Migratory birds as disseminators of ticks and the tick-borne pathogens Borrelia bacteria and tick-borne encephalitis (TBE) virus: a seasonal study at Ottenby Bird Observatory in South-eastern Sweden

Background Birds can act as reservoirs of tick-borne pathogens and can also disperse pathogen-containing ticks to both nearby and remote localities. The aims of this study were to estimate tick infestation patterns on migratory birds and the prevalence of different Borrelia species and tick-borne encephalitis virus (TBEV) in ticks removed from birds in south-eastern Sweden. Methods Ticks were collected from resident and migratory birds captured at the Ottenby Bird Observatory, Öland, Sweden, from March to November 2009. Ticks were molecularly identified to species, and morphologically to developmental stage, and the presence of Borrelia bacteria and TBEV was determined by quantitative real-time PCR. Results A total of 1339 ticks in the genera Haemaphysalis, Hyalomma, and Ixodes was recorded of which I. ricinus was the most abundant species. Important tick hosts were the European robin (Erithacus rubecula), Blackbird (Turdus merula), Tree pipit (Anthus trivialis), Eurasian wren (Troglodytes troglodytes), Common redstart (Phoenicurus phoenicurus), Willow warbler (Phylloscopus trochilus), and Common whitethroat (Sylvia communis). Borrelia bacteria were detected in 25% (285/1,124) of the detached ticks available for analysis. Seven Borrelia species (B. afzelii, B. burgdorferi (s.s.), B. garinii, B. lusitaniae, B. turdi, B. valaisiana, and B. miyamotoi) were identified. B. turdi was recorded for the first time in ticks in Sweden. The number of Borrelia cells per tick ranged from 2.0 × 100 to 7.0 × 105. B. miyamotoi-containing ticks contained a significantly higher median number of Borrelia cells than B. burgdorferi (s.l.)-containing ticks. B. garinii and B. miyamotoi were the most prevalent Borrelia species in tick larvae. Larvae of I. ricinus with B. garinii were removed from seven bird species, particularly S. communis and A. trivialis, which may suggest that the larvae had contracted the Borrelia bacteria from or via these birds. Also, a high percentage of tick larvae containing B. miyamotoi was removed from E. rubecula. All ticks were negative for TBEV. Conclusions The results corroborate the view that the contributions of birds to human disease are substantial, particularly as blood hosts for ticks and for their short-, medium-, and long-distance dispersal. Moreover, several ground-foraging bird species appear to be important for the maintenance and dispersal of Borrelia species. The absence of TBEV in the ticks conforms to other similar studies.

Seasonal migration is common among birds breeding in northern latitudes but shows large variation between and within species in timing and direction of migration as well as distance travelled. Most migratory birds divide the trip into several migration legs, interspersed with time spent on stopover sites along the route. At suitable stopover sites, birds feed and prepare for the next leg of migration, providing opportunities for new ticks to attach and already fed ticks to detach from the birds, thereby following birds along the migratory routes and potentially creating new foci of tick-borne diseases.
The overall aim of this study is to better understand the potential role of birds as disseminators of ticks and tickborne pathogens into Sweden during their northward, spring, and early summer migration and from Sweden during their southward, late summer and autumn migration. In particular, the present investigation was designed to determine the abundance of different tick species and tick stages infesting migratory and other birds captured at one of Sweden's most important bird observatories. Moreover, we aimed at determining the prevalence of Borrelia and TBEV contents in the ticks infesting the birds.

Description of the sampling site, Ottenby Bird Observatory
Ottenby Bird Observatory is located on the southern point of the island of Öland in south-east Sweden. At the observatory, migratory birds have been banded since 1946, and more than 1 million birds have been caught, providing a solid knowledge base on which bird populations that pass through the area [23,24]. Apart from funnelling migratory birds during migration, the area also functions as stopover site and breeding ground for many species of birds. In spring, the vast majority of birds caught at the observatory have just arrived after a night of migration, including passage over stretches of open water (the Baltic Sea), while in autumn, birds caught will be a mixture of birds arriving from areas to the north and east, and those that have spent some time on stopover in the area and restarting migration. The reserve in which the observatory is situated encompasses an area of about 25 km 2 with shore meadows, pastures, and woodland dominated by stands of old oaks (Quercus robur) and birch (Betula sp.). The area is grazed by cattle (Bos taurus), sheep (Ovis aries) and fallow deer (Dama dama).

