Pathogen communities of songbird-derived ticks in Europe’s low countries
Heylen et al. Parasites & Vectors
Pathogen communities of songbird-derived ticks in Europe's low countries
Dieter Heylen 0
Manoj Fonville 2
Arieke Docters van Leeuwen 2
Arjan Stroo 1
Martin Duisterwinkel 5
Sip van Wieren 4
Maria Diuk-Wasser 3
Arnout de Bruin 2
Hein Sprong 2
0 Evolutionary Ecology Group, University of Antwerp , Antwerp , Belgium
1 Centre for Monitoring of Vectors, Netherlands Food and Consumer Product Safety Authority, Ministry of Economic Affairs , Wageningen , The Netherlands
2 Centre for Zoonoses & Environmental Microbiology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment , Bilthoven , The Netherlands
3 EcoEpidemiology Lab, Columbia University , New York , USA
4 Resource Ecology Group, Wageningen University , Wageningen , The Netherlands
5 Independent agricultural entrepreneur , Groningen , The Netherlands
Background: Birds play a major role in the maintenance of enzootic cycles of pathogens transmitted by ticks. Due to their mobility, they affect the spatial distribution and abundance of both ticks and pathogens. In the present study, we aim to identify members of a pathogen community [Borrelia burgdorferi (s.l.), B. miyamotoi, 'Ca. Neoehrlichia mikurensis', Anaplasma phagocytophilum and Rickettsia helvetica] in songbird-derived ticks from 11 locations in the Netherlands and Belgium (2012-2014). Results: Overall, 375 infested songbird individuals were captured, belonging to 35 species. Thrushes (Turdus iliacus, T. merula and T. philomelos) were trapped most often and had the highest mean infestation intensity for both Ixodes ricinus and I. frontalis. Of the 671 bird-derived ticks, 51% contained DNA of at least one pathogenic agent and 13% showed co-infections with two or more pathogens. Borrelia burgdorferi (s.l.) DNA was found in 34% of the ticks of which majority belong to so-called avian Borrelia species (distribution in Borrelia-infected ticks: 47% B. garinii, 34% B. valaisiana, 3% B. turdi), but also the mammal-associated B. afzelii (16%) was detected. The occurrence of B. miyamotoi was low (1%). Prevalence of R. helvetica in ticks was high (22%), while A. phagocytophilum and 'Ca. N. mikurensis' prevalences were 5% and 4%, respectively. The occurrence of B. burgdorferi (s.l.) was positively correlated with the occurrence of 'Ca. N. mikurensis', reflecting variation in susceptibility among birds and/or suggesting transmission facilitation due to interactions between pathogens. Conclusions: Our findings highlight the contribution of European songbirds to co-infections in tick individuals and consequently to the exposure of humans to multiple pathogens during a tick bite. Although poorly studied, exposure to and possibly also infection with multiple tick-borne pathogens in humans seems to be the rule rather than the exception.
Co-infection; Bird; Ixodes ricinus; Borrelia burgdorferi (s; l; ); Borrelia miyamotoi; Rickettsiales
Songbirds are swift transporters of ticks and tick-borne
pathogens, spreading them over long distances on bird
migration and dispersal routes. They are important
pathogen reservoirs and carriers of infected ticks in
areas that are less accessible to mammals, but still
frequently visited by humans, such as islands, green space
and gardens in urbanized areas [
]. Not only their
contribution in the terrestrial cycles of pathogens has
become clear during the past decades, but also their
importance in maintaining tick populations is now
generally recognized [
In Europe, bird-associated Borrelia burgdorferi (s.l.)
species such as B. garinii and B. valaisiana [
been associated with human Lyme borreliosis [
However, limited information is available on the birds’
contribution to the cycles of other human tick-borne
pathogenic agents, as well as the mechanisms of
cooccurrence of more than one pathogenic agent in
individual birds and bird-derived ticks (“co-infection”) .
