Introduction

Hard ticks (Acari: Ixodidae) are common carriers of pathogens that can affect both humans and animals, so it is not surprising that they are considered one of the world’s most important arthropod vectors1. Because of this, research on their ecology and distribution is of utmost importance2. This is especially true nowadays, as ecological systems are transforming rapidly due to climate change3.

The role of birds in the dissemination of ticks is unquestionable. Migratory birds can transport ticks between continents, for instance from Africa into Europe4. Thermophilic tick species, most notably from the genus Hyalomma, have long been considered adventitious, but in recent years these species have been reported in Europe with increasing numbers north of the Mediterranean Basin, and were even able to establish a resident population5,6,7,8. In this context, the importance of synanthropic, resident bird species (e.g. the Blackbird (Turdus merula)) is also crucial, regarding the local dissemination of ticks, as they can introduce ticks to urban areas9. The monitoring of bird-tick relationships is a long-standing and intensively researched field that has greatly contributed to what is known about ticks and the epidemiology of the pathogens they transmit10,11,12.

Research on this topic has been conducted in Hungary for decades, particularly at Ócsa Bird Ringing Station8,13,14,15,16,17. At this station more than 15 thousand birds are caught yearly. The area has several different habitat types (e.g. forest, arable field, reedbed)18 and is an important stop-over site for birds migrating along the Adriatic Flyway through Central Europe. Therefore, it is suitable for the examination of both migratory and resident birds of different habitats.

In order to evaluate factors that influence the association of ixodid ticks with avian hosts, ticks were collected from birds at Ócsa Bird Ringing Station between March 2015 and November 2022. The data collected over the course of eight years ensure a better understanding of ecological and ornithological factors that influence the epidemiological significance of birds as tick carriers.

Results

Identification of tick species and their occurrence on infested birds

During the study period 2395 tick-infested birds were captured, belonging to 51 different species, and a total of 5833 ticks were collected. The most frequent host species were the European Robin (Erithacus rubecula) (ninfested = 521, ncaptured = 14,809), followed by the Blackbird (T. merula) (ninfested = 359, ncaptured = 1525). The mean number of ticks found on infested birds (i.e., intensity of infestation) was 2.44, while the median intensity was 1.

Based on morphological characteristics, eight tick species were identified, namely Ixodes ricinus, Ixodes frontalis, Ixodes lividus, Ixodes festai, Ixodes arboricola, Haemaphysalis concinna, Haemaphysalis punctata and Dermacentor reticulatus (Table 1). Concerning molecular identification of Hyalomma species, one nymph showed 100% identity to sequences of Hyalomma rufipes reported in Africa and in Malta (cox1 gene: OQ540949 from Kenya; 12S rRNA gene: OL352890 from Malta, 16S rRNA gene: MK737649 from Egypt). Two further nymphs proved to be Hyalomma marginatum, one of them with 100% sequence identities to ticks reported from the eastern-middle-western Mediterranean region (cox1 gene: LC508365 from Portugal; 12S rRNA gene: OL352894 from Malta; 16S rRNA gene: KT391060 from Israel) and another with 99.8–100% sequence identities to specimens reported from the western Mediterranean region (cox1 gene: LC508365 from Portugal; 16S rRNA gene: LC508322 from Portugal).

Table 1 Total number of tick species and stages found during the study (2015–2022).
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Altogether, 1872 (78.16%) of the captured, tick-infested birds carried I. ricinus, while 592 (24.72%) of them carried Ha. concinna. Ixodes frontalis was present on 51 birds (2.13%). The remaining seven tick species were carried by a combined total of 13 (0.54%) birds. One hundred thirty-one birds carried two tick species, and on one Song Thrush (Turdus philomelos) three different tick species were present simultaneously (I. ricinus, I. frontalis and Ha. concinna).

The 2395 tick-carrying birds captured during this study are summarized in Table 2, according to the characteristics that were examined. In the table, we also indicated how many bird species belonged to the given category. More detailed information can be found in Supplementary Tables 2 and 3.

Table 2 Total number of birds, and bird species found to be tick-infested between 2015 and 2022, according to their migration habit, habitat and feeding place.
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Number and temporal occurrence of tick species, their host associations according to habitat and migration characteristics

All tick-host associations are listed in Supplementary Tables 1 and 2 and visualized in Fig. 1 and Supplementary Fig. 1 Statistical analyses on ticks and their hosts according to their habitat and migration characteristics were only calculated for the most abundant tick species (I. ricinus and Ha. concinna), due to the fact that the limited numbers of other tick species precluded robust statistical analyses.

Figure 1
figure 1

Tick-host associations visualized on a plotweb.