Bird captures and tick collections
Sampling of birds was approved by the Swedish Board of Agriculture, delegated through the Animal Research Ethics Committee in Linköping (decision 43-09). Birds were captured during the general trapping activities of staff members at the Ottenby Bird Observatory (56° 12′ N, 16° 24′ E). With the approval of the Swedish Museum of Natural History in Stockholm, Sweden, mist nets and Heligoland traps were used to capture birds during the periods 15 March-15 June and 15 July-15 November 2009 as part of the observatory's standardized trapping scheme. Trapped birds were banded, measured, and identified to species level and, when possible, sexed and aged. At the observatory, trapping starts half an hour Keywords: Migratory birds, Borrelia prevalence, Borrelia burgdorferi (sensu lato), Borrelia miyamotoi, Tick-borne encephalitis virus before sunrise and ends at 11 a.m., which means that the length of the trapping day varies with season. To achieve a representative sample of birds for this study, and to comport with other duties at the observatory, we devised a schedule wherein every second day each bird captured during the first four working hours (start 30 min before sunrise) was checked for ticks around the base of the beak and around the eyes and ears, and every other day each bird captured during the last four working hours (7-11 a.m.) was checked. Ticks were removed with forceps and photographed as described below. Ticks were stored individually in empty snap-lid tubes at − 70 °C.

Classification of bird migration categories
As noted above, the majority of birds screened for ticks in this study were caught during migration. We broadly classify the investigated species into residents, shortdistance migrants, partial migrants, and long-distance migrants based on band recovery data from the observatory and Sweden as a whole [25]. The resident bird species category comprises four species-Passer domesticus, Passer montanus, Corvus monedula, and Pica pica-that are sedentary in the area and show little to no migratory propensity apart from local dispersal. The division between short-and long-distance migrants is based on distance between breeding areas and non-breeding areas, where species that mainly winter in Europe are classified as short-distance migrants and species wintering predominantly in Africa or Asia as long-distance migrants. A special category is the partial migrants, a category which here is used to denote species of which some individuals are residents, some are short-distance migrants, and some alternate between these categories depending on the severity of winter or other environmental cues. Thus, the categories used here reflect the populations of the species studied at Ottenby and would in some instance be categorized differently at other study locations in the distribution range.

Determination of developmental stage and species of the ticks
Each tick was photographed dorsally and ventrally using a digital USB microscope (Dino-Lite Long AM4013TL, AnMo Electronics Corp., Taiwan), as previously described [15]. Based on the photographs, each tick was morphologically identified to developmental stage (larva, nymph, or adult) and sex of adults. To identify the genus and species of the ticks, each specimen was analysed using molecular methods. All ticks were analysed by a PCR method targeting the tick mitochondrial 16S rRNA gene, as previously described [26].

Detection and quantification of Borrelia bacteria and TBEV by real-time PCR analyses
Two microlitres of cDNA per reaction was used in a LUX ™ real-time PCR assay to detect and quantify a 131-bp-long fragment of the Borrelia spp. 16S rRNA gene, using genus-specific primers and a serial dilution of plasmid standard [27]. Two microlitres of cDNA per reaction was used in a multiplex TaqMan ™ real-time PCR assay to detect and quantify all three subtypes of TBEV, using primers, probes, and a serial dilution of plasmid standard [28]. PCR reagents and templates were added to a 96-well real-time PCR plate by using a CAS1200 pipetting robot (Corbett Robotics Inc.). The real-time PCR analyses were carried out using the BioRad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

