Tick Dispersal and Borrelia Species in Ticks from Migratory Birds: Insights from the Asinara National Park, Sardinia, Italy

Abstract

Rapid environmental changes driven by human activities are contributing to a significant decline in global biodiversity, with avian species being particularly affected due to their migratory behavior. As highly mobile hosts, birds facilitate the geographic dispersal of ectoparasites, including ticks, which serve as vectors for numerous zoonotic pathogens. This study, conducted in collaboration with the Faunistic Observatory of the Asinara National Park between 2021 and 2023, aimed to investigate the potential role of migratory birds in tick dispersal and the presence of Borrelia spp. DNA. Birds were captured using mist nets during pre-breeding (April–May) and post-breeding (October–November) migration periods. Ticks were systematically collected and identified at the species level, and molecular analyses were performed using real-time and conventional PCR to detect the presence of Borrelia spp. DNA. Results showed a distinct seasonal variation in tick species composition. In autumn, Ixodes ricinus was predominant (99%), whereas Hyalomma species were more frequently observed in spring (78%). Molecular screening revealed Borrelia spp. DNA in 26.1% of the collected ticks, with Borrelia garinii being the most prevalent species. These findings underscore the ecological significance of migratory birds in the dissemination of ticks and tick-borne pathogens, highlighting their potential role in shaping disease transmission dynamics across different geographic regions. This study provides valuable insights into the seasonal fluctuations in tick populations associated with migratory avifauna and the epidemiological risks posed by these interactions. Continued surveillance of migratory birds as vectors of zoonotic pathogens is essential for informing public health strategies and mitigating the risks of emerging infectious diseases, but further investigation is needed to clarify the actual role of migratory birds in the transmission of Borrelia spp.

1. Introduction

In recent years, the incidence of vector-borne infectious diseases (VBDs) transmitted by arthropod vectors has been rising, posing a significant global public health challenge that affects both human and veterinary medicine [1]. The VBDs are driven by a complex interplay of environmental and socio-economic factors [2]. Key contributors include climate change, globalization, the intensification of trade in live animals and animal-derived products, shift in agricultural practices, and urbanization, all of which facilitate the introduction and establishment of vector populations in new geographical regions. This, in turn, exacerbates the spread of zoonotic diseases associated with these vectors [3].

According to the World Health Organization (WHO), VBDs account for over 17% of all infectious diseases in humans and animals, with more than half of the global population is at risk of infection [4]. These diseases affect a wide range of environmental landscapes, ranging from rural to urban and peri-urban settings, often disproportionately affecting socioeconomically disadvantaged communities [2]. In Europe, approximately 77,000 cases of VBDs are reported annually, while in Italy, the number of notifiable VBDs is increasing. In Sardinia, rickettsioses belonging to the Mediterranean Spotted Fever (MSF) group remain endemic, with an incidence rate of 10 cases per 10,000 inhabitants per year (ISS/2016) [5,6]. The mechanisms by which arthropod vectors overcome geographic barriers have become a crucial research area. Migratory birds play a fundamental role in the ecology of ectoparasites and the dissemination of pathogenic microorganisms. Each year, billions of birds undertake long-distance migrations, utilizing various stopover sites for feeding and resting [7]. These locations serve as hotspots for the attachment and detachment of ectoparasites, thereby establishing new natural foci for disease transmission. As highly mobile hosts, migratory birds facilitate the spread of pathogens beyond their original geographic location [8].

The role of migratory birds in the epidemiology of VBDs remains an active field of study. Researches have demonstrated their involvement in the transmission of several pathogens, including avian influenza, West Nile virus, and Borrelia burgdorferi, the causative agent of Lyme disease [9,10]. Furthermore, the physiological stress associated with long-distance migration may compromise immune function, increasing birds’ susceptibility to infections and enhancing their potential as reservoirs and dispersers of pathogens [11].

Despite advances in molecular or immunological diagnostics, VBDs remain underdiagnosed, complicating epidemiological assessment. Therefore, the identification of VBDs in new geographic areas must be accompanied by a comprehensive understanding of their ecological and public health implications.

