Received: 19 December 2018 | Revised: 10 April 2019 | Accepted: 17 April 2019

DOI: 10.1111/zph.12584

ORIGINAL ARTICLE

Flaviviruses in migratory passerines during spring stopover in a

desert oasis

Tasnim Ayadi1 | Abdesslem Hammouda1 | Ceclie Beck2 | Thierry Boulinier3 |

Sylvie Lecollinet2 | Slaheddine Selmi1

1
Unité de Recherche ‘Ecologie de la
Faune Terrestre’, UR17ES44, Faculté des Abstract
Sciences, Université de Gabès, Gabès, Bird migration has long been hypothesized as the main mechanism for long‐distance
Tunisia
2 dispersal of flaviviruses, but the role of migratory birds in flaviviruses spillover is not
UPE, ANSES, Laboratoire de Santé
Animale de Maisons‐Alfort, UMR1161 well documented. In this study, we investigated the eco‐epidemiology of West Nile
Virologie, INRA, ANSES, ENVA, Maisons‐
virus (WNV) and Usutu virus (USUV) in trans‐Saharan passerines during their spring
Alfort, France
3
Centre d′Ecologie Fonctionnelle et
stopover in southern Tunisian oases. To do, we combined oral swab analysis and sero‐
Evolutive, CNRS‐Université de Montpellier logical tools to assess whether migratory birds could be reaching these stopover sites
UMR 5175, Montpellier, France
while infectious or have been previously exposed to viruses. All sampled birds tested
Correspondence negative for oral swab analysis. However, anti‐WNV and anti‐USUV antibodies were
Tasnim Ayadi, Unité de Recherche ‘Ecologie
de la Faune Terrestre’ (UR17ES44), Faculté
detected in 32% and 1% of tested birds, respectively. Among WNV‐seropositive spe‐
des Sciences, Université de Gabès, Gabès, cies, the Golden oriole (Oriolus oriolus) showed the highest anti‐WNV occurrence
Tunisia.
Email: tasnimayadiii@gmail.com
probability. In this species, anti‐WNV occurrence was twice larger in males than fe‐
males. Inter‐specific and intraspecific morphological, physiological and behavioural
Funding information
DGRST‐CNRS, Grant/Award Number: 14/
differences could explain these results. Although our findings did not show evidence
R0901 for passerines migrating while infectious, they did not exclude an existing enzootic
WNV transmission cycle in Tunisian oases. Further investigations including larger
samples of migratory birds are needed for a better understanding of this issue.

KEYWORDS
migration, oasis, passerines, Tunisia, Usutu virus, West Nile virus

1 | I NTRO D U C TI O N countries (Benjelloun, Harrak, & Belkadi, 2016; Chaintoutis et al.,

West Nile virus (WNV) and Usutu virus (USUV) are sub‐Saharan
African flaviviruses belonging to the Japanese encephalitis virus
group (family Flaviviridae), which are associated with human enceph‐
alitis (Gaibani & Rossini, 2017; Nikolay, 2015). They are maintained
in nature by a transmission cycle involving mosquitoes as vectors
and birds as reservoir hosts (Nikolay, 2015). Since the 1950s, WNV
and USUV have spilled over from their original ecological niches into
the Mediterranean basin and Europe, establishing new endemic foci
when conditions are favourable (Gaibani & Rossini, 2017; Nikolay,
2015). In the last few decades, WNV outbreaks and sporadic cases in
humans, horses and birds occurred in several European and African
2015; Nikolay, 2015). Likewise, USUV has been responsible for sev‐
eral infection cases in birds and humans in different African and
European areas (Gaibani & Rossini, 2017; Lelli et al., 2008). In Tunisia,
WNV circulation has been recorded since the 1960s (Hammami et
al., 2017), while USUV circulation has only been reported recently
in horses (Ben Hassine et al., 2014) and laughing doves (Ayadi et al.,
2017).
Long‐distance spillover of WNV and USUV has been linked
with bird dispersal (Gaibani & Rossini, 2017; Nikolay, 2015;
Rappole & Hubalek, 2003). The detection of virus RNA in swabs
and tissues of infected migratory birds, as well as serological and
phylogenetic data have given strong supports to this hypothesis