Determination of Borrelia species by conventional PCR and nucleotide sequencing
To determine Borrelia burgdorferi (s.s) species of the samples positive in the LUX real-time PCR assay, a nested conventional PCR assay using primers targeting the intergenic spacer region (IGS) between 5S and 23S rRNA genes was applied, as described in [29]. Samples that failed to produce PCR products with this assay were instead analysed with primers targeting the IGS between 16S and 23S rRNA genes to determine other Borrelia spp., as described in [30,31]. Tick samples, positive for Borrelia spp. in the LUX real-time PCR assay, which failed to produce PCR products with the 5S-23S IGS assay and 16S-23S IGS assay, were denoted 'untypeable' .
Sequencing of all the PCR products was performed by Macrogen Inc. (Amsterdam, The Netherlands). Chromatograms were initially edited using BioEdit software v7.0 (Tom Hall, Ibis Therapeutics, Carlsbad, CA) and sequences were examined using Basic Local Alignment Tool (BLAST). The appearance of dual peaks in chromatograms was interpreted as at least two Borrelia species in the sample and thus denoted as 'mixed' . An additional file shows the aligned sequences (see Additional file 1).

Statistical analyses
Data were presented as medians with interquartile range (IQR) for numerical variables and as percentage for categorical variables. The numerical variables (i.e. the number of Borrelia spp. cells of different Borrelia spp. detected in the ticks) were analysed using Kruskal-Wallis test, which, if significant, was followed by Dunn's multiple comparison test. The categorical variables (e.g. developmental stages of the tick; tick species; spring vs. autumn, etc.) were analysed using chi-square test, but when the expected frequency was < 5 in at least one of the cells of the contingency table, Fisher's exact test with a confidence interval (CI) of 95% was used instead. Statistical analyses were performed, and graphs and figures were drawn using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA). P-values ≤ 0.05 were considered statistically significant.

Ticks collected from birds and seasonal dynamics of infestation pattern
A total of 4601 bird individuals (4788 bird captures) of 65 species was examined for ticks at least once during the study period, i.e. 15 March-15 June and 15 July-15 November 2009, at the Ottenby Bird Observatory. A total of 749 bird individuals (759 bird captures) of 35 species was infested with at least one tick specimen (Table 1). Calculations based on bird captures showed that the monthly prevalence of birds infested with one or more ticks during the study period ranged from 4.90 to 31.5%, with the lowest and highest infestation prevalence in the second half of May and second half of September, respectively ( Fig. 1).
A total of 1339 ticks was collected from the infested birds. Due to missing photos of ticks and because some ticks were damaged when removed from their hosts, or lost in the nucleic acid extraction process, the genus, species, or developmental stage could not be determined in 10.8% (n = 145) of the ticks (Table 1).
Larvae and nymphs of the most prevalent tick species, i.e. I. ricinus, were collected during the whole collection period but mainly from mid-March to mid-May and from early August to mid-October (Fig. 2a). Of all collected I. ricinus ticks, larvae were more prevalent in autumn Most ticks (46%) were found on the European robin (Erithacus rubecula), which was also infested with the highest number of tick species (n = 4) ( Table 1). Among all ticks identified to species, I. ricinus infested the broadest range of bird species, i.e. 32 species (Table 1; Fig  3a). Most of the I. ricinus nymphs were removed from E. rubecula and Common blackbird (Turdus merula) ( Table 1; Fig. 3b). Most I. ricinus larvae were removed from E. rubecula, Eurasian wren (Troglodytes troglodytes), and Tree pipit (Anthus trivialis) (Fig. 3c).

Prevalence of Borrelia bacteria and TBEV in the ticks
Of the ticks for which the genus, species, or developmental stage could be determined (n = 1194), 94.1% (n = 1124) were available for analyses of contents of Borrelia bacteria and TBEV.
Of all ticks analysed, 25.4% (285/1,124) were positive in real-time PCR for Borrelia spp. (Table 2). All 1124 ticks were negative in real-time PCR for TBEV. A significantly higher proportion of nymphs ( Of all samples that were determined to Borrelia species level and where the developmental stage of the corresponding tick could be determined (n = 137), 23.4% (n = 32) Borrelia species were recorded in larvae while 76.6% (n = 105) were recorded in nymphs ( , respectively). There were no significant differences in prevalence between nymphs and larvae among the other Borrelia species.
High proportions of I. ricinus larvae containing B. garinii were removed from Common whitethroat (Sylvia communis) and A. trivialis (Fig. 5a). Also, a high percentage of tick larvae containing B. miyamotoi was removed from E. rubecula (Fig. 5b).