Currently, data on the role of migratory birds in the circulation of vector-borne pathogens in Sardinia are scarce. This study aims to enhance knowledge regarding the distribution of ectoparasites in migratory birds traveling through the island along their seasonal routes between sub-Saharan Africa and northern Europe. By elucidating the role of migratory birds in pathogen dissemination, this research will contribute to a more comprehensive understanding of VBD epidemiology in the region.

2. Materials and Methods

2.1. Ethical Statement

All procedures related to bird capture and handling followed ethical guidelines for wildlife research and adhered to national and international regulations for animal welfare. This study did not involve any invasive procedures, and captured birds were carefully handled, measured, and released immediately after data collection. The research group was trained in ethical handling techniques.

2.2. Bird Capture and Study Area

Birds were captured for ringing at the Faunal Observatory of Asinara National Park (Figure 1) during the periods of April–May and October–November 2021, corresponding to the spring and fall migration seasons. Birds were captured using mist nets placed at ground level. After capture, they were identified based on morphological features, and when possible, their sex and age were determined. Each bird was then fitted with a uniquely coded ring for individual identification, enabling precise tracking of migration patterns and potential recaptures.

Asinara Island, located in the northwestern part of Sardinia, covers an area of 50.90 km², making it the fifth largest island in Italy (excluding Sicily and Sardinia) and the third largest in Sardinia. The island features a hilly landscape with peaks reaching 408 m, a rugged coastline, and mainly shrubland vegetation, with sparse holm oaks and low scrub. As part of Italy’s national park network, Asinara is a wildlife and marine reserve. The coastline is varied, with steep western cliffs and a lower eastern coast with several beaches. The island contains artificial freshwater basins and a landscape composed mainly of schist, with some granite formations.

2.3. Ectoparasite Sampling

Each captured bird was carefully examined, and the plumage was blown to inspect for the presence of ticks. Any ticks found, particularly on the head and neck regions, were carefully removed using fine forceps and placed into plastic vials for transport to the Entomology Laboratories at the Istituto Zooprofilattico Sperimentale (IZS) in Sassari for further analysis. Tick species identification followed the methods described by Estrada- Peña et al. [12]. They were classified by developmental stage (larvae, nymphs, or adults) and engorgement status (fed or unfed). Molecular analysis, including DNA sequencing, was then performed to confirm species identification.

2.4. DNA Extraction

DNA was extracted from individual ticks using the Mag-Max automatic extractor Express96 (Applied Biosystems, Foster City, CA, USA) in 96-well plates. Prior to extraction, the ticks were homogenized using 5 mm tungsten beads in 200 µL of phosphate-buffered saline (PBS) in a TissueLyser (TissueLyser II, Qiagen Inc., Hilden, Germany) for 4 min at 30 Hz. The homogenate was then mixed with 140 µL of Buffer ATL and 20 µL of Proteinase K, followed by DNA extraction according to the manufacturer’s protocol. The extracted DNA was quantified using a NAnoDrop^TM spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then stored at −20 °C until use.

2.5. Tick Molecular Identification

Molecular identification of ticks was performed by amplifying the mitochondrial 16S rDNA gene. The gene fragment was amplified via PCR analysis using primer sequences listed in

Table 1. Sequences of primers and probes used in rt-PCR and PCR amplification assays for detection of Borrelia spp. using 16S rRNA and FLA gene targets.

Pathogen	Gene Target	PCR Assay	Primer/Probe Sequence	Reference
Borrelia spp.	16S rRNA	PCR	16S+1_F 5′-CTGCTCAATGATTTTTTAAATTGCTGTGG-3′	[13]
			16S+1_R 5′-CCGGTCTGAACTCAGATCAAGT-3′
	16S rRNA	rt-PCR	16S For 5′-GGATATAGTTAGAGATAATTATCCCCGTTTG-3′	[14]
			16S Rev 5′-CATTACATGCTGGTAACAGATAACAAGG-3′
			16S Borr Probe 6FAM-CAGGTGCTGCATGGT-BHQ1
	FLA	PCR	FLA1 F 5′-AGAGCAACTTACAGACGAAATTAAT-3′	[15]
			FLA2 R 5′-CAAGTCTATTTTGGAAAGCACCTAA-3′

.