Understanding the mechanisms underlying co-infections
in ticks is important, as co-infections in hosts in which
tick bites are relatively low (e.g. humans) can result from
the attachment of a single co-infected tick rather than
sequential bites of multiple singly-infected ticks [
Simultaneous infections of multiple pathogen species
can lead to increased pathogenicity, can affect pathogen
proliferation dynamics in the hosts, can influence the
host’s immune responses, can affect the distribution of
pathogens in the host body and can complicate the
diagnosis and treatment of disease [
Here, we investigated the (co-)occurrence of
tick-bornepathogens of humans and domesticated animals, for
which songbirds are believed to potentially contribute to
their maintenance, either as transmission facilitator (i.e.
via local or systemic infections) or as vehicles of infected
ticks. The pathogens considered are B. burgdorferi (s.l.)
], Anaplasma phagocytophilum [
‘Candidatus Neoehrlichia mikurensis’ [
3, 17, 30, 31
] and Borrelia miyamotoi [
Among the avian B. burgdorferi (s.l.) species, B. garinii
is responsible for human neuroborreliosis, while B.
valaisiana has low pathogenicity, if any at all, for humans
]. The epidemiological importance for humans of B.
turdi is currently unknown. Borrelia miyamotoi is a
member of the relapsing fever group of Borrelia spirochaetes
and can be hosted by rodents [
]. Rickettsia helvetica
belongs to the spotted fever group and is an obligate
intracellular bacterium, potentially causing cardiac and
neurological problems in humans [
phagocytophilum is an obligate intra-cellular rickettsia-like
bacterium that can infect neutrophils causing granulocytic
anaplasmosis in humans, livestock and companion
animals . The rickettsia-like bacterium ‘Ca. N.
mikurensis’ is associated with febrile patients [
] and has been
found in tissues of wild rodents [
34, 39, 40
The scope of our study is to identify the members of
the pathogen community in tick species derived from
songbird species in Europe’s Low Countries (Belgium
and the Netherlands), to define their infection
prevalence, and to investigate whether the occurrences of
different pathogenic agents are independent of each other.
Bird trapping and collection of ticks
From 2012 to 2014, trained and experienced
birdbanders opportunistically collected ticks from songbirds
that were caught using Japanese mist nets in seven
locations in the Netherlands (Eemshaven: 53°26′18.91″N, 6°
50′7.77″E; Hijkerveld: 51°52′16.15″N, 4°28′49.84″E;
Schiermonnikoog: 53°29′21.74″N, 6°13′51.27″E; Almere
Oostvaardersdijk: 52°24′20.19″N, 5°10′40.45″E;
Ankeveen: 52°15′51.19″N, 5°5′53.71″E; Nunspeet: 52°23′
29.33″N, 5°49′15.12″E; and Oud Naarden: 52°18′16.93″
N, 5°11′32.29″E) and four in Belgium (Merksplas: 51°
21′29.48″N, 4°51′48.77″E; Vorselaar: 51°12′9.08″N, 4°
46′15.17″E; Wilrijk: 51°10′5.91″N, 4°23′39.43″E; and
Brecht: 51°20′58.75″N, 4°38′15.80″E). No information
was obtained on the number of birds without ticks.
Songbirds could be classified in nine categories following
their foraging habitats (see Table 1) based on the
information provided in a reference work [
] and the expert
knowledge of two experienced bird-watchers of the
University of Antwerp (J. Elst and D. Heylen). Immediately
after collection, the ticks of an individual bird were
immersed in a single tube filled with ethanol (80%),
which was subsequently stored at -20 °C until species
identification and DNA extraction. Ticks were identified
to species and life stage by trained and experienced
technicians who used various taxonomic keys [
DNA extraction, qPCR assays and sequencing procedures
DNA was extracted from ticks using a Qiagen DNA
extraction procedure. For the detection of B. burgdorferi
(s.l.), a duplex qPCR was used, based on the detection
of fragments of ospA and flagellin genes [
conventional PCR assay, targeting the 5S-23S intergenic region
(IGS), was performed for B. burgdorferi (s.l.) species
]. Conventional PCR assays were
carried out in a Px2 thermal cycler (Thermo Electron
Corporation, Breda, the Netherlands) and visualized on a
2% agarose gel. Both strands of PCR products were
sequenced by BaseClear (Leiden, the Netherlands),
according to the company’s protocol and using the same forward
and reverse primers as in conventional PCR. BLAST
analyses and in-house molecular epidemiological databases
(Bionumerics 7.1. - Applied Math, Belgium) were used to
identify Borrelia burgdorferi (s.l.) species. These databases
contain all our DNA sequences from (field) isolates,
together with (reference) sequences from GenBank [
For detection of B. miyamotoi, a qPCR assay was used
that targets a region of the flagellin gene, specific for B.