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Ixodes ricinus (ntotal = 3971, nlarvae = 1229, nnymphs = 2742): Ixodes ricinus subadults occurred most frequently on resident and/or short-distance migrating birds with forest or forest and meadow habitats (Tables 3, 4), This category includes the two species on which the most ticks were found: Blackbirds (nticks = 1073, nbirds = 326), and European Robins (nticks = 924, nbirds = 490) (Table 5). Ixodes ricinus ticks were present on birds year-round, with nymphs reaching their peak in the first half of the sampling periods (1st of March–30th of June), while larvae were the most abundant in the second half (1st of July–31 of October). This was consistent over the course of eight years. The difference between the numbers of nymphs and larvae regarding their half-yearly activity was significant (p < 0.0001) (Supplementary Table 1, Supplementary Fig. 2).

Table 3 Number of ticks according to the typical habitat of their avian hosts.
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Table 4 Number of ticks according to the migration habit of their avian hosts .
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Table 5 The five most common avian hosts of Ixodes ricinus (A) and Ha. concinna (B).
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Ixodes frontalis (ntotal = 102, nlarvae = 52, nnymphs = 38, nfemale = 12): The European Robin was the most common host of this tick species (nticks = 52, nbirds = 21). The second most frequently identified host species was the Blackbird (nticks = 18, nbirds = 4). Ixodes frontalis occurred most frequently on resident and/or short-distance migrating birds with forest habitat (Tables 3, 4). All stages of I. frontalis were the most abundant in the second half of March every year, during the eight-year-long study period (Supplementary Fig. 3).

Ixodes lividus (ntotal = 13, nfemales = 12, nnymphs = 1): All 13 I. lividus ticks were collected from four Sand Martins (Riparia riparia).

Ixodes festai (ntotal = 8, nfemales = 6, nmales = 2): In total eight ticks of this species were collected. Five ticks were removed from two Blackbirds and three ticks from two Dunnocks (Prunella modularis). It is important to mention, that the two males collected were not feeding, but were in copulation with females.

Ixodes arboricola (ntotal = 1, nnymphs = 1): Only one nymph was collected. It was removed from a Great Tit (Parus major).

Haemaphysalis concinna (ntotal = 1706, nlarvae = 698, nnymphs = 1008): The majority (933 ticks, 54.7%) of individuals of this tick species were found on Savi’s Warbler (Locustella luscinioides) (nbirds = 238), making this bird the most common host of Ha. concinna. This tick species was also frequently collected from Eurasian Reed Warbler (Acrocephalus scirpaceus) (nticks = 157, nbirds = 82). The five most common hosts of Ha. concinna are listed in Table 5. Both Ha. concinna larvae and nymphs were active in the summer. However, the peaks of their abundance showed differences over the study period. In particular, the peak of infestation of birds with Ha. concinna larvae preceded that of its nymphs in 2015 and 2022, but the opposite trend was observed in 2017 and 2018. In other years, the peak larval and nymphal infestations occurred simultaneously (Supplementary Fig. 2).

It was found that the host habitat (reed, forest, meadow, meadow/forest) of I. ricinus and Ha. concinna was significantly different (p < 0.0001) (Table 3). We also compared the migratory habits (resident, short-distance migrants, middle-distance migrants, resident or short distance migrants, resident or middle-distance migrants, long-distance migrants) of the hosts of I. ricinus and Ha. concinna. The difference was strongly significant (p < 0.0001) (Table 4). According to our findings, Ha. concinna occurred most frequently on long-distance migrant and reed-associated birds, while I. ricinus was the most abundant on short-distance migrants, with forest habitat. (Tables 3, 4).

Haemaphysalis punctata (ntotal = 28, nlarvae = 28): A single Common Quail (Coturnix coturnix) was found with 28 feeding larvae.

Hyalomma marginatum (ntotal = 2, nnymphs = 2): One engorged nymph was collected from European Robin, and another from Song Trush (T. philomelos). Both were collected in the first half of April (in 2015, and 2016, respectively).

Hyalomma rufipes (ntotal = 1, nnymph = 1): Only one engorged nymph was found, which was feeding on a European Pied Flycatcher (Ficedula hypoleuca) in the second half of April 2015.

Dermacentor reticulatus (ntotal = 1, nfemale = 1): One female of this species was found on a Blackbird (Turdus merula), though it had not started to feed.

Host associations of ticks according to bird weight and feeding level characteristics

The mean intensity of infestation with I. ricinus nymphs was the highest among bird species with typical body weight above 100 g, whereas this was the lowest among bird species measuring below 10 g (Supplementary Fig. 4). The same was not true in the case of Ha. concinna nymphs, because the mean intensity of tick-infestation was the highest on birds weighing between 10.1 and 20 g (Supplementary Fig. 4).