2.6. Molecular Detection of Borrelia spp. and Sequencing

The extracted DNA was screened for the presence of Borrelia species using TaqMan Real-Time PCR assays. Additionally, conventional PCR was performed to amplify a 482-bp fragment of the flagellin gene (FLA) of Borrelia spp. The sequences of the primers are listed in Table 1.

Following amplification, sequencing was performed on both FLA-positive samples to confirm Borrelia detection and 16S rRNA gene sequences to determine tick species using the Big Dye Terminator Kit (dRhodamine Terminator Cycle Sequencing Ready Reaction; Applied Biosystems) following the manufacturer’s protocol. Separate reaction mixtures were prepared for forward and reverse sequencing. The obtained sequences were aligned using ChromasPro software (version 1.34; Technelysium Pty Ltd., Tewantin, QLD, Australia), and the consensus sequence was compared against the GenBank database using BLASTn tool [16]. Chromatograms were carefully examined for misreads or ambiguous base calls. Only high-confidence sequences with ≥98% identity were included in the dataset. For comparative sequence analysis, CLUSTALW 2.1 software [17] was utilized to generate pairwise and multiple sequence alignments, as well as determining sequence similarities. Meanwhile, the BioEdit platform [18] was used to construct sequence identity matrices.

Phylogenetic analysis was performed using the Maximum Likelihood method and Kimura 2-parameter model [19] for nucleotide substitutions. The tree with the highest log likelihood (−837.86) is shown (Figure 2). The percentage of replicate trees in which the associated taxa clustered together (1000 replicate) [20]. The initial tree for the heuristic search was selected by choosing the tree with the superior log-likelihood between a Neighbor-Joining (NJ) and a Maximum Parsimony (MP) tree. The NJ tree was generated using a matrix of pairwise distances computed using the p-distance [4]. The MP tree had the shortest length among 10 MP tree searches, each performed with a randomly generated starting tree. The evolutionary rate differences among sites were modeled using a discrete Gamma distribution across 8 categories (+G, parameter = 0.1477). The analytical procedure encompassed 19 coding nucleotide sequences using 1st, 2nd, 3rd, and non-coding positions. The partial deletion option was applied to eliminate all positions with less than 95% site coverage resulting in a final data set comprising 385 positions. Evolutionary analyses were conducted in MEGA12 [21] utilizing up to 6 parallel computing threads.

2.7. Sequence Accession Numbers

Sequences of Borrelia FLA genes were deposited in the GenBank using the National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) BankIt v3.0 submission tool (http://www.ncbi.nlm.nih.gov/BankIt/, accessed on 3 February 2025).

3. Results

3.1. Analysis of Bird Species and Ixodida Distribution

A total of 961 ticks (862 in fall and 99 in spring) were collected from 461 birds (405 in fall and 56 in spring). Information about the migratory routes and the timing of migration for the different avian species from this study are provided in

Table 2. Migratory routes and timing of the bird species in this study.