Zoonoses Public Health. 2019;66:495–503. wileyonlinelibrary.com/journal/zph

© 2019 Blackwell Verlag GmbH | 495
496 | AYADI et al.
(Charrel et al., 2003; Engel et al., 2016; Perez‐Ramirez, Llorente,
& Jimenez‐Clavero, 2014; Reisen, Wheeler, Garcia, & Fang, 2010;
Tran et al., 2017). Twice a year, Palearctic trans‐Saharan migra‐
tory birds move thousands of kilometres, spanning the African and
European continents (Altizer, Bartel, & Han, 2011). While winter‐
ing in or passing through areas where WNV and USUV are known
to be enzootic, some migratory birds may get the infection and
spread the viruses (Dusek et al., 2009; Malkinson & Banet, 2002).
However, only individuals belonging to a competent reservoir
species and migrating through at least part of the infectious pe‐
riod might do it (Komar et al., 2003). Otherwise, birds chronically
infected might undergo viral relapse owing to the stress associ‐
ated with migration (Malkinson & Banet, 2002). Hence, they could
spread the viruses into naïve local populations along their routes
(Malkinson & Banet, 2002).
Along major migratory flyways linking sub‐Saharan Africa and
Europe, Tunisian oases are attractive stopover sites for migratory
birds. In these areas, a dense mixture of resident and migratory bird
species co‐occur twice a year (Selmi, 2000; Selmi & Boulinier, 2003;
Wasfi et al., 2016). Since competent mosquitoes are also abundant
within Tunisian oases (Wasfi et al., 2016), transmission of WNV and
USUV might be promoted. In previous studies, we have reported
serologic evidence of WNV and USUV circulation in the resident
avifauna in these areas (Ayadi et al., 2017; Hammouda et al., 2015).
However, the potential role played by migratory birds in the ecology
of these viruses in Tunisian oases is still poorly known, and two hy‐
potheses could be proposed. First, the viruses might be periodically
re‐introduced by infected birds from sub‐Saharan African areas.
Alternatively but not exclusively, the viruses might be circulating si‐
lently in an endemic sylvatic bird–mosquito cycle, while migratory
birds might allow their further spillover in other stations along their
routes. Hence, a suitable approach to assess the likelihood of these
two hypotheses in free‐living migratory passerines can be to com‐
bine both oral swab analysis and serological tools.
Oro‐faecal swab analyses allow assessment of whether a bird is
infectious through detection of the RNA of viruses in their secre‐
tions, especially as viral shedding occurs simultaneously with viremia
and lasts longer (Guerrero‐Sanchez et al., 2011; Komar et al., 2003).
In particular, oral swab samples generally contain more virus parti‐
cles than cloacal ones (Guerrero‐Sanchez et al., 2011). Thereby, birds
identified as positive could be suspected to be involved in infecting
local mosquitoes or passively shedding the viruses in nature, but
this may depend on the viremia (Grisenti et al., 2013; Komar et al.,
2003; Wheeler, Vineyard, Barker, & Reisen, 2012). Serological tools
allow assessment of whether a bird has been in contact with the vi‐
ruses during its lifetime through the detection of specific antibodies
(Nemeth et al., 2009). They do not give information on when and
how the infection occurred, but they can be useful in investigating
the ecological factors shaping bird exposure to viruses (Ayadi et al.,
2017; Figuerola et al., 2008; Hammouda et al., 2015; Nallar et al.,
2016).
Passeriformes are known to be one of the main reservoir hosts
of WNV owing to their high and long‐lasting levels of viremia
Impacts
• Migratory birds have been hypothesized to be the main
natural spreaders of several flaviviruses of public health
importance.
• We investigated the role of trans‐Saharan passerines in
the eco‐epidemiology of West Nile and Usutu viruses
during their spring stopover in Tunisian oases. We com‐
bined oral swab analysis and serological tool in free‐liv‐
ing migratory passerines.
• No evidence of birds migrating while infectious has been
recorded. However, this does not exclude the likelihood
of a silent local enzootic West Nile virus transmission
cycle. Serology results underlined important insights
into patterns of bird exposure to West Nile virus.
(Perez‐Ramirez et al., 2014). Positive WNV serology has been re‐
ported in migratory passerines belonging to several families, such
as Corvidae, Oriolidae, Sylviidae and Muscicapidae (Jourdain et al.,
2007; Strand, Lundkvist, Olsen, & Gustafsson, 2018). However,
WNV oro‐faecal shedding with viremia titres high enough to allow
bird‐to‐bird transmission has only been recorded in the Corvidae and
Icteridae families, which mainly includes resident species (Komar et
al., 2003; Perez‐Ramirez et al., 2014). Passeriformes are also known
to be highly susceptible to USUV infection (Ashraf et al., 2015).
Positive USUV serology has been reported in some migratory pas‐
serines, such as Sylvia communis, Sylvia borin, Ficedula hypoleuca and
Hirundo rustica (Ashraf et al., 2015; Meister et al., 2008). However,
few investigations have been carried out to detect USUV oro‐faecal
shedding in migratory passerines, and the results have always been
negative (Grisenti et al., 2013).
In this study, we aimed to assess the potential role of trans‐
Saharan passerines in the eco‐epidemiology of WNV and USUV
in Tunisian oases. We first explored whether migratory birds
reached these spring stopover sites while infectious using oral
swab analyses. Second, we assessed the seropositivity of the sam‐
pled birds towards WNV and USUV, and we investigated whether
the prevalence of antibodies varied among and within species.
Because body size and behaviour might shape species exposure
to mosquito vectors, and thus to infections (Janousek, Marra, &
Kilpatrick, 2014), the occurrence probability of antibodies and
their detection duration may vary among species. At the intraspe‐
cific level, sex‐biased antibody prevalence could be expected due
to possible sex‐related differences in behavioural, morphologi‐
cal and even physiological characteristics (Burkett‐Cadena et al.,
2011; Travi et al., 2002). Moreover, since migration demands are
energetically traded off against immune functions (Altizer et al.,
2011), a bird's serological status may be related to its energetic
condition. Birds that were recently exposed to infections are ex‐
pected to exhibit lower body condition compared with those that
did not suffer immunological challenges.
AYADI et al. | 497

