 25,122 km 2 , which accounts for 8.02% of the territory of Poland ( Figure 1).

Lublin Province
A grid with sides corresponding to 15.8 km of terrain was applied to the study site, resulting in 101 squares with a mean area of 250 km 2 each, representing 1% of the landmass of Lublin Province ( Figure 2).
Next, satellite photos available in Google Maps were used to establish a potential tick collection site in each square. Grasslands undergoing ecological succession located at the margins of or in close vicinity to forests, and with access to watercourses, were regarded as habitats preferred by these ticks [43]. In each square of the grid, one potential approximately 2500 m 2 site of tick collection was established. The next step involved visual inspection of the previously established tick collection sites. In sites that met the assumed criteria, one 100 m 2 plot was delineated ( Figure 4) for subsequent collection of ticks. In the event of discrepancies between the satellite images of the area and the actual situation, the procedure of selection of the sites was repeated (the selection of plots 34, 55, 62, and 75 was repeated once).

Lublin Province
A grid with sides corresponding to 15.8 km of terrain was applied to the study site, resulting in 101 squares with a mean area of 250 km 2 each, representing 1% of the landmass of Lublin Province ( Figure 2).
Next, satellite photos available in Google Maps were used to establish a potential tick collection site in each square. Grasslands undergoing ecological succession located at the margins of or in close vicinity to forests, and with access to watercourses, were regarded as habitats preferred by these ticks [43]. In each square of the grid, one potential approximately 2500 m 2 site of tick collection was established. The next step involved visual inspection of the previously established tick collection sites. In sites that met the assumed criteria, one 100 m 2 plot was delineated ( Figure 4) for subsequent collection of ticks. In the event of discrepancies between the satellite images of the area and the actual situation, the procedure of selection of the sites was repeated (the selection of plots 34, 55, 62, and 75 was repeated once).

Tick Surveillance
D. reticulatus ticks were collected in Polesie National Park in 2014-2019. In 2014-2018, the ticks were collected at equal 14-20 day intervals. Month-long intervals were used for collection of ticks in 2019. Each time, the ticks were collected between 11:00 and 14:00 with the flagging method, in accordance with the procedure described by Nowak-Chmura [44]. Throughout the study period in Polesie National Park, the ticks were collected by the same person. The vegetation was swept with a 1 m 2 white sheet each time for 30 min. A 100 m long and 1 m wide transect was covered, which corresponds approximately to 100 m 2 . The sheet was inspected approximately every 2 min and the attached ticks were removed using forceps, placed in a container, and transported to the central laboratory.
The investigations of the tick density in Lublin Province were conducted during seasonal peaks of tick activity in this region indicated in the literature [43,45,46], based on the results of studies of the seasonal activity of D. reticulatus ticks in Polesie National Park (Table A1) and concurrent tick surveillance of the D. reticulatus activity in different areas of Lublin Province. The tick surveillance was conducted in the spring from 18 to 31 March 2019, and from 12 to 24 October 2019 in the autumn. Plot 93 was excluded during the autumn due to ongoing construction. Ticks were collected with the standard flagging method. Vegetation was swept with 1 m 2 white flannel fabric. Each time, the ticks were collected from a 100 m 2 plot. This area was flagged over a distance of 10 m and then the flag was checked for the presence of ticks on both sides ( Figure 5). This was repeated 10 times to ensure

Tick Surveillance
D. reticulatus ticks were collected in Polesie National Park in 2014-2019. In 2014-2018, the ticks were collected at equal 14-20 day intervals. Month-long intervals were used for collection of ticks in 2019. Each time, the ticks were collected between 11:00 and 14:00 with the flagging method, in accordance with the procedure described by Nowak-Chmura [44]. Throughout the study period in Polesie National Park, the ticks were collected by the same person. The vegetation was swept with a 1 m 2 white sheet each time for 30 min. A 100 m long and 1 m wide transect was covered, which corresponds approximately to 100 m 2 . The sheet was inspected approximately every 2 min and the attached ticks were removed using forceps, placed in a container, and transported to the central laboratory.
The investigations of the tick density in Lublin Province were conducted during seasonal peaks of tick activity in this region indicated in the literature [43,45,46], based on the results of studies of the seasonal activity of D. reticulatus ticks in Polesie National Park (Table A1 in Appendix A) and concurrent tick surveillance of the D. reticulatus activity in different areas of Lublin Province. The tick surveillance was conducted in the spring from 18 to 31 March 2019, and from 12 to 24 October 2019 in the autumn. Plot 93 was excluded during the autumn due to ongoing construction. Ticks were collected with the standard flagging method. Vegetation was swept with 1 m 2 white flannel fabric. Each time, the ticks were collected from a 100 m 2 plot. This area was flagged over a distance of 10 m and then the flag was checked for the presence of ticks on both sides ( Figure 5). This was repeated Int. J. Environ. Res. Public Health 2020, 17, x 5 of 17 that the entire surface area had been swept. The ticks were collected once in the spring and once in the autumn, each time from exactly the same 100 m 2 area. In both stages, the current weather conditions, temperature, and relative air humidity were measured using Data Logger R6030 (Reed Instruments, Wilmington, NC, USA). In the laboratory, the species and sex of collected specimens were identified with the use of a stereoscopic microscope Zeiss STEMI DV4 (Carl Zeiss Light Microscopy, Göttingen, Germany) and an identification guide compiled by Estrada-Peña et al. [47].