Quantity of Borrelia cells in the ticks
According to the LUX real-time PCR assay, the number of Borrelia cells per tick containing a typeable Borrelia species ranged from 2.0 × 10 0 cells to 7.0 × 10 5 cells (Fig. 6), with a median of 1.1 × 10 3 . No significant difference in number of Borrelia spp. cells between larvae and nymphs was observed (medians 2.5 × 10 3 , and 1.1 × 10 3 , respectively). Ticks

Tick infestation prevalence on birds
In the present study, tick infestation rates were highest in late April and late September (Fig. 1), when as many as 30% of investigated passerine birds were infested with at least one tick. However, ticks were detected on birds in all study months, with infestation rates in the range of 4.90-31.5%. Observed infestation rates are likely influenced both by the phenology of migration of particular bird species and by tick and host biology. For instance, the early season (March-April) is dominated by shortdistance migrants, including important ground foraging tick host species such T. merula and E. rubecula, while    [4]. Similarly, in Switzerland, the prevalence of tick infestation, mainly of I. ricinus on migratory birds was significantly higher on birds migrating southwards than northwards [9]. In our study, the highest tick infestation prevalence occurred in April and August-September (Fig. 1).

Birds as reservoirs and transmission hosts for Borrelia species
A few xenodiagnostic investigations have demonstrated that birds are competent transmission hosts for certain Borrelia species. This suggests that birds may harbour infectious Borrelia spirochaetes in their skin or blood and consequently can act as competent reservoirs. Another possibility is that birds may be competent transmission hosts in non-spirochaetemic co-feeding transmission among one or more Borrelia-containing ticks, feeding adjacent to one or more susceptible ticks. A third possibility is that certain birds may act in both ways of Borrelia transmission. Kurtenbach et al. (2002) showed that pheasants are competent transmission hosts for B. garinii and B. valaisiana, but not for B. afzelii [36]. Comstedt et al. (2006) obtained evidence that several species of birds act as transmission hosts for B. garinii, thus transmitting the bacteria to larvae of I. ricinus, which were ingesting blood from their avian host [2]. The present study may suggest that many I. ricinus larvae contracted B. garinii and B. miyamotoi spirochaetes while feeding on some of their avian hosts (see below). However, while transovarial transmission is rare in B. burgdorferi (s.l.) [37], it is well known that such transmission is a common trait in B. miyamotoi, which results in B. miyamotoi-containing larvae capable of transmitting the spirochaetes to their hosts [37]. Thus, it is most likely that B. miyamotoi-containing larvae had been transovarially infected whereas the B. garinii-containing larvae had contracted the spirochaetes from their avian hosts.