2 | M ATE R I A L S A N D M E TH O DS and virus controls, as well as virus back titration controls, were in‐
cluded in every MNT assay. After incubation of the plates at 37°C
2.1 | Sample collection for 1.5 hr, 2 × 10 4 Vero cells in 100 µl of DMEM were added to
all the wells. Plates were incubated at 37°C for 3 days, and then,
Data used in this work were collected during spring 2016 (April–
cytopathogenic effects were observed under a light microscope. A
May), in Kettana oasis (33°45′N, 10°13′E), in south‐eastern
sample was considered negative if infection occurred regardless of
Tunisia. In this oasis, vegetation is composed of a dense mixture of
plasma concentration. It was considered positive if cells were pro‐
cultivated and spontaneous plants, including notably palm trees,
tected at the 1:10 plasma dilution; its titre was calculated as the
fruit trees (mainly olive, pomegranate, apricot and fig trees) and
inverse of the latest dilution at which cells were protected (Beck et
herbaceous plants. Water surfaces required for mosquito larval
al., 2015). On plasma samples with low volumes, only MNTs have
development, which are also used by birds for drinking, are con‐
been conducted and positive MNT samples have been automati‐
tinuously available.
cally assigned as ELISA‐positive too. Owing to cross‐neutralization
Birds were captured by means of mist nets (Karr, 1981) placed
between flaviviruses, especially within the same serocomplex,
around fruit trees highly frequented by migrant passerines, notably
virus neutralization test end point titres require comparison. The
blackberry and apricot trees. For each captured bird, we measured
flavivirus can then be identified as the virus with the highest titre,
body weight (in g) using a spring scale (PESOLA) and tarsus length (in
that is the one that shows a titre at least four times higher than
mm) using a digital calliper to the nearest 0.01. Right and left sides
that of the other (Beck et al., 2015; Yeh et al., 2012).
were measured, and the mean value was calculated. Whenever pos‐
For oral swab analysis, RNA extraction was conducted using sil‐
sible, birds were sexed according to plumage colour. Subsequently, a
ica‐membrane RNeasy spin columns (RNeasy® mini Kit, QIAGEN)
0.1–0.5 ml blood sample was taken from the brachial vein using a ster‐
following the manufacturer's instructions. For the screening of WNV,
ile heparinized syringe, and oral swabs were collected using sterile
we carried out a real‐time RT‐PCR using primers that amplify 5′NC
swabs with transport medium (Sigma Virocult® swab). The obtained
and C regions of WNV lineage 1 and 2 strains (Linke et al., 2007).
blood samples and swabs were maintained in a cooler (4°C) while in
To control the quality of RNA samples, we added primers specific to
the field before being transferred and stored into the laboratory till
β‐actin RNA to mix. We used TaqMan classic probes FAM‐TAMRA
analysis could be performed. Before release, each sampled bird was
(for WNV) and VIC‐TAMRA (for beta‐actin) for revelation. To deter‐
marked with a patch of paint on the head to avoid resampling. All
mine calibration curve and evaluate PCR efficiency, we used a pos‐
captured birds were handled by the same observer (T. Ayadi).
itive control and prepared serial dilutions with dilution factor of 10
from an initial suspension of the positive control with an initial titre
2.2 | Laboratory analyses of 106pfu/µl. We also carried out a generic pan‐flavivirus RT‐PCR
for screening of flavivirus presence. We used QIAGEN one‐step RT‐
Blood samples were brought to the laboratory on the day of collec‐
PCR kit and primers that amplify a region of the NS5 gene which is
tion, where they were centrifuged (10 min at 5,000 g). The plasma
well conserved within this genus (Grisenti et al., 2013). Primers used
samples were then stored at −20°C until immunological analyses
were WNV9040 (5′TACAACATGATGGGVAARAGAGAGA‐3′) and
could be performed. Screening of plasma for flavivirus antibodies was
WNV10124 (5‐AGCATGTCTTCYGTBGTCATCCAYT‐3), resulting in
conducted using an ELISA kit designed to detect antibodies against
a 1,084‐bp amplification product (Weissenbock et al., 2002). In a
the structural pre‐membrane (prM) and envelope (E) proteins of WNV
total PCR mix of 25, 1 µl of RNA was added to 1× buffer, 1 µl of
(ID Screen® West Nile Competition, IDvet). Tests were performed
the QIAGEN OneStep RT‐PCR Enzyme Mix, 2 µl of each primer 10
according to the manufacturer's instructions. Because WNV E pro‐
and 400 µM of each dNTP (QIAGEN one‐step RT‐PCR). Finally, the
tein shares conserved epitopes with other flaviviruses, this ELISA tool
products of the RT‐PCR were analysed by electrophoresis with a 1%
can be conveniently used as a surrogate test to detect antibodies to
agarose gel and visualized by staining with ethidium bromide. Positive
many flaviviruses, particularly those belonging to the Japanese en‐
and negative controls were included in the analyses. Both RT‐PCR as‐
cephalitis serocomplex (Beck et al., 2013). This test is known to show
says can detect viral loads lower than 10 TCID50/ml (data not shown).
high sensitivity and specificity, exceeding 95% (Beck et al., 2015).
ELISA‐positive samples were further investigated through
virus‐specific microneutralization tests (MNTs) against flaviviruses 2.3 | Statistical analysis
reported in the area where the plasma was collected (Beck et al.,
The collected data were first used to estimate the prevalence (and
2015, 2013). Heat‐inactivated plasma, serially diluted (1:5–1:320)
associated 95% confidence interval) of anti‐WNV and anti‐USUV an‐
in Dulbecco's modified Eagle's medium (DMEM), was mixed with
tibodies in the sampled species. We also checked whether the occur‐
an equal volume (50 µl) of DMEM containing 100 tissue cul‐
rence probability of antibodies differed among species, by means of
ture infectious dose 50 of WNV strain IS‐98‐ST1 (provided by
a generalized linear mixed model (GLMM), with a logit link function
the National Reference Centre for Arboviruses in France at the
and binary distribution. In this GLMM, only species with sufficient
Institut Pasteur, Paris) or USUV strain It2012 (206795‐3, gener‐
sample size were included (see results). For these species, separate
ous gift from IZSLER). Each plasma was tested in duplicate. Cell
498 | AYADI et al.

