Tick Infestation in Migratory Birds of the Vistula River Valley, Poland

Migratory birds play an important role in the eco-epidemiology of tick-borne diseases due to their ability to carry ticks for long distances. The aim of the present study was to investigate the prevalence and factors influencing the intensity of tick infestation in migratory birds. The study was conducted in a locality situated in the Vistula River valley, eastern Poland, during autumn, when the high migratory activity of birds is registered in the region. The birds were captured using ornithological nets and identified at the species level. In the next step, they were carefully inspected for attached ticks. Tick infestation was observed in 4.43% of the captured birds. The highest mean intensity of tick infestation was observed in birds foraging on the ground or in low shrubs and by long- and medium-distance migrants, i.e., Turdus merula (2.73), T. philomelos (2.04), and Erithacus rubecula (1.58). Ixodes ricinus was found to infest the birds most frequently. However, other tick species, i.e., I. trianguliceps, I. crenulatus (synonym I. canisuga), and I. apronophorus, rarely found in eastern Poland, were also found parasitizing birds. The occurrence of I. persulcatus, I. frontalis, and I. acuminatus (synonym I. redikorzevi) was confirmed in the region for the first time. The results of the study suggest that captured bird species are susceptible to tick infestation and could play an important role in the circulation of some tick-borne pathogens. They also play a significant role in the spread of ticks. The ecology and ethology of birds, including their foraging styles and migratory habits, are factors determining the risk of exposure of birds to tick attacks.


Introduction
Birds are hosts of a wide variety of parasites representing different taxonomic groups, such as protozoa, helminths, and arthropods [1][2][3]. Given their potential to transport ticks and their role as reservoirs of many pathogenic microorganisms posing a threat to human health, birds play an important role in the eco-epidemiology of infectious diseases transmitted by tick vectors [4][5][6][7][8][9]. In this aspect, the role of migratory birds is particularly important, as they can cross geographical barriers and spread ticks over long distances, even thousands of kilometers [10]. In contrast, ixodid ticks, such as Ixodes scapularis Say, 1821, are able to cover a short distance of 2-5 m in natural conditions, depending on the developmental stage [11]. The role of migratory birds in the spread of new alien tick species in Europe, e.g., the Hyalomma genus in Germany, has also been confirmed [12]. Similar observations have been reported in Great Britain [13]. In turn, tick species that are

Study Area
The field study was conducted in cooperation with the Kaliszany Ornithological Station (51 • 04 45 N 21 • 48 04 E), eastern Poland, which is part of the National Bird Ringing Station Network and the European Bird Migration Network (EBMN). The research station is located on a river island within the Vistula River Gorge of Lesser Poland. The river is a natural bird migration corridor, while the island is a stopover site for migrating birds. The station is situated on the bird migration route between northern and northeastern Europe and the southern and southwestern parts of the continent [41].