]. For detection of A. phagocytophilum
and ‘Ca. N. mikurensis’ DNA, a single duplex qPCR
assay was used, which is described elsewhere [
This qPCR assay targets specific regions of genes msp2
(major surface protein 2) for A. phagocytophilum and
groEL (heat shock protein) for ‘Ca. N. mikurensis’. For
detection of R. helvetica, we used a multiplex qPCR
assay, targeting the gltA gene, as described earlier [
All qPCR runs were carried out in a final volume of
20 μl, containing IQ Multiplex Powermix (Bio-Rad,
Hercules, CA, USA), 400 nM of primers and hydrolysis
probes and 3 μl of DNA template. Conditions for PCR
amplification were the following: 95 °C for 5 min, 60
cycles at 95 °C for 5 s and 60 °C for 35 s, followed by a
final incubation step at 37 °C for 20 s. qPCR assays were
carried out on a LightCycler 480 instrument (Roche
Diagnostics Nederland B.V, Almere, the Netherlands)
and analysis was performed by the instrument’s software
(release 126.96.36.199). Quantification cycle (Cq) values were
calculated using the second derivative method.
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Generalized linear mixed effects models (GLMM) were
fitted to test whether co-occurrences of different
pathogen species in individual ticks were independent of
each other (logit-link, binomial-distributed residuals),
taking into account the correlation structure of
cofeeding ticks that were obtained from the same
]. In these models, individual birds were nested
within bird species; both were modelled as random
effects. For the inference of the maximum likelihood
estimates of the fixed effects, Kenward Roger approximation
was used to estimate the denominator degrees of freedom
of the F-distributed test statistics, which takes into
account the correlation of observations within the same
]. For those bird species of which at least 10
individuals were caught, mean tick infestation intensity
(i.e. the average number of ticks in infested individuals)
was calculated. For bird species with at least 10 infected
individuals per tick stage, estimates of proportions of
infected ticks are given by their arithmetic mean ±
standard error (i.e. the square root of the estimated variance/
the square root of the number of bird individuals) in
the main text. Data management and statistical
analyses were performed using SAS v9.2 (SAS Institute,
Cary, North Carolina, USA).
Overall, 375 infested individual birds were trapped,
belonging to 35 different species that could be classified
into nine categories based on foraging habitats (Table 1).
Thrushes (Turdus merula: 173, T. philomelos: 53, T.
iliacus: 33) were trapped most often, representing 69% of
the total number of infected birds. A total of 671 ticks
was collected from the birds, belonging to three species:
Ixodes ricinus (134 larvae, 479 nymphs and 1 adult
female; collected from 334 birds), I. frontalis (40 larvae, 5
nymphs and 11 adult females; collected from 46 birds)
and one Hyalomma spp. nymph from Sylvia borin (Table
1). Six individual birds (2 T. merula, 2 T. iliacus, 1 T.
philomelos and 1 Acrocephalus scirpaceus) were infected
with both I. ricinus and I. frontalis.
For I. ricinus, tick infestation intensity was highest for
Prunella modularis (4.0 ± 1.5; n = 13), followed by T.
iliacus (2.2 ± 0.4; n = 33), Erithacus rubecula (2.0 ± 0.6; n =
13) and T. merula (1.9 ± 0.2; n = 139). For I. frontalis, the
infestation intensity for T. merula was 1.0 ± 0.0 (n = 36).
