West Nile virus host-vector-pathogen interactions in a colonial raptor
Soltész et al. Parasites & Vectors
West Nile virus host-vector-pathogen interactions in a colonial raptor
Zoltán Soltész 0 1 3
Károly Erdélyi 2
Tamás Bakonyi 6 7
Mónika Barna 7
Katalin Szentpáli-Gavallér 2
Szabolcs Solt 5
Éva Horváth 5
Péter Palatitz 5
László Kotymán 4
Ádám Dán 2
László Papp 9
Andrea Harnos 8
Péter Fehérvári 0 8
0 Hungarian Natural History Museum , Budapest , Hungary
1 Lendület Ecosystem Services Research Group, MTA Centre for Ecological Research , Vácrátót , Hungary
2 National Food Chain Safety Office , Veterinary Diagnostic Directorate, Budapest , Hungary
3 Lendület Ecosystem Services Research Group, MTA Centre for Ecological Research , Vácrátót , Hungary
4 Körös-Maros National Park Directorate , Szarvas , Hungary
5 MME/BirdLife Hungary, Red-footed Falcon Conservation Working Group , Budapest , Hungary
6 Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, University of Veterinary Medicine , Vienna , Austria
7 Department of Microbiology and Infectious Diseases, University of Veterinary Medicine , Budapest , Hungary
8 Department of Biomathematics and Informatics, University of Veterinary Medicine , Budapest , Hungary
9 Hungarian Academy of Sciences, Biological Section , Budapest , Hungary
Background: Avian host species have different roles in the amplification and maintenance of West Nile virus (WNV) , therefore identifying key taxa is vital in understanding WNV epidemics. Here, we present a comprehensive case study conducted on red-footed falcons, where host-vector, vector-virus and host-virus interactions were simultaneously studied to evaluate host species contribution to WNV circulation qualitatively. Results: Mosquitoes were trapped inside red-footed falcon nest-boxes by a method originally developed for the capture of blackflies and midges. We showed that this approach is also efficient for trapping mosquitoes and that the number of trapped vectors is a function of host attraction. Brood size and nestling age had a positive effect on the number of attracted Culex pipiens individuals while the blood-feeding success rate of both dominant Culex species (Culex pipiens and Culex modestus) markedly decreased after the nestlings reached 14 days of age. Using RTPCR, we showed that WNV was present in these mosquitoes with 4.2% (CI: 0.9-7.5%) prevalence. We did not detect WNV in any of the nestling blood samples. However, a relatively high seroprevalence (25.4% CI: 18.8-33.2%) was detected with an enzyme-linked immunoabsorbent assay (ELISA). Using the ELISA OD ratios as a proxy to antibody titers, we showed that older seropositive nestlings have lower antibody levels than their younger conspecifics and that hatching order negatively influences antibody levels in broods with seropositive nestlings. Conclusions: Red-footed falcons in the studied system are exposed to a local sylvatic WNV circulation, and the risk of infection is higher for younger nestlings. However, the lack of individuals with viremia and the high WNV seroprevalence, indicate that either host has a very short viremic period or that a large percentage of nestlings in the population receive maternal antibodies. This latter assumption is supported by the age and hatching order dependence of antibody levels found for seropositive nestlings. Considering the temporal pattern in mosquito feeding success, maternal immunity may be effective in protecting progeny against WNV infection despite the short antibody half-life measured in various other species. We conclude that red-footed falcons seem to have low WNV host competence and are unlikely to be effective virus reservoirs in the studied region.
Culicidae; Transmission ecology; Mosquito trap; Arthropod vector; Passive immunity; Host competence; Falco vespertinus; Lineage 2; Antibody
West Nile virus (WNV) is the most widespread member of
the arthropod-borne group of the genus Flavivirus, family
]. Virus strains belonging to genetic lineages
1 and 2 have been causing an increasing number of
epidemics in North America [
] and Europe [
WNV is considered one of the most important pathogens
causing viral neurological disease in humans .