When the numbers of I. ricinus, I. frontalis and Ha. concinna  were compared according to the feeding level of their hosts (only ground level or above ground categories were tested), I. ricinus tended to predominate on ground-feeding birds, whereas Ha. concinna nymphs and larvae were more often found on birds looking for food items above the ground level (p < 0.0001). On the other hand, the difference was not significant between I. ricinus and I. frontalis (p = 0.2584) (Table 6).

Table 6 Number of ticks according to the typical feeding place of their avian hosts.
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Discussion

Although there are many similar previous reports from different countries (e.g.19,20,21,22,23,24), this study presents one of the longest continuous bird tick collections in Europe. Over an eight-year-long period, 5833 ticks of 10 species were collected from 2395 birds of 51 species. The dataset itself provides a valuable contribution to the field due to its size.

The two most abundant tick species collected were I. ricinus (ntotal = 3971) and Ha. concinna (ntotal = 1706). Only subadult stages were found in case of both species. Ixodes ricinus has long been known as the most common tick species that feeds on birds in the Palearctic region12 and is primarily a forests-dwelling tick species25. This fact was confirmed by data presented here, as I. ricinus occurred most frequently on birds with forest habitat. Interestingly, this was not true for Ha. concinna. According to the literature data, Ha. concinna can be found in a broad range of different habitats, mainly moist, wooded ecosystems, but in reedbeds as well25,26,27. However, according to our data, this species mainly parasitized reed-associated birds (1333 Ha. concinna ticks on birds with reed habitat). The difference between the host habitats of I. ricinus and Ha. concinna was strongly significant (p < 0.0001). Furthermore, 933 Ha. concinna ticks were collected from only one bird species, Savi’s Warbler. According to Csörgő et al.28 number of ringed Savi’s Warblers present in Hungary between 1951 and 2006 was 32 083 birds. Interestingly the same numbers of the second and third most common hosts from Ócsa (Eurasian Reed Warbler and Sedge Warbler (Acrocephalus schoenobaenus)) were over 220 000 for each species. Savi’s Warbler had the highest level of median infestation in the case of nymphs, and the second highest in the case of larvae. In our opinion however, larvae are not the greatest indicators in this regard, as a low presence of larvae can be overlooked easily due to the small size of the parasites.

Regarding the migration habit of the hosts of Ha. concinna, and I. ricinus, the results were in line with our previous observation8 on a strongly significant difference between the hosts of the two tick species: Ha. concinna most commonly occurred on long-distance migrants, whereas I. ricinus was most frequently collected from short-distance migrant or resident birds. Haemaphysalis concinna is a thermophilic tick species. Its larvae and nymphs have a similar period of peak activity (mainly the summer)27 and its typical avian hosts are reed-associated long-distance migrants28.

Among the latter, Savi’s Warbler was the most common host in Hungary. According to our hypothesis, the feeding habit of this bird species may explain this phenomenon. Savi’s Warbler shares a very similar ecological niche with Ha. concinna, because it mainly feeds on small invertebrates near the water surface in reedbeds. This behavior is consistent during the migration period28, and lakeshore vegetation was reported to be a preferred habitat type of Ha. concinna in Central Europe27.

Taken together, Ha. concinna subadults apparently share a much more similar ecological niche with Savi’s Warbler, than with other reed-associated songbirds. It is important to mention that this difference is only true among avian hosts and does not extend to mammals and reptiles, which are also favored hosts of Ha. concinna 27, especially roe deer29.

It was previously reported, that birds with larger body mass carry a higher number of I. ricinus nymphs30. In this study, the mean intensities of infestations with I. ricinus and Ha. concinna stages were categorized according to the average body mass of their avian hosts. In the case of I. ricinus, the mean intensity of nymphs showed a trend of increase with the average body mass of the hosts, unlike in the case of Ha. concinna nymphs. However, this could not be supported by the results of statistical analyses, because of the following reasons. First, the majority of Ha. concinna ticks were carried by Savi’s Warblers, which is a small size bird species. Second, I. ricinus occurred most frequently on ground feeding birds which in general have a larger body mass (e.g., Blackbirds). On the other hand, data on the body mass of bird individuals examined for tick-infestation in the present study were not available, and this parameter is known to change significantly even within the same bird species between different seasons.