Bird Capture Procedure and Tick Collection
The study was carried out during the autumn migration in 2021 over 53 consecutive days, with a 9-day break at the beginning of September due to local flooding.
The birds were captured in ornithological nets (Ecotone, Gdynia, Poland) with a total length of 500 m, which were stretched along a transect (a total of 1.5 km) running through riverside thickets dominated by riparian vegetation transforming into a meadow community ( Figure 1). In principle, the nets were inspected every hour, but these periods were shortened to half an hour in unfavorable weather conditions (rainfall, low or very high air temperatures). The captured birds were carefully released from the net and identified to the species [41][42][43]. Additionally, their sex, age, and amount of fat tissue were determined based on their morphological traits where possible. All birds were weighed using an Ohaus compact portable Ohaus balance (Series CL-CL501) and ringed. These steps were performed in accordance with the protocol proposed by Operation Baltic and EBMN, as described by Zaniewicz and Rosińska [44].
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 3 of community ( Figure 1). In principle, the nets were inspected every hour, but these perio were shortened to half an hour in unfavorable weather conditions (rainfall, low or ve high air temperatures). The captured birds were carefully released from the net and ide tified to the species [41][42][43]. Additionally, their sex, age, and amount of fat tissue we determined based on their morphological traits where possible. All birds were weigh using an Ohaus compact portable Ohaus balance (Series CL-CL501) and ringed. The steps were performed in accordance with the protocol proposed by Operation Baltic an EBMN, as described by Zaniewicz and Rosińska [44]. Subsequently, the whole birds' body was carefully inspected for the presence of tick Parasites were removed from the body of the birds with tweezers and placed in an E pendorf tube containing 70% ethanol solution. Samples were transported to the labor tory, where a Zeiss STEMI DV4 stereoscopic microscope (Carl Zeiss Light Microscop Göttingen, Germany) and tick identification keys [45][46][47] were used to identify the speci sex, and developmental stage. In the case of tick species that undergo discussion of th species distinctiveness, synonyms were applied [46][47][48]. When the identification of t tick species was impossible due to damage to the body, the specimens were identified the genus level. Due to the identification of tick species only on the basis of morphologic characteristics, the features typical for the identified species and developmental stages a summarized in Supplementary Tables S1 and S2.

Analysis of the Quantitative Structure of Tick Populations and Their Host Preferences
The quantitative structure of the tick population was analyzed by calculating the ec logical index of dominance (DTs%) and the index of prevalence (PTs%) using the follo ing formulas [49,50]:

100%
where DTs-ecological index of tick dominance, Ts-number of ticks of a given spec collected from all birds, Tt-total number of ticks collected from all birds.

100%
where PTs-prevalence of particular tick species, BTs-number of birds infested by a pa ticular tick species, -total number of examined birds Subsequently, the whole birds' body was carefully inspected for the presence of ticks. Parasites were removed from the body of the birds with tweezers and placed in an Eppendorf tube containing 70% ethanol solution. Samples were transported to the laboratory, where a Zeiss STEMI DV4 stereoscopic microscope (Carl Zeiss Light Microscopy, Göttingen, Germany) and tick identification keys [45][46][47] were used to identify the species, sex, and developmental stage. In the case of tick species that undergo discussion of their species distinctiveness, synonyms were applied [46][47][48]. When the identification of the tick species was impossible due to damage to the body, the specimens were identified at the genus level. Due to the identification of tick species only on the basis of morphological characteristics, the features typical for the identified species and developmental stages are summarized in Supplementary Tables S1 and S2.

Analysis of the Quantitative Structure of Tick Populations and Their Host Preferences
The quantitative structure of the tick population was analyzed by calculating the ecological index of dominance (DTs%) and the index of prevalence (PTs%) using the following formulas [49,50]: where DTs-ecological index of tick dominance, Ts-number of ticks of a given species collected from all birds, Tt-total number of ticks collected from all birds.
where PTs-prevalence of particular tick species, BTs-number of birds infested by a particular tick species, Bt-total number of examined birds. The host preferences of the analyzed tick populations were determined based on an indicator called the preference index according to the formula proposed by Dudich [51].
Due to the possibility of overestimating the result for calculations with a small sample, only bird species with at least 100 captured individuals were included in the analysis: where I p -preference index, TsBs-number of ticks of a given species collected from a given bird species, Bt-total number of examined birds, Ts-number of ticks of a given species, Bs-number of birds of given species, I p < 1-negative interaction; I p = 1-neutral interaction; I p > 1-positive interaction. Additionally, the number of host species infested by a particular tick species (NHSPI) (Baláž and Zigová) [52] and the mean intensity of infestation (MII) of the birds by a particular tick species were calculated as in Bush et al. [53]: where MII-mean intensity of infestation of particular tick species; Ts-number of ticks of a given species; BTs-number of birds infested by a particular tick species.

Ornithophilicity of Ticks
Based on literature reports [54], the degree of ornithophilicity of the tick species was determined using the following scale: + accidentally ornithophilic, ++ relatively ornithophilic, +++ strictly ornithophilic.