Analysis of the Land Structure of the Study Area
The Google Maps and a layer of satellite images of the region [48] were used for calculation of the land use structure in each plot of the superimposed cartographic grid. The area of cultivated fields (arable land), forests, and meadows (grasslands) was calculated in this way. The designation "other" referred to buildings, water reservoirs, and areas that could not be clearly defined.

Statistical Analysis
Differences in the number of active D. reticulatus females and males in each experimental plot were analyzed statistically using the Wilcoxon signed-rank test. The Kruskal-Wallis test was used for statistical analysis of the number of active D. reticulatus females and males in the spring and autumn periods. A multiple regression model and Spearman's rank correlation tests were used to assess the impact of the land use on tick densities. The effect of temperature and relative humidity on tick activity in Polesie National Park was evaluated using the Kruskal-Wallis test.
The statistical analysis was conducted using Statistica 10PL (StatSoft, TIBCO Software Inc, Palo Alto, CA, USA) software, and the significance level for all statistical tests was p < 0.05. In both stages, the current weather conditions, temperature, and relative air humidity were measured using Data Logger R6030 (Reed Instruments, Wilmington, NC, USA). In the laboratory, the species and sex of collected specimens were identified with the use of a stereoscopic microscope Zeiss STEMI DV4 (Carl Zeiss Light Microscopy, Göttingen, Germany) and an identification guide compiled by Estrada-Peña et al. [47].

Analysis of the Land Structure of the Study Area
The Google Maps and a layer of satellite images of the region [48] were used for calculation of the land use structure in each plot of the superimposed cartographic grid. The area of cultivated fields (arable land), forests, and meadows (grasslands) was calculated in this way. The designation "other" referred to buildings, water reservoirs, and areas that could not be clearly defined.

Statistical Analysis
Differences in the number of active D. reticulatus females and males in each experimental plot were analyzed statistically using the Wilcoxon signed-rank test. The Kruskal-Wallis test was used for statistical analysis of the number of active D. reticulatus females and males in the spring and autumn periods. A multiple regression model and Spearman's rank correlation tests were used to assess the impact of the land use on tick densities. The effect of temperature and relative humidity on tick activity in Polesie National Park was evaluated using the Kruskal-Wallis test.
The statistical analysis was conducted using Statistica 10PL (StatSoft, TIBCO Software Inc, Palo Alto, CA, USA) software, and the significance level for all statistical tests was p < 0.05.

Multiannual Monitoring of the Seasonal Activity of D. reticulatus Ticks
In Polesie National Park, a statistically significant (H = 6.98, p = 0.047) tendency towards increased numbers of active D. reticulatus was observed every year in 2014-2018. Higher numbers of active ticks were reported in the autumn. Concurrently, higher numbers of females than males were collected. In 2019, there were changes in the seasonal activity of D. reticulatus, as higher numbers of active ticks were collected in the spring (on average 165.5 individuals per collection event: 97 females and 68.5 males) than in the autumn (on average 46.5 individuals per collection event: 28.5 females and 18 males) ( Table 1). Throughout the study, the seasonal activity (spring-autumn) of D. reticulatus ticks was significantly influenced by the air temperature prevailing during tick collection (H = 12.887, p = 0.002).