The virus is maintained in an enzootic cycle between
vectors and avian hosts, while humans [
], equines [
and other vertebrate taxa are predominantly dead-end
]. Therefore, to assess human infection risks and
predict the spatio-temporal patterns of disease outbreaks
it is vital to better understand the complex avian
hostmosquito vector transmission ecology of WNV [
wide array of bird species have been identified as potential
virus amplifying hosts . Competent arthropod vectors
also belong to a range of taxa [
ornithophilic mosquitoes (Diptera: Culicidae) are established to
be the group predominantly responsible for maintaining
the sylvatic cycle of the virus.
Pathogenicity in birds seems to be rather species-specific,
and the effect of infection ranges from subclinical to rapid
development of fatal neuropathy [
]. WNV can also have
a substantial negative impact on an avian population and
may even demand attention in the conservation
management of high priority species [
]. However, morbidity
and mortality rates do not necessarily reflect the
epidemiological role of a host species . A more sophisticated
approach is to evaluate or quantify host competence, i.e. the
ability of a host to generate infection in another susceptible
]. Recent studies focusing on WNV host
competence were able to pinpoint avian “superspreader” and
“supersuppressor” species in North America, and through
these, they were able to explain the geographical variation
in human spillover rates . In the complex WNV
hostvector system, host competence is a function of the
magnitude and length of viremia, vector contact rates and host
mortality rates [
]. Estimating these parameters for
individual species, however, requires a combination of
laboratory experiments and field studies which may not be
feasible for endangered species. Here, we present a case
study where we implemented a comprehensive study
design using various methods simultaneously to evaluate host
competence of a high conservation value species in WNV
circulation under natural conditions.
The studied avian host was the red-footed falcon
(Falco vespertinus), a species of high international
conservation concern [
]. WNV has been reported to
cause central nervous disease and mortality in a few
sporadic cases for nestlings, but not in adult birds in
Hungary . Red-footed falcons are long-range
transSaharan migrants [
] and may therefore be amongst
the candidate species responsible for large spatial scale
dispersal of WNV. These raptors are also facultative
colonial breeders [
] and therefore potentially more
vulnerable to rapidly spreading infections [
to territorially breeding birds. Nestlings and juvenile
birds are thought to be important in viral amplification
] as they are localized in the nest and presumably
have a less effective immune response against infections.
We, therefore, concentrated on red-footed falcon broods
and their relationship with vectors and WNV.
Despite its importance, host-vector interaction in WNV
transmission ecology studies is often neglected as there are
no widely accepted methods currently available to quantify
it. Instead, vectors collected with traps that attract
mosquitoes through visual and/or olfactory cues (e.g. CDC traps)
are used to assess virus presence, quantify virus prevalence
in vectors and as a proxy to potential vector loads on hosts
]. Albeit these methods are cheap and easy to implement
they only account for vector abundance, or rather the
availability of mosquitoes reacting to the attractant, but
completely fail to provide information on actual host-vector
contact rates. A more promising method is to use live birds
as baits to attract mosquitoes [
]. However, these are
limited in the possible number of bait-species that can be used
and also fail to account for avoidance strategies of hosts.
Tomás et al. [
] suggested and used an easy yet effective
method to quantify biting midges (Ceratopogonidae) and
blackflies (Simuliidae) in Passerine nest-boxes with the help
of a non-invasive adhesive. If also applicable to trap
ornithophilic mosquitoes, this method may revolutionize in situ
WNV transmission ecology studies as it allows to directly
measure the vector species composition and the effects of
nestling characteristics on attracted and blood-fed vectors.
Here, we initially aimed to evaluate whether WNV
vectors can be trapped directly in the vicinity of
redfooted falcon broods and whether we can quantify
attraction patterns, virus prevalence, and blood-feeding
success of vectors attracted by the studied hosts.
Simultaneously, we aimed to estimate WNV status of falcon
broods through estimating seroprevalence and frequency
of nestlings in viremia, to assess the population level
effects of WNV on the host species, and to evaluate the
potential virus reservoir role of red-footed falcons.