TA B L E 1 Antibody prevalence to West Nile virus (WNV) in the sampled migratory passerines using neutralization assays

Range of tarsus Range of body % WNV‐positive

Common name Scientific name n length (mm) mass (g) WNV‐positive and 95% CI

Eurasian golden Oriolus oriolus 52 25.75–27.35 62–79 30 58 (44–71)

oriole
Common Sylvia communis 28 22.28–25.30 12–22 10 36 (18–54)
whitethroat
Garden warbler Sylvia borin 37 23.26–25.95 13–25 8 22 (8–35)
Orphean warbler Sylvia hortensis 31 24.97–26.22 20–27 4 13 (0–23)
Common Phylloscopus collybita 2 20.34–21.42 10–11 0 0
chiffchaff
Great reed Acrocephalus arundinaceus 1 26.71 38 0 0
warbler
European pied Ficedula hypoleuca 4 17.82–19.39 11–14.5 0 0
flycatcher
Spotted flycatcher Muscicapa striata 1 17.98 16 0 0
Common Luscinia megarhynchos 5 29.28–31.63 21–33 0 0
nightingale
Rufous bush robin Cercotrichas galactotes 3 30.01–30.20 23–26 0 0
Tree pipit Anthus trivialis 1 24.31 28 1 100
Total 165 53 32 (25–39)

TA B L E 2 Results of analyses for antibodies to West Nile virus (WNV) by microneutralization test on ELISA‐positive samples

Number of samples with positive antibody titres

WNV antibody titres Oriolus oriolus Sylvia communis Sylvia borin Sylvia hortensis Anthus trivialis

10 5 2 2 1 0
20 7 3 2 0 0
40 4 2 2 1 1
80 6 2 1 1 0
160 3 0 1 0 0
320 3 1 0 1 0
640 2 0 0 0 0
Total 30 10 8 4 1