Ixodes ricinus
I. ricinus is considered to be the primary vector of all human-pathogenic species of B. burgdorferi (s.l.) and of B. miyamotoi occurring in Europe [38]. Moreover, I. ricinus is able to transmit B. turdi to T. merula [32]. Larvae and to a lesser extent nymphs of I. ricinus parasitize small . One bird capture was excluded from the analysis because of missing collection data mammals, birds, and reptiles; all stages can also be found on medium-and large-sized mammals [39,40]. In most investigations, the number of larvae of I. ricinus collected from birds is usually larger than the number of nymphs collected. However, the proportion of nymphs of I. ricinus in relation to that of larvae of I. ricinus is usually higher on birds than on small mammals. For instance, during the summer months of 1991-1994 in a study area near Stockholm, Sweden, median numbers of 16-105 larvae and 0-2 nymphs of I. ricinus were found on infested bank voles, Myodes glareolus [40]. In contrast, from 22,998 birds captured at eight localities in Sweden, Olsen et al. [33] removed 949 I. ricinus ticks; 29.8% were larvae and 70.0% were nymphs. Even though the nymphs are easier to detect than the larvae (some of which may therefore have been missed), it is generally accepted that nymphs constitute a significantly higher proportion of the I. ricinus ticks on birds than on small mammals. This is confirmed by the results of the present study: almost equal proportions of larvae (48.3%) and nymphs (51.7%) of I. ricinus were recorded. Almost equal proportions of larvae and nymphs of I. ricinus were also encountered in similar studies by others [4,[41][42][43]. In the present study 46% of the ticks, most of which were immatures of I. ricinus, were removed from E. rubecula. Other bird species from which high total numbers of ticks were collected were T. merula, T. troglodytes, and A. trivialis. The large numbers of ticks from these bird species partly reflects their abundance, where E. rubecula, T. merula, and T. troglodytes (and to a lesser extent A. trivialis) make up a large proportion of the trapping totals, but it likely also reflects their habits of spending a lot of time on the ground, thereby rendering them vulnerable to attack by questing larvae and nymphs of I. ricinus.
Since the prevalence of tick-associated pathogens is generally higher in tick nymphs than in tick larvae, the high proportion of nymphs feeding on birds suggests that birds receive a substantial proportion of infections from the nymphs. On the other hand, the tick larvae, while feeding on their avian hosts, are likely to contract Borrelia bacteria with nymph-derived spirochaetes. In the present study, in I. ricinus larvae we detected these Borrelia species : B. afzelii, B. garinii, B. valaisiana, B.  burgdorferi (s.s.), and B. miyamotoi. B. burgdorferi (s.l.) spirochaetes are usually not transmitted transovarially [37]. Therefore, we think it probable that the larvae, containing one or two species in the B. burgdorferi s.l. complex (Table 2), had contracted the Borrelia cells directly from their infective avian hosts or through co-feeding transmission. In contrast, B. miyamotoi is often transmitted transovarially. Therefore, we cannot know if the eight I. ricinus larvae positive for B. miyamotoi had contracted the spirochaetes transovarially or from their infective avian hosts or by co-feeding transmission.
I. frontalis parasitizes birds, particularly Passeriformes [44], and is a likely enzootic vector of blood-borne microbes in bird-tick-bird cycles. I. ricinus, which feeds on both birds and mammals, could here potentially function as a bridge vector by transferring pathogens from the bird-I. frontalis enzootic cycle to humans and other mammals. Gilot et al. (1997) describes one occasion when an adult female of I. frontalis attached to a human tick-collector's hand [45]. This is apparently an exceptionally rare behaviour exhibited by this otherwise strictly ornithophagous tick species. In western and central France and in north-eastern Spain, I. frontalis is the most abundant ixodid on passerine birds [46,47]. Heylen et al. (2016) have shown that I. frontalis is able to transmit B. turdi to T. merula, but that I. frontalis appears to be an incompetent vector of B. garinii and B. valaisiana [32].
Agoulon and co-workers showed that the seasonal activity of I. frontalis differs from that of I. ricinus, particularly of the larvae. In I. frontalis larval activity in France was completely absent during the summer [47]. This conforms to our results: immatures of I. ricinus were recorded during both spring-early summer and late summer-autumn while I. frontalis was only present during the first of these seasons.

Haemaphysalis punctata
The adults of Haemaphysalis punctata are usually parasites of medium and large mammals, in particular domestic ungulates, but may occasionally feed on humans [39,44,48,49]. The immatures can be found on small mammals and more rarely on birds and lizards [39,44,48]. In the present study, we recorded 12 larvae of H. punctata, 11 of which were investigated and scored negative for Borrelia spp. (Table 1). Previously, among 967 ticks removed from 465 tick-infested birds out of nearly 23,000  (26) 30 (10) 49 (17) 42 (14) 2 (1) 2 (1) 1 (1) 12 (4)   7 (2) 145 (50) birds examined, only 1 H. punctata, a nymph, was found [2]. An earlier study on questing ticks at three Swedish Baltic Sea islands, spirochaetes, believed to belong to B. burgdorferi (s.l.) were recorded in ~ 2% of H. punctata nymphs [50]. Since the spirochaetes were detected by phase-contrast microscopy, it is not certain that they belong to B. burgdorferi (s.l.). In any case, the low number of spirochaetes detected in the ticks and the low prevalence suggest that H. punctata is not an important Borrelia spp. vector [50]. This conclusion conforms to our results.