The temporal distribution of I. ricinus and Ha. concinna developmental stages was also analyzed. Ixodes ricinus nymphs always reached their peak infestation on birds during the first half of the annual sampling periods throughout the study, whereas larvae reached their highest abundance from July to October. This is likely a consequence of the prolonged development of I. ricinus which takes several years in Central Europe31 implying that nymphs and larvae collected in the same year belonged to different generations. By contrast, the nymphal and larval peak activities of Ha. concinna were always close to each other, i.e., within 0.5–1.5 month over the summer period. This may reflect that a notable portion of these developmental stages belonged to the same generation, particularly when the larval peak preceded the nymphal peak (e.g., in 2015, 2022). This is in line with observations that under temperate climate this tick species can complete one generation in one year if hosts are available32 On the other hand, Ha. concinna larvae were also found on birds as early as March and April (e.g., in 2016 and 2017), suggesting overwintering in the larval stage and a generation time longer than one year27.

During the eight-year-long study period, 102 I. frontalis ticks were collected (nlarvae = 52, nnymphs = 38, nfemale = 12). According to the known literature data, I. frontalis reaches its peak in March and in November33,34. In this study, the vast majority of all developmental stages were collected in the second half of March. The absence of a peak in November is likely because the main sample collection period was finished at the end of October/beginning of November each year. From November tick collections, there are only a scarce and random amount of data. The fact that this tick is the most abundant at the beginning of spring and the end of autumn explains why I. frontalis showed association with resident and/or short-distance migrant birds according to our data (Table 4). Ixodes frontalis was almost exclusively found on birds with forest- or mixed meadow/forest habitats (98/102) (Table 3).

Altogether 8 specimens of I. festai, six females and two males were also collected during the study period. All hosts were Blackbirds and Dunnocks. The same host species were found to be infested in a previous study from another part of Central Europe, Switzerland 35, but literature data are available from multiple other host species as well 12.

Three Hyalomma nymphs were found in total. According to our molecular analyses, two were Hy. marginatum and one was Hy. rufipes. Each of these nymphs had 100% sequence identity with at least one specimen reported from the mid-Mediterranean region (Malta), situated along the Adriatic Flyway crossing Hungary toward the north. Thus, these data are highly relevant to their probable geographical origin. Hyalomma species are important vectors of sevedunral different pathogens, including the Chrimean-Congo haemorrhagic fever virus25. All Hyalomma ticks were collected in April (2015, 2016). Findings of Hyalomma subadults during the spring migration period are not uncommon in Central Europe36 and in Ócsa16. It is important to note, however, that the monitoring of Hyalomma-carrying birds is of great epidemiological importance.

Thirteen I. lividus were identified. This tick is the host-specific parasite of Sand Martin, that feeds extremely rarely on other birds12,25,37. It is not surprising that we have found all I. lividus ticks on Sand Martins. Ixodes lividus have been reported from Hungary long before our study38.

Twenty-eight Ha. punctata larvae were found feeding on a single Common quail. Haemaphysalis punctata has been already known in Hungary, as an uncommon parasite on birds 39.

Only one I. arboricola nymph was collected during this study. This low number is not surprising, since I. arboricola is a nidicolous tick that feeds on nestlings during the summer, and therefore adult birds are mostly infested during winter seasons, when roosting 40. For this study, sample collection period started in March and ended at the beginning of November each year so did not include the peak season for I. arboricola.

Lastly, one D. reticulatus female on a Blackbird was found in 2022. This tick was not (yet) feeding, and its occurrence is believed to be accidental, though D. reticulatus subadults have been found on Blackbirds (among other birds) before 41.

Conclusions

Based on this 8-year survey, the migration distance, the habitat type, and the feeding habit of birds, as well as the seasonal activity of ticks are all important factors determining the role of birds as tick disseminators. Haemaphysalis concinna was the most abundant on long-distance migrant, reed-associated, above-ground feeder birds, in contrast to I. ricinus, which predominated on resident or short-distance migrant, ground-feeder birds with forest or meadow habitat. In the study region, Savi’s Wabler (L. luscinioides) was by far the most common avian host of Ha. concinna larvae and nymphs.

Methods

Sample collection

Sample collection took place from March 2015 to November 2022 at Ócsa Bird Ringing Station (N47.2970, E19.2104), approximately 33 km south of Budapest. Ócsa is situated in a humid continental transitional climate zone. Summers are medium warm and dry, with relatively cold winters. The annual average temperature is 10.1 °C (minimum ca. − 15.6 °C, maximum ca. 34.1 °C). Annual precipitation is around 550–580 mm. Annual sunshine is ca. 2000 h. The mean elevation is 100 m above sea level18. There are a large variety of different habitats in the area of the station: it is surrounded by arable fields, but forests, shrubs, and garden yards, can also be found here. Due to the fact, that Ócsa is situated on the edge of a wetland, open water surfaces and reedbeds are also common18.