GLMMs (with a logit link function and binary distribution) were also
conducted to investigate whether the occurrence probability of an‐
tibodies varied according to sex and an index of body condition (BCI)
as fixed effects. BCI values were obtained as the residuals of a linear
regression of body weight (log‐transformed) on tarsus length (log‐
transformed). GLMMs were conducted using the GLIMMIX proce‐
dure in SAS software (SAS Institute Inc., 2008).
3 | R E S U LT S
A total of 165 birds belonging to 11 migrant passerine species were
sampled (Table 1). All collected oral swabs tested negative for WNV
and flaviviruses by RT‐PCR. The positive control tested always posi‐
tive, and the negative one resulted always negative.
Using ELISA, anti‐flavivirus antibodies (including anti‐WNV and
anti‐USUV antibodies) were recorded in 56 samples, corresponding
to an overall prevalence of 34% (95% CI: 26%–40%). Among these
samples, 53 were WNV‐seropositive, as revealed by the virus MNT,
corresponding to 32% (95% CI: 25%–39%) of sampled birds (Tables 1
and 2). Only two samples were USUV‐seropositive, corresponding to
an overall prevalence of 1% (95% CI: 0%–3%). These two USUV‐se‐
ropositive samples were from the same species, the Garden warbler
(S. borin), with an anti‐USUV titre of 20.
Using data on the four species with sufficiently large samples,
namely the European golden oriole (Oriolus oriolus), Common white‐
throat (S. communis), Garden warbler (S. borin) and Orphean warbler
(Sylvia hortensis), we found that the occurrence probability of anti‐
WNV antibodies differed significantly among species (F3,148 = 6,
p = 0.0004). Pairwise comparisons revealed that anti‐WNV
AYADI et al.
occurrence probability was overall higher in the European golden
oriole than in the three warbler species (Figure 1).
Species‐level analyses showed that none of the investigated
variables was significantly associated with anti‐WNV occurrence
probability in the three warbler species (Table 3). However, individ‐
ual sex provided a significant predictor of the occurrence probabil‐
ity of anti‐WNV antibodies in the European golden oriole (Table 3).
Anti‐WNV occurrence probability was almost twice as large in males
compared with females (Table 3, Figure 2). Furthermore, in all spe‐
cies, there was a tendency towards decreased anti‐WNV occurrence
probability with increasing BCI, but the trends were non‐significant
(Table 3, Figure 3).
4 | D I S CU S S I O N
To our knowledge, this is the first investigation of the possible role
of migratory passerines incoming from wintering grounds in the eco‐
epidemiology of WNV and USUV in North African stopover sites.
Overall, all sampled species tested negative for flaviviruses by oral
swab analyses, but serology results underlined important insights
into patterns of bird exposure to WNV.
In Tunisia, the link between WNV circulation and implication
of migratory birds in potential re‐introductions of the virus is, until
now, conjectural (Hammami et al., 2017; Triki et al., 2001). Negative
results of oral swab analyses in this study did not add arguments
in favour of this assumption. Indeed, to act as WNV re‐introducing
hosts in North African stopover sites, trans‐Saharan migratory birds
might first become infected with the virus shortly before their de‐
parture from their wintering grounds in sub‐Saharan Africa. Second,
they might remain viremic when they reach North African stop‐
over sites in order to be able to infect local mosquito vectors and/
F I G U R E 1 Estimated least square means (±SE) of the
occurrence probability of anti‐West Nile virus antibodies from a
generalized linear mixed model accounting for species as a fixed