Bird Species (Scientific Name)	Migration Type	Breeding Range	Wintering Range	Migration Timing
European Robin (Erithacus rubecula)	Short to medium distance	Continental Europe, east to Ural Mountains, northwestern Africa	Western and southern Europe, northern Africa	Southward in autumn, northward in spring
Common Blackbird (Turdus merula)	Short distance/partial migrant	Europe, North Africa, western Asia	Some populations winter in southern Europe and North Africa	Partial migration; northern populations migrate south in autumn
Song Thrush (Turdus philomelos)	Medium distance	Europe, western Siberia	Southern Europe, North Africa, Middle East	Migrates south in autumn, returns in spring
Woodlark (Lullula arborea)	Short distance/resident	Europe, North Africa	Southern Europe, North Africa	Some move south in autumn, others remain resident
Blackcap (Sylvia atricapilla)	Medium distance	Europe, western Asia	Southern Europe, North Africa	Migrates south in autumn, returns in spring
Garden Warbler (Sylvia borin)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Common Redstart (Phoenicurus phoenicurus)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Little Owl (Athene noctua)	Resident	Europe, North Africa, Asia	Resident	Non-migratory
Willow Warbler (Phylloscopus sibilatrix)	Long distance	Europe, western Siberia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Pied Flycatcher (Ficedula hypoleuca)	Long distance	Europe	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Common Whitethroat (Sylvia communis)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Chiffchaff (Phylloscopus collybita)	Medium to long distance	Europe, western Asia	Southern Europe, North Africa	Migrates south in autumn, returns in spring
Nightingale (Luscinia megarhynchos)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Redstart (Saxicola rubetra)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Little Owl (Athene noctua)	Resident	Europe, North Africa, Asia	Resident	Non-migratory
Willow Warbler (Phylloscopus sibilatrix)	Long distance	Europe, western Siberia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Pied Flycatcher (Ficedula hypoleuca)	Long distance	Europe	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Common Whitethroat (Sylvia communis)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Chiffchaff (Phylloscopus collybita)	Medium to long distance	Europe, western Asia	Southern Europe, North Africa	Migrates south in autumn, returns in spring
Nightingale (Luscinia megarhynchos)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Redstart (Saxicola rubetra)	Long distance	Europe, western Asia	Sub-Saharan Africa	Migrates south in autumn, returns in spring
Tyrrhenian Spotted Flycatcher (Muscicapa striata tyrrhenica)	Short to medium distance	Western Mediterranean islands	Southern Europe, North Africa	Migrates south in autumn, returns in spring
Magpie (Pica pica)	Resident	Europe, Asia, North Africa	Resident	Non-migratory
Southern Grey Shrike (Lanius senator)	Long distance	Southern Europe, North Africa	Sub-Saharan Africa	Migrates south in autumn, returns in spring

.

The European Robin (Erithacus rubecula) was the most frequently captured bird in the fall, both in number of captures and ticks. It accounted for 301 birds (74.3% of fall captures) and 579 ticks (67% of fall ticks). The Common Blackbird (Turdus merula) was the second most captured species, with 54 birds (13.3% of fall captures) and 188 ticks (21.8% of fall ticks). The Song thrush (Turdus philomelos) accounted 34 birds (8.4% of fall captures) and 68 ticks (8% of fall ticks). In spring, the Common Redstart (Phoenicurus phoenicurus) was the most prevalent species, representing 44.6% of spring captures, and it had the highest tick burden accounting for 55% of spring ticks. The Willow Warbler (Phylloscopus sibilatrix) and Pied Flycatcher (Ficedula hypoleuca) each had seven birds (12.5% of spring captures), with 11 and 8 ticks, respectively (see

Table 3. Seasonal trends in bird captures and ticks collected across different species.

Season	Bird Species (Scientific Name)	Number of Birds Captured (n°/%)	Number of Ticks (n°/%)
Fall	European Robin (Erithacus rubecula)	301 (74.3%)	579 (67%)
	Common Blackbird (Turdus merula)	54 (13.3%)	188 (21.8%)
	Song Thrush (Turdus philomelos)	34 (8.4%)	68 (8%)
	Woodlark (Lullula arborea)	7 (1.73%)	17 (2%)
	Blackcap (Sylvia atricapilla)	4 (1%)	4 (0.5%)
	Garden Warbler (Sylvia borin)	2 (0.5%)	2 (0.2%)
	Common Redstart (Phoenicurus phoenicurus)	2 (0.5%)	3 (0.35%)
	Little Owl (Athene noctua)	1 (0.3%)	1 (0.15%)
	Total Fall	405	862
Spring	Common Redstart (Phoenicurus phoenicurus)	25 (44.6%)	54 (55%)
	Willow Warbler (Phylloscopus sibilatrix)	7 (12.5%)	11 (11%)
	Pied Flycatcher (Ficedula hypoleuca)	7 (12.5%)	8 (8%)
	European Robin (Erithacus rubecula)	5 (9%)	10 (10%)
	Common Whitethroat (Sylvia communis)	3 (5.4%)	3 (3%)
	Chiffchaff (Phylloscopus trochilus)	2 (3.5%)	2 (2%)
	Nightingale (Luscinia megarhynchos)	2 (3.5%)	3 (3%)
	Song Thrush (Turdus philomelos)	1 (1.8%)	2 (2%)
	Redstart (Saxicola rubetra)	1 (1.8%)	1 (1%)
	Tyrrhenian Spotted Flycatcher (Muscicapa Striata Tyrrhenica)	1 (1.8%)	1 (1%)
	Magpie (Pica pica)	1 (1.8%)	1 (1%)
	Southern Grey Shrike (Lanius senator)	1 (1.8%)	1 (1%)
	Total Spring	56	99
TOTAL		461	961