Birds were mist-netted for ringing by standard ornithological mist-nets (mesh size 16 mm). During the ringing procedure, birds were examined for the presence of ticks. Ticks were removed with the help of pointed tweezers and were placed and stored in 96% ethanol. Bird ringing and tick collection were constant from the beginning of March to the end of October each year. Only sporadic data were obtained during November and December. During 2020 and 2021, due to COVID-19 restrictions, bird ringing activity was reduced and tick collections were limited. Birds were handled, identified, and released by professional ringers throughout our study. Only data about tick-infested birds were recorded. Some data from 2022 were used for another study in a different context 8.

Morphological identification

Ticks were identified with a stereomicroscope (SMZ-2 T, Nikon Instruments, Japan, illuminated with model 5000-1, Intralux, Switzerland). To identify Ixodes ricinus, I. frontalis, I. lividus, I. arboricola, and Dermacentor reticulatus to the species level and Hyalomma species to the genus level, we used morphological keys provided by Estrada-Peña et al.25 Hyalomma ticks were identified to the species level with molecular methods8. Differentiation of subadults of Haemaphysalis concinna and Ha. punctata was based on the morphological keys by Filippova42. For the identification of I. festai, we used the manuscripts of Contini et al.43 and Hornok et al.14.

DNA extraction and molecular identification of Hyalomma species

These steps were carried out as in Keve et al.8. Purification and sequencing of the PCR products were done by Biomi Ltd. (Gödöllő, Hungary). Quality control and trimming of sequences were performed with the BioEdit program, then alignment with GenBank sequences by the nucleotide BLASTN program (https://blast.ncbi.nlm.nih.gov). New sequences were submitted to GenBank (cox1 gene: OR145129–OR145131, 16S rRNA gene: OR145132–OR145134, 12S rRNA gene: OR145138–OR145139).

Data curation and statistical analysis

All tick-host associations can be found in Supplementary Table 1. Birds were categorized according to their feeding place, minimum and maximum body mass, migration habits, and habitats according to ornithological data and previous reports14,15,28 (Supplementary Table 3). In order to categorize birds according to their feeding places, “Above ground” category was created. Birds belonging to this group are feeding on (e.g.) reed trunks, bushes, or branches that do not touch the ground directly but are not far from it either. For this categorization, the expertise of our co-authors (TCS and DK) were used, as well as the available literature data28.

Data curation and calculation of mean and median tick intensity was done in Microsoft Office Excel. Mean and median intensities were calculated for each tick and developmental stages according to Reiczigel et al.44. For comparing the half-yearly activity of I. ricinus larvae and nymphs, and the migration habits and the habitats of the hosts of tick species (I. ricinus, Ha. concinna), chi-squared tests were used. Chi-squared test was also used to compare feeding places of hosts of I. ricinus and Ha. concinna. The comparison of the feeding places of the hosts of I. ricinus and I. frontalis was done with Fisher’s exact test (R -program 4.3.0.). Results were considered significant if p < 0.05.

The average body mass of each bird species was calculated as the mean of the minimum and the maximum body mass registered and listed in Supplementary Table 3. \((\mathrm{Average\, body\, mass}\hspace{0.17em}=\hspace{0.17em}\frac{\mathrm{Minimum\, body\, mass}+\mathrm{Maximum\, body\, mass}}{2}\)).

The mean intensity of Ha. concinna and I. ricinus infestation was calculated for each group. The results are shown in Supplementary Fig. 4. Intensity was only calculated, if there were 10 or more infested birds in the respective category, to minimize the distortion caused by outliers. Data from one group (≤ 10g Average body mass for Ha. concinna) was therefore excluded (nbirds = 2).

In Supplementary Fig. 2, Relative, semimonthly numbers (RSN) were calculated as follows:

$${\text{RSN}}=\frac{{\text{SMN}}}{{\text{MON}}}\times 100$$

(SMN = Semimothly number of the tick species and stage; MON = number of the tick species and stage from the respective year, between March 01 - October 31).

English bird species names are capitalized following international recommendations (https://bou.org.uk/britishlist/bird-names/). In our figures and tables, HURING codes are used instead of the complete bird names. These abbreviations are clarified in Supplementary Table 3.

Ethical approval

The study was carried out according to the national animal welfare regulations (28/1998). License for bird ringing was provided by the National Inspectorate for Environment and Nature (No 14/3858-9/2012.). License for sample collection was provided by the Central Danube Valley Environmental Protection and Nature Conservation Inspectorate (KDV-KTF) (No KTF:27251-1/2014.). Where applicable, the ARRIVE guidelines were followed.