Pathogens in bird-derived ticks
All 671 ticks were individually screened for the presence
of B. burgdorferi (s.l.), B. miyamotoi, A. phagocytophilum,
‘Ca. N. mikurensis’ and R. helvetica (Table 2). Overall,
50.9% (341/670) of ixodid ticks collected were found
infected with one or more of these pathogens [I. ricinus:
54% (333/614), I. frontalis 14% (8/56)]. We found none of
these pathogens in the one Hyalomma spp. nymph.
We detected B. burgdorferi (s.l.) DNA in 33.9% (227/
670) of all ixodid ticks. The highest proportion of B.
burgdorferi (s.l.) positive larvae was observed in T.
philomelos (91.6 ± 0.8%; n = 12 infested birds), followed by T.
merula (30.5 ± 9.2%; n = 24 birds) and T. iliacus (9.09 ±
9.09%; n = 11 birds). The proportions of positive I.
ricinus were higher in nymphs than in larvae when
collected from T. merula (53.6 ± 4.2%; n = 122 birds) and T.
iliacus (25.4 ± 8.3%; n = 28 birds) but not from T.
philomelos (61.7 ± 7.7%; n = 40 birds). From latter members of
the Turdidae family, we mainly found avian species (B.
garinii, B. valaisiana and B. turdi) in both larvae and
nymphs (Table 3). In Prunella modularis (mean
prevalence: 20.2 ± 8.5%; n = 12 infested birds) only the
mammal-associated B. afzelii (8 infected nymphs
belonging to 5 infested birds) was found. Overall, for the
complete set of Borrelia-infected ticks for which the
Borrelia-genotyping was successful (173 tick individuals
belonging to 15 bird species), avian species were
detected in all developmental stages, while B. afzelii was
detected in nymphs only (Table 3). Borrelia turdi was
found in I. frontalis (2 adult females) and I. ricinus (3
The occurrence of B. miyamotoi in ticks was very low
[prevalence in ixodid ticks: 0.6% (4/670)]. It was only
found in one I. ricinus larva from E. rubecula and one I.
ricinus nymphs from T. merula, Phylloscopus collybita,
T. philomelos each.
For A. phagocytophilum, we detected DNA in 5.1%
(34/670) of all ixodid ticks. The nymphal infection
prevalence in the four bird species with more than 10
infested birds varied between 1.4 ± 0.9% (P. modularis)
and 8 ± 4.9% (T. iliacus). Furthermore, A.
phagocytophilum DNA was found in two I. frontalis females from one
individual blackbird as well.
The overall prevalence of ‘Ca. N. mikurensis’ in the
ixodid ticks was 4.4% (30/670). A high prevalence was
found in I. ricinus nymphs collected from T. philomelos
(22.5 ± 6.7%; n = 40 infested birds), but below 5.5% in the
other Turdidae. Only two larvae, I. ricinus collected
from Troglodytes troglodytes and T. iliacus, carried ‘Ca.
N. mikurensis’ DNA.
Compared to the other rickettsial infections, the
number of ticks with R. helvetica - DNA was high [overall
prevalence: 21.6% (145/670); Table 2]. Infection
prevalence ranged from 10.7 ± 5.4% (T. iliacus; n = 28 infested
birds) to 29.2 ± 7.2% (T. philomelos; n = 40 infested
birds) in those bird species with at least 10 nymphs. But
also in the bird species of which we obtained a smaller
amount of information (i.e. less than 10 infested birds)
high prevalence was registered (e.g. nymphs in E.
rubecula: 41.7 ± 17.5%; n = 8; Sylvia communis: 42.9 ± 20.2%;
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n = 7; Phoenicurus phoenicurus: 28.6 ± 18.4%; n = 7).
Rickettsia helvetica-positive I. ricinus larvae were
collected from 12 different songbird species. In general,
larval prevalence was lower, but still high (range in
Turdidae: 14.6 ± 7.0% – 25.0 ± 13.1%). In addition, this
bacterium was detected in an I. frontalis larva collected
from T. merula and two I. frontalis adult females from
C. caeruleus and T. philomelos.
Over 10% (13.4%, 90/671) of bird-derived ticks
contained DNA of more than one pathogenic agent. At the
bird level, 19.7% of individual birds (74/375) carried
ticks with a co-infection. In I. ricinus larvae and nymphs,
the most common pathogen combinations were ‘B.
burgdorferi (s.l.) + R. helvetica’ (larvae: 6 ticks; nymphs: 46
ticks over 44 birds), followed by ‘B. burgdorferi (s.l.) +
‘Ca. N. mikurensis’ (larvae: 0 ticks; nymphs: 17 ticks
over 17 birds) and ‘B. burgdorferi (s.l.) + A.
phagocytophilum’ (larvae: 0 ticks; nymphs: 17 ticks over 11 birds).
