This study surveyed mosquitoes and tested for West Nile virus (WNV) circulation in De Oostvaardersplassen nature reserve in the Netherlands, considered high-risk for WNV introduction and transmission. Thirteen mosquito species from five genera were collected, including potential WNV vectors. No WNV was detected among pools of up to five mosquitoes tested using real-time RT-PCR. While suitable conditions for WNV establishment exist, this study found no evidence of current virus circulation in the local mosquito population.

1. 69
European Mosquito Bulletin 28 (2010), 69-83
Journal of the European Mosquito Control Association
ISSN 1460-6127; w.w.w.e-m-b.org
First published online 23 April 2010
A study of the circulation of West Nile virus in mosquitoes in a potential high-risk area for
arbovirus circulation in the Netherlands, “De Oostvaardersplassen”.
Chantal Reusken1*
, Ankje De Vries1
, Wietse Den Hartog2
, Marieta Braks1
and Ernst-Jan Scholte1,2
1.
Laboratory for Zoonoses and Environmental Microbiology, Centre for Infectious Disease Control
Netherlands, Bilthoven, the Netherlands.2.
National Centre of Vector Monitoring, Plant Protection
Service, Wageningen, the Netherlands.
*Corresponding author. Laboratory for Zoonoses and Environmental Microbiology.
Centre of Infectious Disease Control Netherlands, RIVM. P.O. Box 1; 3720BA Bilthoven
the Netherlands; e-mail: chantal.reusken@rivm.nl
Abstract
The Dutch national nature reserve “De Oostvaardersplassen” is considered a potential high-risk area for
West Nile virus (WNV) circulation in the Netherlands: over 6000 hectares of wetland accommodating a
diverse and abundant mosquito population, and a wide variety of wildlife. This wildlife includes migratory
birds arriving from WNV endemic areas in Africa and Central Europe. Here we have conducted a
combined mosquito and virological survey as a first step to assess the risks for public and veterinary
health of this nature reserve as ecosystem for enzootic WNV circulation. Thirteen different mosquito
species, covering five different genera were collected. Remarkable is the presence of Anopheles
algeriensis, a mediterranean species. Eleven of the 13 species are reported field or laboratory vectors for a
variety of infectious pathogens, including WNV, Usutu virus, Tahyna virus, Sindbis virus, Batai virus,
Lednice virus, Francisella tularensis and Dirofilaria immitis. Although seven potential WNV vector
species were collected, viz. Anopheles maculipennis s.l., Culex modestus, Cx. pipiens s.l., Ochlerotatus
cantans and Coquillettidia richiardii, no evidence for WNV circulation in mosquitoes was found in this
study.
Keywords: Anopheles algeriensis, West Nile virus, Culicidae, ecology
Introduction
West Nile virus (WNV) is the aetiologic agent of West Nile Fever (WNF), a vector-borne disease endemic
in Africa, East Asia, North-, Central- and South America and Southern Europe (Dauphin et al., 2004).
The virus was first isolated from a woman with febrile illness in the West Nile province of Uganda in
1937 (Smithburn et al., 1940) and is classified in the genus flavivirus of the family of the Flaviviridae
(Burke & Monath, 2001). West Nile virus is maintained in an enzootic cycle with birds as amplifying
reservoir hosts and mosquitoes as transmitting vectors. Humans and horses are sensitive dead-end hosts
(Hurlbut et al., 1956). The majority of human WNV infections are asymptomatic. American studies
estimated that approximately 20% of human infections will result in a mild disease characterized by fever,
headache, myalgia and fatigue. Less than 1% of all infected persons develop severe neuroinvasive disease,
with a case fatality of approximately 10% (Kramer et al., 2007).