Quantitative Characteristics of the Population of Captured Birds
The population of the captured birds was characterized using the ecological index of dominance (DBs%) [49,50].
where DBs-ecological index of bird dominance, Bs-number of birds of a given species, Bt-total number of birds The prevalence of tick infestation in a particular bird species (PBs%) and the mean intensity of tick infestation in a given bird species (MIIB) were calculated as follows [49,50,53].
where PBs-prevalence of infested birds in captured individuals, BsT-number of birds of a given species infested with ticks, Bs-number of captured birds of a given species

MIIB = TBs BsT
where MIIB-mean intensity of tick infestation in a given bird species, TBs-number of all tick species collected from a particular bird species, BsT-number of birds of a given species infested with all ticks

Behavior of Captured Birds
The captured birds were classified into ecological types based on the foraging style criterion proposed by Ciebiera et al. [36]: Type 0-birds foraging mainly on the ground and Type 1-birds foraging mainly in shrubs. Additionally, they were classified according to the distances covered during the seasonal migrations. Group 1 comprised sedentary/local migrants, i.e., birds staying in the same region throughout the season or migrating over short distances, usually not reaching the Mediterranean basin for overwintering. Group 2 included medium-distance migrants, usually overwintering in the Mediterranean Basin. Group 3 comprised long-distance migrants, i.e., those covering long distances between continents and usually overwintering in central or southern Africa. The birds were also characterized in terms of migration behavior: d-diurnal-daytime migration, n-nocturnalnight migration, and n/d-beginning of the flight at night and continuation during the day [36].

Statistical Analysis
The type of distribution of the analyzed data was checked using the Shapiro-Wilk test. Due to the lack of normality of the distribution, non-parametric tests were used for the statistical calculations.
The statistical significance of differences in the prevalence of tick infestation between the bird species was tested using the chi-square test. The significance of the differences in the mean intensity of tick infestation depending on birds' behavior was analyzed using the Mann-Whitney U test. The relationships between birds' body weight, amount of fat tissue, and tick infestation intensity were tested using Spearman's rho correlation.
In all statistical tests, the results at p < 0.05 were considered significant. The statistical analysis was performed using GraphPad 8.1 software (GraphPad Software, San Diego, CA, USA).

Prevalence of Tick Infestation in Migratory Birds
In total, 3903 birds of 55 different species were captured during the field study. Among the captured bird species, Erithacus rubecula Linnaeus, 1758 (1142); Sylvia atricapilla Linnaeus, 1758 (638); Turdus merula Linnaeus, 1758 (495); and T. philomelos Brehm, 1831 (341) were the most frequent. Tick infestation was confirmed in only 11 of 55 captured bird species (Tables 1 and S3). The most common locations for tick attachment were the eyelid and chin ( Figure 2). The total prevalence of tick infestation was 4.43% (173 infested of 3903 captured birds), with a statistically significant difference in prevalence of tick infestation between individual bird species (χ 2 = 46.12, p < 0.001).

Tick Species and Tick Parasitic Stages Infesting Migratory Birds
Seven species of Ixodes were identified among the 335 ticks collected from the birds.  Table 2).
Only juvenile stages of ticks with a predominance of nymphs (65.37%) over larvae (34.63%) were found in the entire study group (Figure 3). The ticks collected from the captured birds were characterized by considerable ecological dominance (DTs) of I. ricinus (86.56%) (Tables 2 and 3). Moreover, the highest mean infestation intensity (MII) was determined for this tick species (Table 3). I. ricinus ticks were collected from 9 bird species, accounting for 80.12% of all birds captured in the nets (Tables 2 and 3). In contrast, I. crenulatus, I. apronophorus, and I. acuminatus were found in only one bird species each (Table