). Overall, the European Robin accounted for 67% of the fall ticks, while the Common Blackbird and Song Thrush together contributed nearly 30%. In the spring, the Common Redstart accounted for 55% of the ticks collected, despite being a smaller proportion of the total bird captures (44.6%).

A total of 961 ticks were collected across two seasons, with autumn accounting for 90% of the total sample (862 ticks). In the fall, the most common species were Ixodes ricinus (77.2%) and Ixodes frontalis (13.6%). Most of the ticks collected in autumn were in the larval stage (69%), followed by nymphs (27%) and adults (4%).

Spring contributed 10% to the total tick sample (99 ticks), with Hyalomma rufipes (17.2%) and Hyalomma spp. (63.7%) being the most frequently found species. The spring sample had a higher number of adults, particularly in Hyalomma rufipes (82%), while larvae accounted for 30% and nymphs for 14%.

In both seasons, larvae were the most prevalent life stage, comprising 65% of all ticks collected. Nymphs accounted for 26% while adults represented just 9% of the total sample (

Table 4. Seasonal distribution and developmental stage distributions of ticks from migratory birds.

Season	Tick Species	Number of Ticks Collected (%)	Larva (n°/%)	Nimph	Adult
Fall	Hyalomma marginatum	5 (0.5%)	2 (40%)	2 (40%)	1 (20%)
	Ixodes inopinatus	3 (0.2%)	3 (100%)	0	0
	Ixodes ventalloi	5 (0.5%)	4 (80%)	1 (20%)	0
	Ixodes frontalis	117 (13.6%)	83 (71%)	25 (21%)	9 (8%)
	Ixodes ricinus	60 (77.2%)	461 (53.4%)	179 (20.8%)	26 (3%)
	Ixodes spp.	66 (7.7%)	39 (59%)	24 (36%)	3 (5%)
TOTAL		862 (90%)	592 (69%)	231 (27%)	39 (4%)
Spring	Amblyomma marmoreum	1 (1%)	0	0	1 (100%)
	Hyalomma marginatum	11 (11.1%)		3 (27%)	8 (73%)
	Hyalomma rufipes	17 (17.2%)	2 (12%)	1 (6%)	14 (82%)
	Rhipicephalus bursa	1 (1%)	0	0	1 (100%)
	Ixodes frontalis	4 (4%)	2 (50%)	1 (25%)	1 (25%)
	Ixodes inopinatus	1 (1%)	1 (100%)	0	0
	Ixodes ventalloi	1 (1%)	0	1 (100%)	0
	Hyalomma spp.	63 (63.7%)	25 (40%)	7 (11%)	31 (49%)
	TOTAL	99 (10%)	30 (30%)	14 (14%)	55 (55%)
TOTAL		961	622 (65%)	245 (26%)	94 (9%)

).

3.2. Molecular Analysis for Detection of Borrelia spp.

Out of the 862 ticks analyzed from the autumn collection for Borrelia spp. by rt-PCR, 246 samples (28.5%) tested positive. In the spring collection, 99 ticks were analyzed, with five samples (5%) testing positive.

Molecular typing was only possible for some of the Borrelia-positive samples. The FLA gene was successfully amplified in 22.3% (56/251) of the samples, allowing for molecular typing via sequencing (

Table 5. Distribution of tick species and life stages found on different bird species, along with the corresponding Borrelia sequencing results.