Hyalomma marginatum
Each spring Hyalomma marginatum is introduced to northern Europe when larvae or nymphs are carried by birds migrating from southern Europe to Sweden and neighbouring countries [39,51]. This is reflected in our data by the fact that H. marginatum was only present on birds captured during April and May (Fig. 2b). During the summer of 2018, which was exceptionally warm, there were several records of adult H. marginatum and H. rufipes in Sweden [52]. These adult ticks are believed to originally have infested avian hosts in the Mediterranean region, then feeding and becoming nymphs while still on their hosts flying northwards for several days. Subsequently, the blood-fed nymphs left their hosts after their arrival in southern or central Sweden. Due to the warm summer of 2018 such H. marginatum and H. rufipes nymphs in Sweden were able to reach the adult stage before the end of the summer. However, these tick species are presumably not yet permanent members of the Swedish tick fauna. H. marginatum and H. rufipes are of great importance as primary vectors of CCHFV [53,54]. In the present study, one larva, two nymphs, and one adult female of H. marginatum were recorded. All were negative for Borrelia. It is noteworthy that not only larvae but even nymphs and adult females can be transported into Sweden by birds. The fact that one adult female tick   was encountered on a Fringilla coelebs suggests that, if/ when the climate in southern Sweden becomes optimal for H. marginatum and H. rufipes, they will presumably establish indigenous populations here.

Borrelia garinii and Borrelia valaisiana
Birds are reservoirs and transmission hosts for humanpathogenic B. garinii and B. valaisiana [5,11,38,55]. As shown in Table 2, most Borrelia-containing Ixodes larvae were positive for B. garinii. We believe that these larvae had contracted the B. garinii bacteria from their avian hosts. However, in a few cases where one or more Borrelia-containing nymphs were feeding together with larvae on the same bird, it cannot be excluded that the larvae contracted the spirochaetes by co-feeding transmission. The majority (69%) of the I. ricinus larvae with B. garinii were removed from either S. communis (42%) or A. trivialis (27%), which may indicate that these bird species may be competent reservoirs and/or transmission hosts for B. garinii. Among the species in the B. burgdorferi (s.l.) complex present in Europe, B. garinii has the greatest potential to cause severe late neurological disease manifestations. Therefore, from a public health point of view it can be considered to be the most important Borrelia species in Europe. This also underlines the view that birds have a considerable, although indirect, impact on the epidemiology of tick-borne human diseases. B. valaisiana has been found to be common in ticks removed from birds in Europe [2]. Surprisingly, only a few (n = 3) Borrelia-containing I. ricinus larvae removed from the birds in the present study proved to contain B. valaisiana spirochaetes. However, it should be noted that 46 larvae (58%) contained untypeable Borrelia bacteria. It is possible that some of them were due to B. valaisiana.

Borrelia afzelii
Borrelia afzelii was present in several nymphs of I. ricinus, but only in one larva. B. afzelii is generally considered to use small mammals as its vertebrate reservoir [38]. However, there are many reports describing the presence of B. afzelii in I. ricinus larvae removed from birds [38,56,57]. The reason(s) for the presence of B. afzelii in tick larvae attached to birds need(s) further investigations. The presence of B. afzelii in Ixodes nymphs removed from birds is most likely because these ticks, in their larval stage, had fed on B. afzelii-infected small mammals.

Borrelia lusitaniae
There are a few cases of human Lyme borreliosis where B. lusitaniae is considered the etiological agent. It has lizards of the Lacertidae family as its vertebrate reservoir and I. ricinus as its vector [58,59]. B. lusitaniae is considered to have originated in Portugal from where it has spread to other countries in southern Europe and to North Africa and to scattered localities in Central, Eastern and South-Eastern Europe. DNA of this reptileassociated Borrelia species has even been demonstrated on a few occasions in adults and nymphs of I. ricinus that had fed on humans in southern and south-central Sweden [27]. The main method by which this Borrelia species has spread is thought to be by birds infested with B. lusitaniae-containing ticks. It has been recorded in Switzerland from I. ricinus larvae and nymphs feeding on E. rubecula and from larvae feeding on Turdus philomelos, T. merula and Phoenicurus phoenicurus [9].