Tick Species and Tick Parasitic Stages Infesting Migratory Birds
Seven species of Ixodes were identified among the 335 ticks collected from the birds.
Only juvenile stages of ticks with a predominance of nymphs (65.37%) over larvae (34.63%) were found in the entire study group (Figure 3). The ticks collected from the captured birds were characterized by considerable ecological dominance (DTs) of I. ricinus (86.56%) (Tables 2 and 3). Moreover, the highest mean infestation intensity (MII) was determined for this tick species (Table 3). I. ricinus ticks were collected from 9 bird species, accounting for 80.12% of all birds captured in the nets (Tables 2 and 3). In contrast, I. crenulatus, I. apronophorus, and I. acuminatus were found in only one bird species each ( Table 2). Health 2022, 19, x FOR PEER REVIEW 7

nviron. Res. Public
The bird species differed in the number of infesting tick species. The greatest nu of different species of ticks was collected from E. rubecula (5 out of the 7 identified sp of ticks) ( Table 2). Only one of the tick species collected from the birds (i.e., I. frontalis strictly ornithophilic (Table 3).   FT-foraging type, 0-birds foraging mainly on the ground, 1-birds foraging mainly in sh MD-migration distance, 1-sedentary/local migrants, 2-medium-distance migrants, 3-lon  The bird species differed in the number of infesting tick species. The greatest number of different species of ticks was collected from E. rubecula (5 out of the 7 identified species of ticks) ( Table 2). Only one of the tick species collected from the birds (i.e., I. frontalis) was strictly ornithophilic (Table 3).

Intensity of Tick Infestation Depending on Bird Behavior
The highest mean infestation intensity (2.05 ticks/bird) was observed in the ground or low plant feeding bird species (type 0), whereas the value of this parameter in the group of birds feeding mainly in shrubs was 1.18 tick/bird (type 1); these differences were statistically significant (p = 0.0047) (Figure 4). Significant differences in the intensity of tick infestation were also observed between sedentary/local migrant birds (type 1) and longdistance migrants (type 3) (p = 0.0058) and between medium-distance migrants (type 2) and long-distance migrants (type 3) (p = 0.0425). In turn, the differences in the intensity of tick infestation between species exhibiting diurnal (1.0 tick/bird), nocturnal (1.94 tick/bird), and nocturnal/diurnal (1.96 tick/bird) migration behavior were not statistically significant (Figure 4). strictly ornithophilic.

Intensity of Tick Infestation Depending on Bird Behavior
The highest mean infestation intensity (2.05 ticks/bird) was observed in the ground or low plant feeding bird species (type 0), whereas the value of this parameter in the group of birds feeding mainly in shrubs was 1.18 tick/bird (type 1); these differences were statistically significant (p = 0.0047) (Figure 4). Significant differences in the intensity of tick infestation were also observed between sedentary/local migrant birds (type 1) and longdistance migrants (type 3) (p = 0.0058) and between medium-distance migrants (type 2) and long-distance migrants (type 3) (p = 0.0425). In turn, the differences in the intensity of tick infestation between species exhibiting diurnal (1.0 tick/bird), nocturnal (1.94 tick/bird), and nocturnal/diurnal (1.96 tick/bird) migration behavior were not statistically significant (Figure 4).

Tick Host Preferences
The collected tick species differed in terms of the host preference index (Ip). The strongest tick-host relationship was observed between T. philomelos, T. merula, E. rubecula, and ticks. In turn, the most frequent tick species collected from birds, i.e., I. ricinus, exhibited a preference for the infestation of T. merula (3.42). The lowest values were recorded for the P. major and I. ricinus pairs (0.06) ( Table 4).

Tick Host Preferences
The collected tick species differed in terms of the host preference index (I p ). The strongest tick-host relationship was observed between T. philomelos, T. merula, E. rubecula, and ticks. In turn, the most frequent tick species collected from birds, i.e., I. ricinus, exhibited a preference for the infestation of T. merula (3.42). The lowest values were recorded for the P. major and I. ricinus pairs (0.06) ( Table 4). Table 4. Index of host preferences of ticks collected from the birds (only bird species with at least 100 captured individuals were included in the analysis).