effect. Only species with sufficiently large samples were included
in the model. Results of t tests for pairwise comparisons are also
provided
| 499
or birds by oro‐faecal shedding of the virus. However, owing to the
shortness of the viraemic phase (<7 days according to Oesterle et al.
(2009); Perez‐Ramirez et al. (2014), oro‐faecal shedding of the virus
(<10 days according to Komar et al. (2003)) and also to possible non‐
optimal source material, detection of the virus RNA in oral swabs is
not likely easy in migrant birds. Likewise, birds recently infected or
suffering from a relapse of the infection during migration might not
have been able to cross the Sahara and reach North Africa. Previous
studies reported that birds undergoing recent infections might ex‐
perience several consequences such as higher mortality rates along
their routes (Altizer et al., 2011), migratory restlessness (Owen et al.,
2006) and delayed migration (Altizer et al., 2011). For these reasons,
the probability of sampling individuals reaching our grounds while
infectious was expected to be low.
On the other hand, serology results showed an overall anti‐
WNV prevalence of 32%. From the 11 tested species, seropositive
birds belonged to the Eurasian golden oriole, Common whitethroat,
Garden warbler, Orphean warbler and Tree pipit. These findings
are consistent with previous reporting of positive WNV serology
in these passerine species during spring migration (Jourdain et al.,
2007). However, anti‐USUV antibodies were recorded only in two
Garden warblers, corresponding to an overall seroprevalence of 1%.
This low anti‐USUV antibodies prevalence might be due to the high
vulnerability of the tested passerines to USUV infections (Gaibani
& Rossini, 2017). Indeed compared with WNV, USUV has a higher
virulence for birds and results in greater mortality rates (Gaibani &
Rossini, 2017).
Using serological data on the most commonly sampled species,
we found that bird exposure to WNV varied significantly among spe‐
cies. Overall, European golden orioles were more exposed to WNV
compared with warblers. Knowing that the European golden oriole
(62–79 g) is more than twice larger than the three studied warbler
species (12–27 g), this result is consistent with the general ten‐
dency of positive association between seroprevalence rate and bird
body size (Figuerola et al., 2008; Victoriano Llopis et al., 2016; Yan,
Gangoso, Martinez‐de la Puente, Soriguer, & Figuerola, 2017). Larger
body is often linked with larger areas of unfeathered parts, such as
eye rings and tarsus, that are often targeted by blood‐sucking mos‐
quitoes (Yan et al., 2017). Large species may also have a higher an‐
nual survival rate and run a greater number of seasons of exposure,
thus potentially a population containing on average older individuals.
It is also linked with higher CO2 output (Nagy, 1987), higher quantity
of olfactory cues and body temperature release (Takken & Verhulst,
2013), which attract more mosquito vectors. Additional differences
between the European golden oriole and the three sampled warbler
species, concerning notably plumage colour and habitat use, may
also explain, at least partly, its greater exposure to viruses.
With regard to plumage colour, it has been shown that birds
with greater percentages of attractive plumage colours such as yel‐
low, white, light green and light brown are more frequently bitten
by Culex pipiens mosquitoes (Yan et al., 2017). Because the seeking
activity of mosquitoes usually peaks under conditions of poor vis‐
ibility such as sunrise, sunset or at night (Becker et al., 2010), they
500 | AYADI et al.