Season	Bird Species (n.)	Tick Species	Tick Life Stage	Sequencing Result
Fall	Redbreast (10)	I. ricinus (9)	Larva (9)	B. valaisiana (1) B. garinii (7) B. afzelii (1)
		I. frontalis (1)	Larva (1)	B. garinii (1)
	Song Thrush (13)	I. ricinus (11)	Larva (8)	B. garinii (7) B. valaisiana (1)
			Nymph (3)	B. garinii (3)
		I. frontalis (2)	Larva (2)	B. turdi (1) B. garinii (1)
	Blackbird (33)	I. ricinus (27)	Larva (17)	B. valaisiana (4) B. garinii (13)
			Nymph (10)	B. garinii (9) B. valaisiana (1)
		I. frontalis (5)	Adult (1)	B. turdi (1)
			Larva (4)	B. garinii (4)
		I. inopinatus (1)	Larva (1)	B. garinii (1)

).

Borrelia spp. positivities were detected only in ticks collected during the fall, specifically from European Robin, Song Thrush, and Blackbird species. The ticks collected were primarily of the Ixodes genus, with Ixodes ricinus being the most common species. The majority of these ticks were in the larval stage, with nymphs being less frequent and adults only found on Blackbirds. Ixodes ricinus was the predominant tick species found on all bird hosts, with Ixodes frontalis and Ixodes inopinatus occurring in smaller numbers. The most commonly detected Borrelia species were B. garinii and B. valaisiana, with less frequent detections of B. afzelii, and B. turdi (Table 4).

The study identified ten distinct sequence types from 46 tick samples collected from various bird hosts, based on genetic variation and the detected Borrelia species. The details of these sequence types and their associated GenBank accession numbers are summarized in the accompanying table (Table 5). The majority of sequences matched B. garinii, with other species including B. valaisiana, B. turdi, and B. afzelii (see

Table 6. Genetic analysis of Ixodes spp. ticks collected from various bird hosts: Genbank Acession Numbers and Blast results.

Sequence Type	Host	Collection Host	Gen Bank Accession Numbers	Blast Analysis
ST1	I. ricinus 105	European Robin	PV037008	B. valaisiana
	I. ricinus 240	Blackbird	PV037009
	I. ricinus 349A	Blackbird	PV037010
	I. ricinus 349B	Blackbird	PV037011
	I. ricinus 350D	Song trush	PV037012
ST2	I. ricinus 141	European Robin	PV036970	B. garinii
	I. ricinus 308B	Common Blackbird	PV036971
	I. frontalis 185b	Common Blackbird	PV036973
	I. frontalis 185d	Common Blackbird	PV036974
	I. ricinus 186	Common Blackbird	PV036977
	I. ricinus 199a	Common Blackbird	PV036978
	I. frontalis 272a	European Robin	PV036984
	I. ricinus 351b	Common Blackbird	PV036991
	I. ricinus 314b	Common Blackbird	PV036994
	I. ricinus 315A	Common Blackbird	PV036995
	I. ricinus 317B	Common Blackbird	PV036997
	I. ricinus 317E	Common Blackbird	PV036998
	I. ricinus 28B	Common Blackbird	PV037002
	I. ricinus 41	Song Thrush	PV037003
	I. ricinus 42	Song Thrush	PV037004
	I. ricinus 70	Song Thrush	PV037005
ST3	I. ricinus 233A	European Robin	PV036972	B. garinii
	I. ricinus 185H	Common Blackbird	PV036976
ST4	I. frontalis 185F	Common Blackbird	PV036975	B. garinii
ST5	I. ricinus 200A	Common Blackbird	PV036979	B. garinii
	I. ricinus 210B	European Robin	PV036983
ST6	I. ricinus 201	Common Blackbird	PV036980	B. garinii
ST7	I. ricinus 206A	Song Thrush	PV036981	B. garinii
ST8	I. frontalis 206B	Song Thrush	PV037013	B. turdi
	I. frontalis 10A	Common Blackbird	PV037014
ST9	I. ricinus 263C	European Robin	PV068046	B. afzelii
ST10	I. ricinus 342A	European Robin	PV036987	B. garinii
	I. frontalis 345C	Common Blackbird	PV036988
	I. frontalis 345D	Common Blackbird	PV036989
	I. ricinus 347B	European Robin	PV036990
	I. ricinus 351C	Song Thrush	PV036992
	I. ricinus 351D	Song Thrush	PV036993
	I. ricinus 317A	Common Blackbird	PV036996
	I. ricinus 317F	Common Blackbird	PV036999
	I. ricinus 317H	Common Blackbird	PV037000
	I. ricinus 317Q	Common Blackbird	PV037001
	I. ricinus 43A	Common Blackbird	PV037006
	I. ricinus 43B	Common Blackbird	PV037007

).