Borrelia turdi
Birds are considered as reservoirs and transmission hosts for B. turdi. In Europe, I. frontalis is considered to be the primary vector of B. turdi [11,32]. We did not record any among 16 I. frontalis specimens positive for B. turdi. However, 2 among 549 nymphs of I. ricinus were positive for this species. To our knowledge, B. turdi has not previously been recorded from ticks or birds captured in Sweden. However, B. turdi was first recorded in Europe by Hasle et al. (2011) in Norway [3], where they detected B. turdi in 0.4% of I. ricinus larvae and 0.4% of I. ricinus nymphs collected from northward-migrating passerine birds. B. turdi was then detected in I. frontalis, I. ricinus and H. punctata removed from birds in northern Spain [60], from I. ricinus removed from T. merula in Poland [5], from I. frontalis removed from T. merula captured in the Archipelago of the Azores [35], from I. frontalis feeding on birds in Belgium [61], and from I. frontalis feeding on T. merula, T. philomelos, P. major, and T. troglodytes in Portugal [62]. Prevalences of 7% to 56% of B. turdi in I. frontalis have been recorded [62]. B. turdi has also been detected in skin biopsies from T. merula, which is a competent transmission host for this bacterium [11,62]. There is not yet any indication that B. turdi infects humans.

Borrelia burgdorferi (s.s.)
Only one I. ricinus larva and one I. ricinus nymph were positive for B. burgdorferi (s.s.). Several studies have shown that birds are competent reservoirs and transmission hosts for North American strains of B. burgdorferi (s.s.). [38]. B. burgdorferi (s.s.) is responsible for the majority of cases of Lyme borreliosis in North America. In Europe, B. burgdorferi (s.s.) is recorded rarely in ticks removed from birds [2,5,38,57]. However, as pointed out by Franke et al. [56], this may just reflect that this species is generally rare in many European regions, rather than suggesting that birds are incompetent reservoirs for European strains of B. burgdorferi (s.s.).

Borrelia miyamotoi
Borrelia miyamotoi is transmitted transovarially from the adult female tick to her offspring, which results in B. miyamotoi-containing tick larvae [37]. Transovarial transmission is much rarer or absent in the species within the B. burgdorferi (s.l.) complex [37]. In the present study, eight I. ricinus larvae were positive for B. miyamotoi. Presence of spirochaetes in tick larvae infesting birds can occur if the avian host is infected with spirochaetes, if cofeeding transmission from a Borrelia-containing tick to an adjacent susceptible tick larva takes place or if transovarial transmission of spirochaetes from an adult female tick to her offspring occurs. Detection of spirochaetes in unfed Ixodes larvae is an indication of transovarial transmission, which is a prevalent trait in B. miyamotoi [37,63]. This species is known to have rodents as one of its vertebrate reservoirs, but birds are considered to be another of its reservoirs [63,64].
It is reasonable to assume that the infectiousness of a bacterial pathogen is, in general, positively correlated to the concentration of cells of the bacterium in the infective medium. Our results revealed a significantly higher median density of cells in B. miyamotoi-containing ticks than in B. burgdorferi (s.l.)-containing ticks. In another study, based on ticks infesting humans, Wilhelmsson et al. also recorded a higher median density of spirochaetes in B. miyamotoi-containing ticks than in B. burgdorferi (s.l.)-containing ticks [27]. We conjecture that this may be an evolutionary trait which compensates for the generally lower prevalence of B. miyamotoi in the tick population compared to that of B. burgdorferi (s.l.). Similarly, it has previously been shown that in rodents infected with either B. miyamotoi or B. burgdorferi, the concentration of cells in the blood was generally much higher in the B. miyamotoi-infected rodents [63]. It is important to realise that, since transovarial transmission is a common trait in B. miyamotoi, which results in B. miyamotoi-containing tick larvae, many of which will infest birds, this Borrelia can be dispersed to distant localities.

Tick-borne encephalitis virus in ticks infesting birds
Proven, competent vertebrate transmission hosts of TBEV are many species of small mammals (rodents, insectivores) while, in view of recorded viremia and/or virus isolations, some ungulates (goat, sheep) and many bird species [65,66] may be considered suspected or presumed transmission-competent hosts for this virus. Moreover, there are a number of records of TBEV from ticks infesting birds [20,34,66,67]. The prevalence of this virus in host-seeking I. ricinus and in ticks removed from hosts in TBEV-enzootic regions is usually < 1% [68]. The absence of TBEV in all bird-derived ticks investigated by us thus conforms to similar studies, which have recorded a low or zero prevalence of this virus.