Field work was carried out during June and July each year
from 2010 to 2012, at the Vásárhelyi-plains (46°28'16"N,
20°36'17"E) protected an area of the Körös-Maros
National Park Directorate in southern Hungary. The study
site holds 4 artificial nest-box colonies where over 100
pairs of red-footed falcons breed each year [
], along with
numerous kestrels (Falco tinnunculus), jackdaws (Corvus
monedula) and long-eared owls (Asio otus) [
parameters of birds using the nest-boxes was collected
within the scope of an ongoing long-term research
]. Therefore, detailed information such as egg
laying dates, clutch size, brood size, and fledging success
was readily available. All broods selected for the study
were breeding in the same type of standard nest-box. The
area is renowned for a large saline lake and other wetlands
that are important stop-over and wintering sites of various
wader and geese species and common cranes (Grus grus).
The surrounding habitat is characterized by a mosaic of
grasslands and arable fields, with an extensive network of
channels and drainage ditches, providing ample
possibilities for blood sucking dipterans to breed.
We used a modified version of the methods described by
Tomás et al. [
] to estimate the number of vectors
attracted by red-footed falcon broods. Initially, we applied
5 ml gel adhesive (Johnson’s Baby Oil Gel with Chamomile;
Johnson & Johnson, Dusseldorf, Germany) on one side of
10 × 15 cm transparent (0.2 mm) plastic sheets. We then
secured these sheets (with the gel facing upwards) on the
inner side of the nest-boxes’ roofs for 24 h. The sheets were
secured with board pins in a fashion to create an arc, thus
allowing ample space for flying arthropods to get trapped
(Fig. 1). As opposed to other adhesives, the advantage of
the gel is that it is easily dissolved with petrol, leaving
trapped arthropods unharmed. This allows trapped
Culicidae specimens to be reliably identified, sexed and it is also
possible to evaluate their feeding status. All collected
animals were stored in 70% ethanol after the gel was dissolved.
The species, sex and feeding status (blood-fed or not) was
identified under a stereomicroscope (Olympus SZ-50,
Olympus Co. Tokyo, Japan).
Sampling was carried out in three consecutive breeding
seasons from 2010 to 2012. Each year we first randomly
selected empty nest-boxes within each colony. These were
used to test whether hematophagous vectors are attracted
to either the nest-box or the adhesive gel alone (56
samples collected) by comparing the results obtained here
with that in active nests. At the same time, we used a
stratified random sampling approach to select broods
where mosquito traps were placed. Strata considered were
colony and breeding stage, the latter classified into five
groups; before hatching (incubation), 1st week, 2nd week,
3rd week and 4th week after hatching. Red-footed falcons
incubate throughout the night and also brood young
nestlings at night. Presumably, mosquitoes react to the
incubating parent rather than the eggs themselves. Thus our
analyses may be interpreted as a comparison between a
single adult (incubation), an adult with small nestlings (1st
week) and older nestlings alone.
Weather is a key factor controlling mosquito activity
] and consequently WNV infection rates .
Therefore, we only selected three days each year for
sampling to minimize variation caused by changes in
ambient temperature and humidity. On each date broods
in various breeding stages were sampled, thus creating a
cross-section of the host population. Altogether a total
of 309 samples collected in 166 clutches were used in
the analyses (Table 1).
Assessing WNV prevalence in vectors
WNV prevalence has been shown to cumulate in
vertebrate hosts late in the summer [
], therefore to minimize
laboratory costs and to maximize the probability of
detecting WNV presence we only used mosquitoes trapped in
the late breeding stage in 2011. We first grouped the
samples by species, sex and according to the nest-box, the
animals were trapped in. Specimens that were blood filled
were excluded from the analyses to avoid inconclusive
results. A total of 779 mosquitoes of three species (Culex
pipiens, Culex modestus and Coquillettidia richiardii)
were investigated, the majority being Cx. pipiens (725
individuals). We then selected every 6th specimen from
these subsets for individual WNV identification. The
remaining animals were pooled (5 × 4 = 20 individuals/
pool) and also analysed [Cx. pipiens: 145 individuals, 29
pools; Cx. modestus: 3 pools (20 + 20 + 7 individuals); Cq.