The phylogenetic tree confirmed the presence of multiple Borrelia species in Ixodes ricinus and Ixodes frontalis populations in Italy. The Borrelia garinii clade exhibited strong monophyly with sequences from Poland, Norway, and Russia, alongside Italian strains ST2, ST3, ST4, ST5, ST6, and ST10; this suggests a broad host adaptability and potential for widespread transmission. The Borrelia turdi clade, a well-supported evolutionary relationship, included ST8 from Italy, which clustered closely with Borrelia turdi from Turkey. In the Borrelia valaisiana clade, ST1 from Italy grouped with Polish sequences, while the Borrelia afzelii clade exhibited a highly supported topology including ST9 from Italy and sequences from Poland, confirming the genetic stability and distinct evolutionary lineage of this species. The strong bootstrap values across major clades reinforce the reliability of these phylogenetic relationships.

4. Discussion

Migratory birds play a crucial role in the geographic spread of ticks infected with Borrelia spp., as they travel across biogeographical regions and can transport infected ticks over long distances, thereby facilitating the expansion of both tick populations and the pathogens they carry [9]. Avian hosts may directly infect feeding ticks or facilitate transmission via co-feeding transmission, where uninfected ticks acquire pathogens from infected individuals feeding nearby. These mechanisms underscore the importance of migratory birds in amplifying tick-borne pathogens [22].

This study, which tested ticks collected from migratory birds passing through Sardinia (a key stopover site along the Afro-Palearctic flyway), provides insight into their role in tick-borne Borrelia spp. dispersion. Tick infestation rates were significantly higher during autumn migration (mid-September to October), with almost all passerines captured hosting at least one tick. In contrast, during the spring migration (mid-April to May) infestation rates ranged from 2% to 54%, with an overall lower tick burden. These seasonal differences reflect variations in bird migration phenology, tick life cycle dynamics, and host susceptibility to parasitism [23,24].

In autumn, most captured species were short-distance migrants, with the European Robin (74.3%), Common Blackbird (13.3%), and Song Thrush (8.4%) accounting for the majoriy of captures. Less frequent species, such as the Woodlark, Blackcap, Garden Warbler, and Common Redstart, contributed ≤1% of the total captures. Ticks were predominantly found on the European Robin, Common Blackbird, and Song Thrush (see Table 2), which is consistent with previous studies [25,26,27], indicating that ground-foraging passerine birds are more frequently infested due to their prolonged exposure to tick-infested environments. This behavior increases their exposure to Ixodes ricinus larvae and nymphs, the most prevalent stages in autumn [28]. The seasonal variation observed in tick species prevalence may be influenced by differences in avian migratory routes and timing. In autumn, when Ixodes ricinus is predominant, many migratory birds are returning from their breeding grounds in Europe to the wintering areas in Africa, passing through regions where Ixodes ricinus is widespread. Conversely, in spring, Hyalomma spp. is more frequently observed, reflecting the northward migration of birds from Africa to Europe, where Hyalomma ticks are more common.

Ixodes ricinus larvae accounted for 69% of the collected ticks, significantly higher than nymphs (27%), aligning with other studies on tick distribution and infestation rates [25,29]. In contrast, Olsén et al. [30] found a higher proportion of nymphs compared to larvae in Sweden, which may reflect differences in tick visibility and collection, as nymphs are generally larger and more conspicuous, while larvae, being smaller, are more easily overlooked or missed during collection.

The higher prevalence of larvae in autumn reflects their peak seasonal abundance, facilitated by the availability of suitable avian hosts. The European Robin, Common Blackbird, and Song Thrush, which are prevalent in autumn, are short- to medium-distance migrants that migrate from breeding grounds in continental Europe and the Ural Mountains to wintering areas in western and southern Europe or northern Africa. Their ground-foraging behavior makes them more vulnerable to higher tick infestation.