TA B L E 3 Results of GLMMs of the

Species Effect Estimate ± SE df t P
occurrence probability of anti‐West Nile
Oriolus oriolus Intercept 1.653 ± 0.659 49 2.51 0.0155 virus antibodies as functions of sex (two
Sex −1.859 ± 0.751 49 −2.48 0.0168 classes: Male = 1 vs. Female = 0) and body
condition index (BCI) as fixed effects
BCI −5.212 ± 4.264 49 −1.22 0.2273
Sylvia communis Intercept −0.589 ± 0.725 25 −0.81 0.4247
Sex −0.021 ± 0.890 25 −0.02 0.9811
BCI −2.057 ± 2.776 25 −0.74 0.4656
Sylvia hortensis Intercept −1.838 ± 0.763 28 −2.41 0.0228
Sex −0.146 ± 1.106 28 −0.18 0.8604
BCI −3.305 ± 6.395 28 −0.52 0.6093
Sylvia borin Intercept −1.307 ± 0.406 35 −3.22 0.0028
BCI −1.504 ± 2.321 35 −0.65 0.5211

Note: In Sylvia borin, individual sex was not considered in the model because it could not be
accurately determined.

F I G U R E 2 Estimated least square means (±SE) of the
occurrence probability of anti‐West Nile virus antibodies in males
and females of the European golden oriole from a generalized linear
mixed model accounting for sex and body condition as fixed factors
F I G U R E 3 Relationships between the occurrence probability of
anti‐West Nile virus antibodies and body condition index (BCI), as
estimated from generalized linear mixed models accounting for the
effects of sex. The curves are obtained using BCI values spanning
the range of values recorded in the four studied species