There was no statistical evidence for an association
between B. burgdorferi (s.l.) and R. helvetica neither in I.
ricinus nymphs (F(1,204) = 0.35, P = 0.55) nor I. ricinus
larvae (F(1,21) = 0.04, P = 0.83) when taking host species
and bird individual into account as a random effects.
The same holds for B. burgdorferi (s.l.) and A.
phagocytophilum (nymphs: F(1,205) = 3.00, P = 0.09; larvae: no
model convergence). We did find a positive correlation
between the occurrence of ‘Ca. N. mikurensis’ and B.
burgdorferi (s.l.) (nymphs: logit absence-presence = -1.02 ±
0.46, F(1,205) = 4.9, P = 0.028; larvae: no model
convergence). The analysis on the level of individual birds in
which for each bird a binary value (“1”: at least one
infected tick, or “0”: no infected ticks) was assigned as
response variable did not change the conclusions (no
significant association between B. burgdorferi (s.l.) and
A. phagocytophilum, nor B. burgdorferi (s.l.) and R.
helvetica: all P-values > 0.33). The same holds for the
association between ‘Ca. N. mikurensis’ and B. burgdorferi
(s.l.) (logit absence-presence = -1.48 ± 0.49, F(1,300) = 8.99, P =
0.003). Due to low sample sizes, associations between
other pathogen combinations could not be analyzed.
The following rare combinations of pathogens in ticks
were found: R. helvetica + ‘Ca. N. mikurensis’ (larvae: 1
tick; nymphs: 9 ticks over 9 birds); A.
phagocytophilum + R. helvetica (larvae: 1 tick; nymphs: 7 ticks over 6
birds); B. burgdorferi (s.l.) + B. miyamotoi (1 nymph); B.
miyamotoi + ‘Ca. N. mikurensis’ (1 nymph). Six nymphs
(over 6 birds) carried DNA of B. burgdorferi (s.l.), R.
helvetica and ‘Ca. N. mikurensis’ and three nymphs were
infected with the combination B. burgdorferi (s.l.) + R.
helvetica + A. phagocytophilum (over 2 birds). Also in
the smaller set of I. frontalis ticks, we found the
coinfection B. burgdorferi (s.l.) + A. phagocytophilum in
two adult females collected from a single T. merula.
We have shown that half of the songbird-derived I.
ricinus ticks, that readily feed on humans, contained DNA
of one or more bacteria that are pathogenic to humans:
B. burgdorferi (s.l.), R. helvetica, A. phagocytophylum, B.
miyamotoi, ‘Ca. N. mikurensis’. The presence of the
DNA in the ticks shows that songbirds carry infected
ticks and may facilitate bacterium transmission.
Transmission facilitation via birds may occur either via the
infection of bird tissue on which ticks feed or via
cofeeding of ticks in close spatial and temporal proximity
to each other. The latter transmission pathway can occur
in the absence of a systemic infection, allowing some
pathogens (e.g. Borrelia species) to evade the hostile
immune system of otherwise incompetent hosts [
Ground-dwelling birds, especially the members of the
family Turdidae, had the highest infestation intensities
and also yielded the highest numbers of infected ticks
overall. They are known to run a greater risk of I. ricinus
exposures, as they forage inside the habitat of this
abundant tick species (i.e. ground and lower vegetation
]. Particularly the blackbird (T. merula) and
the song thrush (T. philomelos), two very common birds
of European woodlands and gardens, contributed
strongly to the number of infected ticks. In line with
other European studies, birds were predominantly
infested by immature I. ricinus stages and rarely by adult
females. Adult I. ricinus are typically found on
mediumsized and larger mammals (e.g. roe deer) on which they
copulate . In contrast but not surprising, we found
substantial numbers of semi-engorged adult female I.