Density of D. reticulatus Ticks
A total of 19,559 adult (11,598 females and 7961 males) D. reticulatus were collected throughout the study period (Table A2). The mean number of ticks collected in Lublin Province was 96.8 specimens/100 m 2 , with a significant dominance of females (on average 57.6 females and 39.2 males/100 m 2 ) (Z = 8.19, p < 0.001). The adult tick population densities were higher in the autumn than in the spring ( Table 2, Table A2). While the density of D. reticulatus in Lublin Province was high, the mean numbers were unevenly distributed ( Figure 6). The highest numbers of ticks were collected in the northern part of the study area (plots 1-18; South Podlasie and Western Polesie), which varied from 86 specimens with a clear dominance of females over males (on average 53 females and 33 males) to 311 specimens/100 m 2 (224 females and 86.5 males). The highest density of D. reticulatus ticks throughout the study period was observed in plot 69, located in Lublin Upland (Figure 4). During a single collection event at the spring activity peak, 516 adults/100 m 2 of the ornate cow tick were collected, including 297 females and 219 males ( Figure 6, Table A2). Low mean numbers of adults, ranging from 10 to 100 specimens/100 m 2 , were collected from nearby sites (located in the same macro-region) ( Figure 6, Table A2). The lowest number of adult D. reticulatus was collected in the central part of the province, in plot 67, with 5 ticks/100 m 2 (with an average of 3 females and 2.5 males) ( Figure 6, Table A2).

Discussion
Population densities of D. reticulatus are dependent upon biotic and abiotic factors. The most important factors include the presence of potential hosts, ecological habitats, climatic factors, photoperiod, excreted semiochemical substances, and hormones controlling the rhythms of seasonal activity, diapause periods, and biology of tick reproduction and development [12,49].
In Eastern Poland, the greatest impact on the seasonal (spring-autumn) and diurnal rhythms of D. reticulatus activity is exerted by air temperatures prevailing during tick collection [43,45]. This dependence is supported by the results of multiyear monitoring of seasonal tick activity in Polesie At the peak of the seasonal (spring and autumn) activity of D. reticulatus ticks in Lublin Province, there were differences in the distribution of tick density. The northern, southern, and western parts of the Lublin region were characterized by a higher density of D. reticulatus ticks in the autumn, while a higher density in the spring was noted in the central and eastern parts of the area (Table A2, Figure 6).
The density of the ornate cow tick based on ecological variables (arable fields, forests, meadows) was not significantly different (F (3.97) = 1.05, p = 0.3728). However, the analysis of Spearman's rank correlation coefficient indicated that the density of D. reticulatus (both females and males) rose with increased meadow area (Rs = 0.281, p = 0.0044).