might be more attracted to hosts of bright colours (Yan et al., 2017).
Golden orioles are of bright yellow (males) to yellowish‐green (fe‐
males) colours, while warbler species have predominately grey
plumage (Cramp, 1992; Mason & Allsop, 2009). Thereby, European
golden orioles would show greater brightness and sharper colour
contrast against dark backgrounds and hence be easily located by
mosquitoes. With regard to habitat, warbler species inhabit shrubby
and open dry habitats, where mosquito populations could be of low
densities, while the European golden oriole rather avoids treeless
habitats and occurs in more vegetated areas where general condi‐
tions, mainly elevated humidity and low wind speed, might promote
high abundance of mosquitoes (Cramp, 1992; Mason & Allsop, 2009;
Service, 1980). Moreover, Golden orioles roost higher compared
with warblers, which might raise their exposure to mosquito bites.
Indeed, the abundance and feeding preference of Culex mosquitoes
have been reported to increase with the roost height (Janousek et
al., 2014). For these reasons, the exposure of European golden ori‐
oles to mosquito bites, and then to WNV, is likely to be enhanced
compared with warblers.
In the European golden oriole, the probability to detect anti‐
WNV antibody was twice as large in males compared with females.
This suggests a higher exposure of males to mosquito bites com‐
pared with females, supporting a previous report of Culex mosqui‐
toes propensity to take nearly two times more blood meals from
males than females (Burkett‐Cadena, Bingham, & Unnasch, 2014).
At least two non‐exclusive hypotheses could be proposed to ex‐
plain this finding. First, sex‐biased avian host use by mosquitoes
might be owing to hormonal differences between sexes (Travi et
al., 2002). Because males have higher levels of testosterone and, in
consequence, stress hormone levels (Evans, Goldsmith, & Norris,
AYADI et al.
2000), they are likely to be more attractive for mosquitoes than
females. Indeed, a recent experimental study on Zebra finches
showed that Culex mosquitoes fed on birds with high stress hor‐
mone levels nearly twice more frequently compared with birds
from a control group (Gervasi et al., 2016). Second, sex‐biased
avian host use by mosquitoes might be due to morphological dif‐
ferences between males and females. Indeed, in the European
golden oriole males have slightly larger bodies and brighter yellow
plumage, which could be linked with higher exposure to mosquito
bites compared with females.
In all tested species, no statistically significant association be‐
tween BCI and anti‐WNV antibody detection was found, suggesting
that previous exposure to the virus unlikely causes huge negative
impacts on bird's body condition. These findings are opposite to
our hypothesis according to which WNV‐seropositive passerines
would have lower BCI because both migration demands and immune
functioning are energetically costly (Altizer et al., 2011). However to
some extent, they still corroborate the findings of previous investi‐
gations focusing on the impacts of exposure to WNV on passerines
(Hill, Siefferman, Liu, Hassan, & Unnasch, 2010; Owen et al., 2006)
and might thus give support for the potential role of these species
to act as asymptomatic carriers of this virus. However, for a better
understanding of WNV infection impact on body condition of migra‐
tory passerines, further morphological and physiological indicators
need to be investigated.
Although our results did not show evidence of trans‐Saharan
migratory passerines reaching southern Tunisian stopover sites
while infectious, an enzootic local WNV transmission cycle in
these oases could not be excluded (Alba et al., 2014). Considering
the potential spillover of the virus from these areas and the ran‐
domness of outbreaks occurrence, WNV circulation in the oasis
system needs to be carefully monitored. Furthermore, our results
provided important insights into patterns of passerines exposure
to WNV. Nonetheless, it should be considered that serology re‐
sults might be biased by fatal infections that could occur in some
species but not in others. Further investigations including larger
numbers of migratory species known to act as natural WNV reser‐
voirs are needed for a clearer understanding of the implication of
migratory species in the eco‐epidemiology of flaviviruses in North
African oases.
AC K N OW L E D G E M E N T S
The authors wish to thank J. Vandekerkhove, S. Lowenski, A. Poux,
C. Loiseau and M. Ayadi who helped in laboratory and fieldwork,
as well as P. Desprès (Institut Pasteur) and D. Lelli (IZSLER) for the
provision of WNV and USUV field strains, respectively. ELISAs were
conducted at PACE Labex CELEB platform. This work was conducted
as a part of the OISAU‐FCAIMED project financed by the DGRST‐
CNRS collaboration programme (grant number 14/R0901). Support
was also provided by OSU OREME and CNRS INEE. Authorizations
for bird sampling were obtained from the Forest Service of the
Tunisian Ministry of Agriculture (permit number 1035/04‐05‐2015).
| 501
C O N FL I C T O F I N T E R E S T S
We declare that we have no conflict of interests.
ORCID
Tasnim Ayadi https://orcid.org/0000-0001-5234-6407
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How to cite this article: Ayadi T, Hammouda A, Beck C,
Boulinier T, Lecollinet S, Selmi S. Flaviviruses in migratory
passerines during spring stopover in a desert oasis. Zoonoses
Public Health. 2019;66:495–503. https​://doi.org/10.1111/
zph.12584​