In contrast, Ixodes ticks were substantially less prevalent in spring (6.1% of the total tick population), reflecting the migratory behavior of long-distance migratory birds such as the Garden Warbler and Willow Warbler, which are insectivorous and primarily forage in flight and are less likely to harbor ticks. However, these birds, which migrate to sub-Saharan Africa, are less likely to be infested with Ixodes ticks during their spring migration but may carry Mediterranean Hyalomma ticks, which were observed in warmer months. The presence of Hyalomma marginatum and H. rufipes suggests a Mediterranean origin, as these ticks infest avian hosts during migration and molt into nymphs while parasitizing birds traveling northward.

Borrelia was detected in 26.1% of the collected ticks, with a higher prevalence in autumn, coinciding with the seasonal movement of ground-feeding birds.

The identified species, including B. garinii, B. valaisiana, B. afzelii, and B. turdi, were primarily found in Ixodes ricinus larvae. Since larvae do not acquire Borrelia spp. via transovarial transmission, they can become infected either by feeding on infected avian hosts or small mammals or through co-feeding interactions with infected ticks.

Genetic sequencing revealed that Borrelia garinii was the predominant species, detected in 89% of Borrelia-positive ticks, most of which were Ixodes ricinus larvae. The majority of I. ricinus larvae were collected from the Common Blackbird (40%), European Robin (12.5%), and Song Thrush (17.7%), suggesting that these bird species play a crucial role in maintaining and disseminating B. garinii within ecosystems. Given that B. garinii is a key agent of neuroborreliosis in humans [31], these findings reinforce the indirect role of migratory birds in shaping the epidemiology of Lyme borreliosis in Europe.

In this study, apart from B. garinii, Borrelia valaisiana was detected in 12.6% of Ixodes ricinus ticks. This pathogen is also associated with avian hosts and has been detected in bird-associated arthropods [32,33], underscoring the role of birds in the transmission of multiple Borrelia species.

Additionally, Borrelia turdi was identified in two Ixodes frontalis ticks (one adult from a Blackbird and one larva from a Song Thrush). Since Ixodes frontalis is the primary vector for B. turdi in Europe [34], these findings further reinforce the role of migratory passerines in the long-distance dispersal of tick-borne pathogens. Borrelia turdi has also been reported in I. ricinus larvae and nymphs collected from migratory passerines in various European countries, including Poland, the Azores, Belgium, and Spain [35], suggesting that this bacterium is widespread in bird-associated tick populations.

Furthermore, an Ixodes ricinus larva from a European Robin tested positive for Borrelia afzeli. This species is typically associated with small mammals as reservoir hosts [36,37], raising questions about the potential role of birds in its transmission. While birds are not considered primary reservoirs for B. afzelii [38], their occasional involvement suggests that they may act as incidental hosts, potentially contributing to the pathogen’s ecological complexity.

These findings highlight the importance of continued surveillance of tick populations, particularly in regions like Sardinia, where studies on tick-borne pathogen circulation have been limited. However, some limitations should be acknowledged. The primary method of bird capture, mist-netting, may have introduced bias by over-representing ground-foraging birds while under-representing arboreal and aerial foragers. Additionally, the lack of direct screening for Borrelia spp. in birds limits our understanding of their role as pathogen reservoirs. Further research should include broader bird species, molecular testing of avian blood, and long-term monitoring to better understand the contribution of migratory birds to tick-borne disease transmission.

5. Conclusions

This study highlights the critical role of migratory birds in the spread of tick-borne pathogens. Acting as both reservoirs and amplifiers, birds transport infected ticks, such as Borrelia spp., to other ticks and vertebrate hosts, including humans. The seasonal variation in tick infestations reveals a higher prevalence in autumn, with ground-feeding passerines being particularly susceptible to tick attachment. The detection of B. garinii in ticks collected from these birds further supports their role in the enzootic cycle of tick-borne diseases. As the first study in Sardinia, this research provides new insights into tick–host interactions and the role of avian migration in pathogen dissemination in Europe. These findings suggest that additional studies are necessary to better understand how the migratory patterns of birds influence the distribution of vector-borne pathogens, such as Borrelia spp. Continuous monitoring of migratory bird populations and their associated ticks, particularly during migration periods, is essential for understanding disease dynamics and implementing effective preventive measures.