Discussion
Population densities of D. reticulatus are dependent upon biotic and abiotic factors. The most important factors include the presence of potential hosts, ecological habitats, climatic factors, photoperiod, excreted semiochemical substances, and hormones controlling the rhythms of seasonal activity, diapause periods, and biology of tick reproduction and development [12,49].
In Eastern Poland, the greatest impact on the seasonal (spring-autumn) and diurnal rhythms of D. reticulatus activity is exerted by air temperatures prevailing during tick collection [43,45]. This dependence is supported by the results of multiyear monitoring of seasonal tick activity in Polesie National Park. Together with relative humidity, it can affect the ticks' questing behavior [50] and locomotor activity [51].
The area of Lublin Province has the highest density of tick populations (mean 96.8 specimens/100 m 2 ) of all studied areas in Poland. The mean number of adult D. reticulatus collected in Lublin Province was 13.1-fold higher than in Mazovia (Central Poland; mean 7.4 ticks/100 m 2 from 2012-2014), 15.6-fold higher than in Lubuskie Province (Western Poland; mean 6.29 ticks/100 m 2 from 2012-2014), and 32.3-fold higher than in the West Pomeranian Province (North-Western Poland; mean 3.04 ticks/100 m 2 from 2012-2014) [20].
The northern and northeastern regions of Lublin Province are characterized by a high density of D. reticulatus ticks ( Figure 6, Table A2). These areas have the largest proportion of grasslands in the entire province, and an increase in their surface area correlates positively and has a significant statistical effect on the number of collected ticks. A significant percentage of land in this area is also occupied by fallows and wasteland, as well as forest islands. The mosaic character of the landscape offers favorable conditions for the development of D. reticulatus populations. Forest areas provide availability of hosts of adult stages, whereas meadows and wasteland are preferable habitats for rodents, which are the hosts of juvenile ticks.
The highest island density of D. reticulatus ticks (516 specimens/100 m 2 ) was observed in plot 69, located in the central part of the region ( Figure 6). Of note, the tick densities in the neighboring sites (located in the same macro-region) were among the lowest in the entire study area, and ranged from 10 to 57 specimens/100 m 2 ( Figure 6, Table A2). Such a high island density of ornate cow ticks is associated with the ecology of the habitats. The site where the maximum number of ticks was caught is surrounded by an abandoned meadow, watercourse, and forest in close proximity, while the forest cover in the entire macro-region is <8% (average for the province: 22%) ( Table A2).
As demonstrated by Mierzejewska et al. [52], the spread of D. reticulatus is associated with the loss of forest area. We share this opinion, with the provision that excessive tree felling does not result in conversion of the land into intensively used agricultural areas. In the study area, the lowest average density of ornate cow ticks was observed in Lublin Upland, a region that is intensively used for agriculture due to its fertile soils. The impact of large forest areas on the density of ornate cow ticks is observed in Lublin Province as well. The number of ticks collected in plots 73 and 74 (forest cover >61%, meadow area 1.93%-4.09%), located within the biggest forest of Lublin Province ("Janów Forest"), was significantly lower than the average number for the region of 29 and 23 specimens/100 m 2 in spring and 46 and 30 specimens/100 m 2 in autumn, respectively (Table A2). The results of our studies show that D. reticulatus populations can persist and steadily develop only in areas with appropriate proportions of forests and meadows/fallow lands. Nevertheless, isolated populations of D. reticulatus ticks can exist in habitats where these proportions are disturbed.
D. reticulatus ticks mostly exhibit two distinct peaks of seasonal activity in spring and autumn. In the French Alps, the European part of Russia, and North-Eastern Poland, more ticks are active in spring [12,53,54]. The data published so far indicate that the number of ticks collected in autumn in Eastern Poland is three-fold higher than in spring [43,45,46]. Our research results show that the seasonal activity may change within the same population, even if it is recognized as stable ( Table 1). The D. reticulatus population in Polesie National Park, monitored by us for the last 6 years (Table 1), exhibited a clearly higher number of active ticks in autumn in 2014-2018 and dominance of the spring peak of activity over autumn in 2019, with a ratio of 3.5:1 (Table 1). Since the experimental field is located within a protected area, the anthropogenic impact is limited. We believe that the size and dynamics of the D. reticulatus seasonal activity are influenced mostly by the availability of preferred hosts occurring in the investigated area, such as roe deer (Capreolus capreolus), deer (Cervus elaphus), elk (Alces alces), and wild boar (Sus scrofa). Our unpublished observations demonstrate the highest degree of ornate cow tick infestations in wild boars. These animals occur in the entire province and their population size has been growing over recent years, by as much as 178%, or from 6400 to 17,800 specimens within 5 years. For comparison, there were 2300 elks and 7600 deer in the area at the same time [55].
In 2017-2018, a decrease in the size of wild boar populations was observed in Lublin Province due to confirmed outbreaks of African swine fever (ASF). Before April 2018, the mortality caused by this viral disease was 1942 wild boars in Eastern Poland [56]. Furthermore, during this period, the wild boar population was reduced preventively. At the turn of 2018/2019, the estimated size of the wild boar population in the Province declined to "no more than several hundred individuals" (personal communication with the Lublin Hunting Association). This led to a significant decrease in the number of animals that are one of the main hosts of D. reticulatus [57,58]. In our opinion, this situation is reflected in the structure of the population size and the dynamics of seasonal activity of D. reticulatus populations in Eastern Poland. It also explains the differences in the spatial distribution of the density of this tick species in Lublin Province. Considering the over 2-year survival of adult specimens in a habitat [12], the predominance of tick activity in spring in areas where an autumn peak was noted previously [43] could be associated with insufficient numbers of hosts for the adult ornate cow ticks in this period. Nevertheless, impacts from other factors on this phenomenon should not be excluded, and further observations and investigations are required.
The high density of D. reticulatus ticks in Lublin Province is associated with a greater risk of tick attack on animals, and sporadically on humans, than in other country regions, and therefore an increased threat of transmission of tick-borne pathogens. Various pathogens have been detected in ornate cow ticks, including Anaplasma phagocytophilum, Rickettsia raoultii, Borrelia burgdorferi s. l., Babesia spp., and TBE virus [59,60]. An increase in the tick population density may result in an increased incidence of tick-borne diseases in future [61]. Numerous cases of canine babesiosis and domestic animal borreliosis have already been reported [62][63][64].

Conclusions
D. reticulatus ticks occur throughout the entire area of Lublin Province. This region is characterized by a very high density of the ornate cow tick. However, it is unevenly distributed. Changes in land use may influence the size of local D. reticulatus populations. An increase in the surface of meadows, grasslands, and fallow land contribute to a rise in the number of ornate cow ticks, whereas afforestation of new areas may reduce their population size.
The local D. reticulatus population exhibits differences in its peaks of seasonal activity. The highest risk of D. reticulatus infestations of animals and humans occurs in spring in the eastern and central parts of the region, and in autumn in the northern and western parts of the province.