Bird Species Tick Species
Ixodes spp.  I p < 1-negative interaction; I p = 1-neutral interaction; I p > 1-positive interaction. When no calculation was performed due to the absence of particular tick species on particular bird species, the record was marked with "-".

Ixodes crenulatus
A weak negative but statistically significant correlation was observed between the bird's body weight and the prevalence of tick infestation only in E. rubecula (r s = −0.2563, p = 0.0265). There was no significant correlation between the amount of fat tissue and tick infestation in any bird species.

Discussion
Migratory birds play a significant role in introducing new tick species to areas previously considered tick-free [12][13][14]45], thereby posing a risk of local outbreaks of tick-borne diseases [55,56]. The results of the present study show that I. ricinus is the most frequently collected tick species from birds during their autumn migration (86.56% of all collected ticks) in eastern Poland. It is also the most widespread tick species in Europe [45], and its dominance on migratory birds has been reported in other studies conducted on the continent [7, 15,18,23,25,35,36]. In the present study, we identified tick species that, to the best of our knowledge, had not been reported from eastern Poland (I. persulcatus, I. frontalis, I. acuminatus) or had been reported only sporadically (I. trianguliceps, I. crenulatus, I. apronophorus) [46,57,58]. Among the ticks identified in the current study, I. frontalis and I. ricinus are the most frequent tick species collected from birds [38,46]. I. persulcatus specimens parasitizing birds were confirmed, among others, by studies in Sweden, on birds captured at their stopover sites in Yakutia (Asia) and even from migrating birds in Japan [59][60][61]. In the case of I. acuminatus, the main spectrum of hosts is small mammals of the genera Microtus, Sorex, and Apodemus [46,62], while birds are considered exceptional hosts for this species [48]. In turn, Filippova [63] considered birds to be important hosts, mainly for the I. acuminatus larvae. These observations are also confirmed by the results of studies from Romania [64]. In turn, I. trianguliceps is a hydrophilic tick species that occurs in wet mixed forest habitats. This tick species is mostly found near the burrows of potential hosts, mainly small mammals [47,65]. Such behavior promotes ticks to attack birds that usually forage on the ground or in low parts of plants (foraging type 0 and/or 1) ( Table 2). Feeding of this tick species on birds has also been confirmed [46,47]. Similar host preferences are observed in I. crenulatus and I. apronophorus occurring in river valleys, which are habitats of birds [47].
Particularly noteworthy is the relatively large number of collected larvae (2) and nymphs (10) of I. persulcatus (Table 2), whose compact range covers northeastern Europe [66,67]. In the present study, this species was collected from short-distance (T. merula) and medium-distance (E. rubecula, T. philomelos) migrants (Table 2), who migrated from the north and northeast of the continent to the Mediterranean region in autumn. Considering the feeding time of juvenile stages of ixodid ticks [68] and the distance covered daily by, e.g., E. rubecula [41], it can be suggested that these birds may play a significant role in the introduction of ticks in the eastern Poland region. However, our earlier study on the occurrence of ticks in this region did not confirm the presence of I. persulcatus [27,28]. Due to the northern range of its occurrence and the progressive climate change, this tick species may not find favorable habitat conditions in eastern Poland. Another noteworthy finding is the absence of D. reticulatus, i.e., the dominant tick species in eastern Poland [27,69], on the birds examined in this study. This is most probably related to the host specificity of these ticks. D. reticulatus larvae and nymphs typically infest small rodents [70] and are only sporadically found on birds [56,71].
In contrast to the results reported by other authors [4,72], we identified the presence of only Ixodes ticks on the birds (Table 2, Figure 3). This is probably associated with the location of the region along the birds' autumn migration route from the north and northeast (dominated by I. ricinus and, to a lesser extent, by I. persulcatus) [73] to the south of the continent [41]. In Central Europe, in addition to the dominant Ixodes ticks, birds are most frequently infested by H. marginatum Koch, 1844; Haemaphysalis punctata Canestrini and Fanzago, 1878; Hae. concinna Koch, 1844; and less often by D. reticulatus [22,36,74].