richiardii: 1 pool (7 individuals)]. Samples were tested for
the presence of WNV nucleic acid (RNA) by a
reversetranscriptase - polymerase chain reaction (RT-PCR) using
the primer pair FL1-f and FL1-r as described earlier [
Specific PCR products were sequenced directly from both
ends with the same primers [
WNV seroprevalence in red-footed falcon nestlings
We used 42 broods that were also selected for mosquito
abundance sampling to assess WNV seroprevalence in the
studied population. The samples were chosen to represent
both the spatial heterogeneity of breeding pairs (colonies)
and temporal heterogeneity of the egg laying dates. Blood
samples of 0.8–1.0 ml were taken by basilic venipuncture
from fledgelings (n = 139) reaching the second half of the
breeding stage (17–24 days) in 2011. Sera were separated
from coagulated blood samples and stored at -20 °C until
processing. The remaining serum and cellular elements
were stored at -80 °C until being further processed by
simultaneous RNA and DNA extraction using the Roche
High Pure Viral Nucleic Acid kit (Lewes, United
Kingdom). Serum samples were tested for the presence of
antiWNV antibodies using the ID Screen® West Nile
Competition ELISA kit (ID VET, Montpellier, France), according
to the manufacturers’ instructions. This diagnostic kit
detects IgY antibodies by competitive ELISA directed against
the Pr-E envelope protein of the West Nile virus. We used
2 ELISA plates for the analyses. Obtained optical density
(OD) values were transformed into OD ratios (i.e.
Competition Rate = (OD sample / OD Negative control) × 100). These
ratios were then handled and analysed with two different
approaches. First, we elaborated the recommendations of
the manufacturer and defined cut off levels of the test as
positive (competition rate ≤ 40%); doubtful (40% <
competition rate ≤ 50%); and negative (competition rate > 50%).
Secondly, we considered OD ratio as a proxy for antibody
levels (e.g. [
]) and analysed these values on a
continuous scale (see Statistical analyses). Blood samples were
also tested for the presence of WNV nucleic acid (RNA)
with the same technique described above.
To understand the relationship between blood-sucking
dipteran abundance and host traits we used generalized
linear mixed effects models (GLMM) with Poisson
distribution and log link function [
]. In the next step,
we used GLMMs with binomial distribution and logit
link function to assess how the ratio of blood filled
parasites is affected by these variables. Random factors were
the date of sampling, Nest ID and Colony ID for all
aforementioned models. All dipteran species were
analysed in separate models.
To estimate the WNV seroprevalence in red-footed
falcon nestlings, we applied the methods described in
Messam et al. [
]. We used the same procedure to
estimate WNV prevalence in the vector species, using only
the non-pooled samples for the analysis.
We used linear mixed effects (LME) models [
to estimate the relationship between WNV ELISA OD
ratios and nestling characteristics. First, we selected
ELISA-positive nestlings and modelled their OD ratios
as a function of nestling age measured in days. We then
selected all nestlings in broods with at least one
WNVseropositive nestling and assessed their hatching order
based on weight (measured with 300 g spring scale to
the nearest 2 g), the length of central tail feathers
(measured with a ruler to the nearest 1 mm), wing chord
(measured with a ruler to the nearest 1 mm) and wing
bone (measured with a caliper to the nearest 0.1 mm)
lengths recorded at the time of sampling. This hatching
order was subsequently used as a predictor for nestling
OD ratios. The Nest ID and the ELISA Plate ID were
used as random factors in case of both LME models. We
also ran the models with the full set of nestlings and
obtained numerically similar results.