frontalis on the birds; all developmental stages I.
frontalis feed on birds [
We found a strong association of B. garinii, B.
valaisiana and B. turdi with avian reservoir hosts,
which has previously been shown by numerous
European field studies concluding that birds act both as
competent reservoirs and transmitters for these species
10–12, 29, 58–60
]. Given that vertical transmission of
B. burgdorferi (s.l.) spirochetes in I. ricinus ticks only
rarely occurs [
], their presence in larvae (Tables 2,
3) indicates that they were acquired either via (local)
infection in the host or via co-feeding with an infected
nymph. Borrelia turdi, recently discovered in Europe
and strongly associated with I. frontalis, was also found
in I. ricinus nymphs. Transmission experiments have
shown that I. ricinus can transmit B. turdi to naïve
avian hosts and, seen the extreme host range of this
tick species, I. ricinus could potentially act as a bridging
vector towards mammals, including human . Recent
experimental and observational studies based on larval
and nymphal infections show non-homogeneous
distributions of the avian Borrelia species in bird-derived ticks,
indicating differential transmission and amplification of
these species depending on the avian reservoir hosts and
tick species [
12, 60, 63
An interesting outcome of our study and previous
field studies is that several of the ground-dwelling birds
(T. merula, T. philomelos, E. rubecula and P. modularis)
were frequently infested with ticks that carried B.
afzelii, a species that is associated with rodent reservoir
]. The fact that all B. afzelii-infected ticks
were nymphs suggests that these individuals had fed as
a larva on a B. afzelii-infected mammal, moulted and
maintained their infections when feeding on the birds.
Findings of bird-derived infected larvae in other studies
have led to speculations that particular strains of B.
afzelii can also use bird hosts for transmission .
PCR-based screening outcomes, like ours and others,
should, however, be interpreted with caution as they
are based on the detection of specific DNA sequences
and do not necessarily mean that viable, infectious
microorganisms are present. A recent experimental study
investigating transmission of B. afzelii in blackbirds and
great tits showed that nymph-to-adult transstadial
transmission of B. afzelii DNA could occur. However, the
positive signal in the adult ticks turned out not to be viable
and infectious spirochetes, as shown by the BSK culture
]. It is, therefore, necessary to identify the B.
afzelii strains found in bird-derived ticks from the wild
and test via culture-based infection methods and tick
transmission experiments whether they are still
infectious and transmittable after being exposed to bird
blood during tick feeding.
Also for the more sporadically observed
mammalassociated pathogens (B. miyamotoi and ‘Ca. N.
mikurensis’) that we detected in bird-derived ticks, including
larvae (Table 2), experiments are needed to investigate
whether viable bacteria survive the birds immune system
and/or are transmitted during tick feeding. Studies in
the USA and Europe have implicated small rodent
species as the reservoir hosts for B. miyamotoi [
a limited number of studies reported B. miyamotoi
infections from bird-derived ticks as well [
17, 29, 68
]. For the
rodent-associated ‘Ca. N. mikurensis’  observed in
bird-derived I. ricinus larvae and nymphs of our study
and others [
], a role for songbirds as transmission
facilitators could be expected.
Compared to the other A. phagocytophilum and ‘Ca.
N. mikurensis’, the number of ticks with R. helvetica
DNA was high. A good comprehension of the
transmission dynamics of rickettsial bacteria in songbirds is still
lacking. Within infected ticks, a proportion of the
bacteria could have a maternal origin, as R. helvetica, in
contrast to Borrelia burgdorferi (s.l.), can be transmitted
]. However, the experimental study
of Heylen et al.  using great tits (Parus major)
exposed to a community of pathogens, clearly shows rapid
R. helvetica transmission via co-feeding (cf. mammals
]) and/or fast systemic infection (as found in
mammals experimentally injected with different rickettsial
species ). Our and other’s finding of R. helvetica in
bird-derived ticks, including larvae [
3, 29–31, 51, 74, 75
reinforces the presumption that songbirds can become
bacteraemic and effectively facilitate the transmission of
this pathogen via host tissue [
Further, our study provides evidence that
grounddwelling birds, especially thrushes, are important hosts
in the transmission cycles of A. phagocytophilum.