The present study demonstrated the presence of ticks in 11 of the 55 identified bird species (20.00%), and the individuals representing the tick-infested species accounted for 82.09% (3204) of all captured birds (Table 1 and Table S3). Many tick-free bird species were captured in small numbers, excluding any reasoning about the lack of infestation. However, in this group there were also some species represented by a considerable number of individuals, and most of them were the ones foraging mainly in the tree canopy or higher bushes where ticks do not occur, e.g., Cyanistes caeruleus Linnaeus, 1758; Regulus regulus Linnaeus, 1758; Muscicapa striata Pallas, 1764; Periparus ater Linnaeus, 1758; or Fringilla coelebs Linnaeus, 1758 (Table S3). The presence of ticks in 61.11% of captured bird species was reported in studies conducted in west-central Poland, whereas tick infestation was observed in 55.55% of birds captured on the Baltic Sea coast [36,37]. These findings confirm the important role of birds as tick hosts and the involvement of birds in the spread of ticks. Although birds are not considered primary ticks' hosts and the main reservoir of tick-borne pathogens in the environment [70,75], the broad spectrum of infested species increases the risk of transmission of pathogens; hence, birds should be considered an important link in the circulation of tick-borne pathogens.
In the present study, 4.43% of all captured birds were infested by ticks ( Table 1). The lower prevalence of tick infestation than that in other regions of Poland [36,37] is probably associated with the fact that the study was conducted only in autumn (limitation related to coordination with ornithological station dates), while tick infestation rates are usually higher in spring [46,59,76].
The results of the present study indicate the dominance of nymphs (65.37%) over larvae (34.63%) infesting birds (Figure 3) in contrast to findings reported by other authors indicating the predominance of larvae in autumn [36,77,78]. In our opinion, these differences may be related to fluctuations in the seasonal activity of ticks.
The highest ecological index of dominance, the highest mean intensity of tick infestation, and a high host preference index were determined mainly in the case of shortand medium-distance migrants and species foraging on the ground or in low shrubs, i.e., T. merula, T. philomelos, E. rubecula, and S. atricapilla (Tables 1, 2 and 4; Figure 4). Similar dependencies between the tick infestation levels and the distance covered by birds during seasonal migrations were also observed in, e.g., Scandinavia [77,79]. The birds' foraging style should be regarded as an important factor influencing tick infestation intensity, especially bearing in mind that most of the tick species collected in this study are classified as relatively ornithophilic, including I. ricinus [54] (Table 3). Birds foraging on the ground are much more likely to be attacked by juvenile tick stages than those foraging in shrubs ( Table 2). The present study showed ticks attached only to the heads of the birds. The most common location was the region around the eye and beak (Figure 2), which had direct contact with the ground during foraging. Moreover, the head is a safe place for the tick to attach, as it cannot be groomed via the beak or claws [80].
Our results also showed a significant (but weak) negative correlation between bird's body weight and tick infestation intensity in the case of E. rubecula. This relationship may have been related to the fact that parasite infection increases hosts' stress and may lead to a decrease in body mass in strongly infested individuals [1]. In addition, the immune status of the host may also influence the intensity of tick infestation in birds [81]. In addition, there are probably no genetic resistance mechanisms contributing to tick infestation in birds [81,82].

Conclusions
In conclusion, I. ricinus is the most common tick species infesting the examined bird species during their autumnal migration in eastern Poland. Similarly, captured Passeriformes species, such as T. merula, T. philomelos, E. rubecula, and S. atricapilla, should be regarded as the preferred tick hosts. They play an important role in the spread of rare tick species in this region. Avian ecology and ethology, foraging styles, and length of migration distance have an impact on the exposure of birds to tick attacks and indirectly contribute to the maintenance and circulation of tick-borne pathogens in the environment.