We used the decrease of deviance and the likelihood
ratio test (LR) to select non-significant variables in case
of all models described above. All analyses were carried
out in R, version 3.2.3 [
] using the following packages;
], epiR [
], epitools [
], nlme [
], lme4 [
], effects [
] lmeans [
Host-dependent mosquito attraction and blood-feeding success patterns
We trapped a total of 11,592 mosquitoes belonging to 4
species from red-footed falcon nest-boxes, namely Culex
pipiens Linnaeus, 1758 (n = 10,203), Culex modestus
Ficalbi, 1889 (n = 1332), Coquillettidia richiardii Ficalbi,
1889 (n = 56), Ochlerotatus dorsalis (Meigen, 1830)
(n = 1). All trapped individuals were females. As the
latter two species had orders of magnitude lower
abundance compared to Cx. pipiens and Cx. modestus we
excluded them from further analyses. We did not trap
any mosquitoes in control nest-boxes; however, other
arthropods like canopy dwelling or non-parasitic nest
substrate feeding species were trapped in small numbers.
Brood size and the breeding stage had a significant effect
(Poisson GLMM, LR χ2 test; brood size χ2 = 132.06, df = 1,
P < 0.001, breeding stage χ2 = 181.39, df = 1, P < 0.001) on
the number of Cx. pipens in nest-boxes. Considering the
latter variable, the number of individuals significantly
increased after hatching, peaked at the 2nd week, and from
here on it significantly decreased until fledging. (Table 2,
Fig. 2a). Meanwhile, the ratio of blood-fed Cx. pipiens
individuals was also significantly affected by breeding stage
(Binomial GLMM, LR χ2 test, breeding stage χ2 = 29.83, df = 1,
P < 0.001; brood size χ2 = 0.05, df = 1, P = 0.82); the
probability of finding a blood-engorged mosquito was highest in
the 1st and 2nd week after hatching, and later significantly
decreased for the second half of the nestling stage (Table 3,
Fig. 2b). In the case of Cx. modestus, brood size significantly
increased while breeding stage did not affect (Poisson
GLMM, LR χ2 test; brood size χ2 = 24.6, df = 1, P < 0.001;
breeding stage χ2 = 2.17, df = 1, P = 0.71) the number of
attracted mosquitoes (Table 2, Fig. 3a). The pattern of the
ratio of blood-fed Cx. modestus (Fig. 3b) individuals
resemble the pattern of Cx. pipiens (Fig. 2b). However, the data
did not allow to completely replicate the same analysis as
only 2 blood-fed Cx. modestus individuals were trapped in
the incubation period (Table 3, Fig. 3b).
WNV prevalence in vectors
We found 1 pooled and 6 individual Cx. pipiens (n = 145)
samples to be WNV-positive, corresponding to a 4.2%
(95% CI: 0.9–7.5%) virus prevalence assuming perfect test
detection (calculated only for individual samples). The
positive samples were found in three nest-boxes, each in a
different colony, indicating WNV carrying mosquito
presence throughout the study site. One nest-box had 5
WNV-positive individual Cx. pipiens samples while the
remaining 1 individual and 1 pooled sample were from
different nest-boxes and different colonies. Sequence
analysis determined that the detected virus belongs to the
genetic lineage 2 of WNV and it is closely related to the
WNV isolates from the study year and previous years [
WNV seropositivity in red-footed falcons
Our results showed relatively high seroprevalence among
red-footed falcon nestlings; 35 of the sampled 134
individuals were ELISA-positive, while 10 were classified as
doubtful (25.4% ± 3.7% SE, CI: 18.8–33.2%). However, we
did not detect WNV by RT-PCR in any of the nestling
blood samples, suggesting that none of the individuals
were viraemic at the time of sampling. Of the 42 broods,
ablood-feeding success was not estimated during incubation due to sample size constraints
Abbreviation: SE standard error
16 had at least one seropositive nestling. We found no
evidence of large scale spatial pattern of the observed
seropositivity, as the ratio of seropositive broods did not differ
significantly between colonies (Fischer’s exact test:
P = 0.94). Moreover, we could not detect any temporal
pattern either, as there was no significant difference in
mean egg laying date between broods without seropositive
and broods with at least one seropositive nestling (Welch
t-test: t = -0.1095, df = 34.601, P = 0.91). However, the
OD ratio significantly decreased with increasing nestling
age among ELISA-positive nestlings (LME LR χ2 test;
nestling age χ2 = 4.97, df = 1, P = 0.02) indicating that
older seropositive nestlings have lower antibody levels.
