Tick infestation prevalence on birds
In the present study, tick infestation rates were highest in late April and late September, respectively (Figure 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 short distance migrants, including important ground foraging tick host species such T. merula and E. rubecula, while the later part of spring (May – June) is dominated by long distance tropical migrants, that to a larger extent comprise insectivorous species that feed less on the ground. In fact, in the present study, most ticks were removed from E. rubecula, T. merula, T. troglodytes and A. trivialis (Table 1). These results conform to several similar studies, which have revealed that passerine bird species that frequently feed on the ground are more often infested with I. ricinus ticks [2, 3, 4, 12, 16, 32, 33, 34, 35]. In a recent study in Denmark, E. rubecula, T. merula and P. phoenicurus constituted less than one third of the birds examined for ticks, but carried 77% of all I. ricinus ticks collected; 78% of all 179 I. ricinus ticks were collected from birds in the autumn [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 sensu lato (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 to transmit 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.
Tick species
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 mammals, birds and reptiles; all stages can also be found on medium- and large-sized mammals [39, 40]. The proportion of nymphs of I. ricinus in relation to that of larvae of I. ricinus is usually much 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 infested bank voles, Myodes glareolus [40]. In contrast, from 22,998 birds captured at eight localities in Sweden, Olsen et al. (1995) removed 949 I. ricinus ticks; 29.8 % were larvae and 70.0 % were nymphs [33]. 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. 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 are T. merula, T. troglodytes and A. trivialis. The large numbers of ticks from these bird species is partly reflecting 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, we detected in I. ricinus larvae 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 regard it most likely 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.
Ixodes frontalis
Among the Ixodes ticks that were identified to species level, 2.0 % were I. frontalis (8 larvae, 15 nymphs, and one adult female tick). Untypeable Borrelia spp. were detected in 3 among 15 I. frontalis nymphs.
I. frontalis parasitises birds, particularly Passeriformes [41] 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 [42]. 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 [43, 44]. 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 [44]. This conforms to our results: immatures of I. ricinus were recorded both during 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, 41, 45, 46]. The immatures can be found on small mammals and more rarely on birds and lizards [39, 41, 45]. 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 birds examined, only one 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 [47]. 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 [47]. 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, 48]. 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 [49]. 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 were in Sweden 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 [50, 51]. 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 by birds into Sweden. The fact that one adult female tick was encountered on a Fringilla coelebs suggests that, if/when the climate in southern Sweden has become 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 human-pathogenic B. garinii and B. valaisiana [5, 11, 38, 52]. As shown in Table 2, most Borrelia-containing Ixodes larvae had B. garinii. We believe that these larvae had contracted their B. garinii- 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. A majority (69%) of the I. ricinus larvae with B. garinii were removed from either S. communis (42%) or A. trivialis (27%), 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 have 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, 53, 54]. 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 as the etiological agent. It has lizards of the Lacertidae family as its vertebrate reservoir and I. ricinus as its vector [55, 56]. 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 reptile-associated 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 in Europe first recorded 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 has then been detected in I. frontalis, I. ricinus and H. punctata removed from birds in northern Spain [57], 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 [58], and from I. frontalis feeding on T. merula, T. philomelos, P. major and T. troglodytes in Portugal [59]. Prevalences of 7 % to 56 % of B. turdi in I. frontalis have been recorded [59]. B. turdi has also been detected in skin biopsies from T. merula, which is a competent transmission host for this bacterium [11, 59]. There is not yet any indication that B. turdi infects humans.
Borrelia burgdorferi sensu stricto
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, 54]. However, as pointed out by Franke et al. (2010), this may just reflect that this species is generally rare in many European regions, rather than to suggest that birds are incompetent reservoirs for European strains of B. burgdorferi s.s [53].
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 co-feeding 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, 60]. This species is known to have rodents as one of its vertebrate reservoirs, but birds are considered to be another of its reservoirs [60, 61].
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. (2013) 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. In similarity to this 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 [60]. 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 [62, 63] 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, 63, 64]. The prevalence of this virus in host-seeking I. ricinus and in ticks removed from hosts in TBEV-enzootic regions is usually <1 % [65]. 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.