Bacteraemia of this pathogen has been shown to develop in
], which is likely the reason for the reports
of infected bird-derived ticks here and other locations
25, 26, 29, 30, 76
]. Probably not all birds are equally
competent in the transmission; in a great tit (Parus
major) experiment by Heylen et al. [
] no transmission
facilitation occurred despite the presence of A.
phagocytophilum in challenge nymphs. Our finding of infections
in a bird-specialized tick (I. frontalis) that is never found
on other vertebrate hosts (two infected adult females
cofeeding with infected I. ricinus nymphs on the same
blackbird individuals) gives further indication that birds
facilitate A. phagocytophilum transmission, either via
cofeeding transmission or systemic infections. Although
the host-specific strains of A. phagocytophilum were not
identified, the avian ecotype IV that has been isolated by
Jahfari et al. [
] from blackbird tissues and
blackbirdderived ticks is to be expected.
The co-infections found in individual ticks and birds
strongly suggest that simultaneous transmission of
different bacterium species can occur and that birds are
permissive for multiple pathogens, as experimentally
shown in Heylen et al. [
]. Especially, the fact that
coinfections were found in (sets of ) larvae provides the
strongest indication. However, larvae could also acquire
pathogens via the maternal line from other hosts than
the individual songbird from which they were collected,
through vertical transmission (e.g. in B. miyomatoi and
R. helvetica) [
69, 70, 77
]. In larvae, the most frequent
observed co-infection was B. burgdorferi (s.l.) with R.
helvetica (E. rubecula, T. troglodytes, T. philomelos), but
also A. phagocytophilum with R. helvetica and ‘Ca. N.
mikurensis’ with R. helvetica (both from T. iliacus)
were observed. Only for one bacterial combination, ‘Ca.
N. mikurensis’ + B. burgdorferi (s.l.), we found that the
occurrence of the one pathogen is more likely when
another pathogenic agent is present. Interestingly, also in
mammals, this combination of pathogens was much
higher than expected from the prevalence of each
]. This positive association could be the
result of variation in general susceptibility among birds,
but could also indicate transmission facilitation, as has
been suggested in other studies on tick-borne
18, 34, 78
]. The pathways that lead to
facilitation can only be elucidated with experimental studies
in which pathogen-driven physiological, cellular and
biochemical interactions are disentangled.
Our findings highlight the contribution of songbirds to
co-infections in individual ticks. In addition, not only
avian but also mammalian bacterium species are
transported via bird-derived ticks, highlighting the need to
experimentally test whether latter pathogens are viable and
infectious in birds. Furthermore, future studies should
focus on the reservoir competence of members in the bird
community and how the different vector-bird-niches
contribute to the pathogen transmission dynamics.
We would like to thank Marieta Braks (RIVM) for critically proofreading the
manuscript and Joris Elst (University of Antwerp) for sharing his expert
knowledge on the foraging habits and habitats of local songbirds.
This study was financially supported by the Dutch Ministry of Health, Welfare
and Sport (VWS), the Fund for Scientific Research - Flanders Belgium (FWO)
(grant G0.049.10) and the University of Antwerp (KP BOF UA 2015). This work
was done under the frame of EurNegVec Cost Action TD1303. Dieter Heylen
is a postdoctoral fellow at the Fund for Scientific Research - Flanders Belgium
(FWO). The funders had no role in study design, data collection, interpretation
and analysis, decision to publish or preparation of the manuscript.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Representative
sequences were submitted to the GenBank database with the accession
DH and AdB analysed data and wrote the final manuscript. DH collected tick
from trapped birds in Belgium and AS, SW and MD from birds in the Netherlands.
DH, SW and MF performed identifications of tick species and tick stages. ADvL,
MF and SJ performed and analysed laboratory tests. HS organized and supervised
the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was carried out according to the national animal welfare regulations.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
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