We also found a significant decrease in OD ratios
corresponding with within brood hatching order among broods
with at least a single ELISA-positive nestling (LME LR χ2
test; hatching order χ2 = 5.36, df = 1, P = 0.02) (Fig. 4).
This shows that nestlings hatching later in a brood had
higher antibody levels.
Here, we comprehensively investigated vector attraction
patterns, blood-feeding success rate, WNV prevalence and
host serum seroprevalence under natural conditions in a
colonial raptor. Initially, we verified that our modified
version of the trap described by Tomás et al. [
effective in collecting Culicidae species and that the trapped
individuals were attracted by the hosts. The two most
common mosquito species (Cx. pipens and Cx. modestus)
attracted by red-footed falcon broods are well known
WNV vectors [
]. The number of these vector
individuals showed a positive linear relationship with the
number of nestlings in a brood, indicating that each
nestling may receive similar vector loads regardless of brood
size. Mosquitoes use multiple olfactory cues and skin
emanations to locate vertebrate hosts . Presumably, larger
broods produce increased host stimuli attracting the
insects from a larger area, hence the observed pattern.
We also demonstrated nestling age dependent vector
attraction and blood-feeding success rate. First, our
results indicated that Cx. pipiens is disproportionately
attracted to nestling age categories where the adult birds
are absent compared to age categories where adult
presence is presumed (incubation and the 1st week after
hatching). It is possible that albeit an incubating adult is
larger than the nestlings, a single bird with a complete
plumage may be emitting less intensive cues compared
to a brood of semi-grown nestlings Secondly,
bloodfeeding success rates are considerably higher for both
Culex species in the first two weeks after hatching, and
subsequently drop in later breeding stages. This decrease
in both attraction and blood-feeding success coincides
with the development of body and flight feathers. The
gradual shift from downy feathers to juvenile plumage
presumably decreases the body surface where
mosquitoes may feed [
] and by acting as a better insulator
may also decrease host attractiveness. Furthermore, it
may also be adaptive for mosquitoes to select for young
nestlings as the probability of successful blood-feeding
may be higher compared to that on fledglings.
It has to be emphasized that our method to quantify
mosquito attraction and blood-feeding patterns hinder
the estimation of true host-vector contact rates.
Nonetheless, it allows us to speculate that younger red-footed
falcon nestlings are at higher risk of infection by
We also showed that the very mosquitoes attracted to the
nests harbour WNV. Although the virus was only present
in Cx. pipiens, this is likely due to the fact that this species
was an order of magnitude more abundant than Cx.
modestus. The obtained prevalence estimate (~4%, 95% CI: 0.9–
7.5%) is in the range of that found in North America for
Lineage 1 strains [
] but somewhat higher than
estimated for Hungary in general [
] and the Czech Republic
]. However, this estimate does not indicate large scale
amplification of the virus, despite the fact that the attributes
of the studied host-vector system (coloniality, a large
number of potential vectors and hosts) would, in theory, allow
for effective virus circulation and accumulation.
Nonetheless, it is likely that this prevalence estimate indicates a
stable WNV sylvatic cycle at the study site.
Although we confirmed WNV presence in vectors
directly attracted by red-footed falcon nestlings, the virus
was not present in detectable amounts in blood samples
of the exposed birds. Despite the lack of viremia, we found
that a considerable proportion (~25%) of these nestlings
was WNV-seropositive. These seemingly contradicting
observations may arise if either all seropositive nestlings
were infected soon after hatching and/or the duration of
detectable viremia was remarkably short. This scenario is
however unlikely based on estimates in viremia magnitude
and length of large falcons [
] and a comparative study
on multiple species [
]. More probable is that the
majority, if not all, seropositive nestlings had maternally derived
antibodies against WNV [
]. This is also
corroborated by two additional results. First, age dependent
increase in ELISA OD ratios, indicate that Ig levels decay
with the days elapsed from hatching as opposed to
increasing and/or stagnation in infected birds .
Secondly, ELISA OD ratios decreased with hatching order,
showing that younger nestlings have higher Ig values in
nests with at least one seropositive nestling. Red-footed
falcons typically hatch 1–2 days apart in a clutch,
therefore, both results show that the time-scale of maternally
derived antibody decay can be measured in days [
However, antibody decrease in free ranging birds with the
active immune response is detectable over months [
Nemeth et al. [
] argued that the rapid decay of maternal
antibodies in nestling house sparrows is unlikely to offer
effective protection to offspring [
If successful blood-feeding probability is nestling
agedependent, it is possible that the efficiency of passive
immunity is not exclusively linked to the half-life of
Understanding vector-borne arboviral infection systems in
avian hosts requires a comprehensive approach that
entails studying host-vector and vector-pathogen
interactions. Using a specific trap placed in the vicinity of the
brood, culicid vectors directly attracted by the studied
hosts can be quantified in natural conditions, and these
samples can also be used to estimate viral loads of vectors.
WNV has an established sylvatic cycle and poses a direct
threat to red-footed falcon nestlings. The array of soft
evidence presented here points to the prediction that either
nestlings have very short viremia or that a large
proportion of red-footed falcon females breeding in the studied
population allocates WNV antibodies to their eggs. This
also leads to the prediction that the breeding adults have
been infected by the virus either at the breeding site or
during their wintering period in Africa. Considering the
host-vector contact rate patterns, yolk transfer of
antibodies may be an efficient measure to protect nestlings
with naive immune systems from the clinical effects of a
WNV infection. The highest risk of contact with a
potential WNV vector is in the first two weeks after hatching
when maternal antibodies are expected to be sufficiently
high based on estimated decay patterns in other birds. In
any case, our results do not allow us to explicitly rule out
either of the following two competing theories, i.e. that
the observed patterns are either caused by very short
viremia or by maternal transfer of antibodies. However,
red-footed falcon nestlings are likely to have low WNV
host competence under both scenarios and are less likely
to be key hosts maintaining the sylvatic cycle of the virus,
at least during their breeding season.
CI: Confidence interval; ELISA: Enzyme-linked immunoabsorbent assay;
GLMM: Generalized linear mixed effects models; LME: Linear mixed effects;
SE: Standard error; WNV: West Nile virus
Field work was greatly assisted by Zsalkin Emese Széles and Dorottya Molnár
for which we are grateful. We also thank Professor Paul Reiter for his most
helpful comments on earlier versions of the manuscript.
This study was partially funded by the grant NKFIH-K120118, EU grant FP7–
261504 EDENext and is catalogued by the EDENext Steering Committee as
EDENext019 (http://www.edenext.eu). The contents of this publication are
the sole responsibility of the authors and do not necessarily reflect the views
of the European Commission. Further funding was provided by the HU-SRB
IPA CBC project (HU-SRB 0901/122/120), NKB 15950, LIFE+ (LIFE11/NAT/HU/
000926) and the OTKA K67900 grant.
Availability of data and materials
The data supporting the conclusions of this article are included within the
article. Raw data used and/or analysed during the current study are available
from the corresponding author on reasonable request.
ZS, PF and KE had designed the study. TB, KE, MB, KSz and ÁD performed
the laboratory experiments and molecular analyses. TB, KE, AH and LP
contributed to the design of the study. ZS, PF, LK, PP, ÉH and SzS carried out
the field work. ZS identified collected mosquito specimens. PF and AH
performed the statistical analyses. All authors contributed to drafting the
manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal handling and sampling protocols were approved by the Ethical
Regulation of MME/BirdLife Hungary (Professional and ethical policies of bird
ringing - 2009) and by the relevant Research Licence Decision of National
Environmental and Nature Conservation Authority (14/3710–2/2009 and 14/
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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