2. 70
The first recognised outbreak in Europe occurred in 1962 in France with clinical symptoms in humans and
horses (Murgue et al., 2001). A major outbreak of human WNV infections took place in 1996 in Romania
(Tsai et al., 1998). In subsequent years enhanced surveillance in Romania identified some WN fever cases
each year between 1997 and 2000 but there were no major outbreaks (Ceianu et al., 2001). In 1997 some
sporadic cases were reported in the Czech Republic (Hubálek et al., 2000). A second severe human
outbreak of WNV took place in the Volgograd region in Russia in 1999 (Lvov et al., 2000, Platonov et al.,
2001). The epidemiological picture of human WNV infections in Europe appears to be changing. In recent
years sporadic cases of human WNV infections have been identified in Hungary, Italy, France, Portugal,
Romania, Russia and Spain (Hubálek et al., 2000, Mailles et al., 2003, Connell et al., 2004, Del Giudice et
al., 2004, Zeller & Schuffenecker 2004, Kaptoul et al., 2007, Krisztalovics et al., 2008, Popovici et al.,
2008, Barzon et al., 2009, Rizzo et al., 2009). In 2008 for the first time outbreaks of human infections
were reported simultaneously from three European countries, viz. Hungary, Italy and Romania
(Krisztalovics et al., 2008, Popovici et al., 2008, Barzon et al., 2009). In addition to the known circulation
of lineage 1 of WNV in Europe, the circulation of lineage 2 of WNV was reported from Hungary in 2005
and Austria in 2008 (Bakonyi et al., 2006, Weissenbock et al., 2009).
Mosquitoes (Diptera: Culicidae) are the principal vectors for WNV transmission. The main route of
introduction of WNV in the regions of Europe with a temperate climate is most likely by infected
migratory birds from Africa (Hubálek & Halouzka, 1999, Rappole et al., 2000, Rappole & Hubálek, 2003,
Koopmans et al., 2008). Moreover, WNV could be introduced in western Europe by birds arriving from
central Europe (Koopmans et al., 2008). For an efficient local transmission, mosquito species are needed
that are capable of sustaining and transmitting the virus among the indigenous bird population.
Ornithophilic mosquito species will be the most relevant species for WNV establishment in an enzootic
cycle in temperate Europe (Rappole et al., 2000, Higgs et al., 2004). For transmission to humans and
horses, vectors are needed that have a more opportunistic feeding behaviour; feeding both on birds and
mammals and serving as bridge vectors. In Europe WNV has been detected in seven different mosquito
species, covering 4 genera, in France, Portugal, Moldavia, Romania, Slovakia, the Czech Republic,
Ukraine and Belarus. Vectors reported positive in the field are Culex pipiens s.l., Cx. modestus, Cx.
univittatus, Coquillettidia richiardii, Ochlerotatus cantans, Anopheles maculipennis s.s. and An.
atroparvus (Hannoun et al., 1964, Filipe, 1972, Labuda et al., 1974, Hubálek & Halouzka, 1996,
Fernandes et al., 1998, Savage et al., 1999, Esteves et al., 2005, Almeida et al., 2008, Hubálek, 2008).
The ornithophilic members of the species complex Cx. pipiens s.l. are the most common vector species for
WNV both in Europe and the USA.
Because WNV in Europe is most likely introduced by bird migration and WNV enzootic circulation and
outbreaks are often focused in or near wetlands where large numbers of birds are bitten by large numbers
of mosquitoes (Koopmans et al., 2008), the Dutch national park “De Oostvaardersplassen”, is considered
a high risk area for introduction and enzootic circulation of WNV in the Netherlands. De
Oostvaardersplassen, located in the province of Flevoland (Figure 1), is one of the many areas of
reclaimed land in the Netherlands. Nowadays, the Oostvaardersplassen is a 6000 hectares nature
development reserve of international importance as wetland and habitat for migratory birds. Over 190
different bird species have been observed in the reserve since 1999, including over 45 bird species of
which evidence for WNV infection exists from foreign field studies (Koopmans et al., 2008). De
Oostvaardersplassen has diverse landscapes including marshes, lakes, ponds, ditches, trenches, plains,
shrubs, and forests, which create a large diversity of potential mosquito breeding sites. This, combined
with the presence of large numbers and variety of hosts (birds and wildlife consisting amongst others of
approximately 500 ‘Heck cattle’, 1100 ‘Konik’ horses and 2300 red deer), makes De Oostvaardersplassen
an area where high mosquito species richness and mosquito abundances are expected.

3. 71
Here we describe a combined mosquito and WNV survey in De Oostvaardersplassen as a first step
towards an assessment of the risks of this nature reserve as ecosystem for enzootic WNV circulation in the
Netherlands.
Materials and methods
Survey design
Adult mosquitoes were collected in the ‘Oostvaardersveld’, an area of approximately 328 ha in the
southeastern corner of De Oostvaardersplassen. The Oostvaardersveld consists of open grassland with
sparse shrubs, shallow ponds, of which the smaller ones contain little or no aquatic vegetation, 5-10m
wide canals with still standing water most of which are lined with reed (Phragmites communis), and
deciduous forest. A group of approximately 100 Konik horses are present, spending most of their time in
an open grassland area in the northeastern part of the Oostvaardersveld. In the middle of this open
grassland lies a shallow, permanent pond without aquatic plants with high numbers of birds (mostly geese
and various duck species). This area of open grassland is surrounded by deciduous forest (mostly willow).
In some parts of this forest, especially near canals, flooding occurs, creating marshes. Due to the high
groundwater level in the entire Oostvaardersplassen, puddles and pools remain in these marshes, even in
relatively dry periods (Kooijman et al., 2005).
Adult mosquitoes were collected using carbon dioxide baited traps of the type Liberty Plus®
(American
Biophysics), with octenol as an additional lure. Where possible, the traps were placed in the lee of trees.
All traps were placed on wind-protected locations, with ample space around the traps’ entry point. In total,
nine traps ran continuously during the experimental period. One mosquito was collected when landing on
one of the researchers.

4. 72
Figure 1. The study area, Oostvaardersveld, where nine traps were placed.
Oostvaardersplassen
A
B
C
D E
F
G
H
I
‘Oostvaardersveld’
Oostvaardersplassen
A
B
C
D E
F
G
H
I
A
B
C
D E
F
G
H
I
‘Oostvaardersveld’5 km
500m

5. 73
Table 1. Presence or absence of several landscape parameters and estimated host availability of the
immediate area surrounding the trap (< 25 m)
a) Consisting mainly of willows (Salix alba), bramble (Rubus fruticosus), nettle (Urtica dioica), and at the margins elder
(Sambucus nigra).
b) Mainly grass (mostly Lolium and Poa spp.), nettle, thistles (Cirsium and Carduus spp.) and occasionally hawthorns
(Crataegus laevigata).
c) Consisting mainly of willow and at the margins dense nettle ‘fields’.
d) 0.50-1.5m deep, with few or no aquatic plants. Often lined with reed (Phragmites communis).
e) The ponds near traps D and E were relatively small, shallow and contained no aquatic plants. The pond near traps G and
H measured approximately 50-200m., and contained some aquatic plant species, such as water lily (Nuphar lutea). No
information is available on submerged aquatic plants in the Oostvaardersplassen.
As WNV outbreaks in Europe in general occur during the summer (July-August-September) we conducted
our survey in August. Mosquitoes were collected twice for two consecutive days; in week 33 (10-12
August 2009) and in week 35 (24-26 August 2009). For the entire duration of the experiment, the nine
traps ran continuously. The traps were placed in the morning of the 10th
of August. The nets from the
mosquito magnets were retrieved from all traps after 24 hrs (on the morning of the 11th
August), and
replaced with empty nets, which were subsequently retrieved 24hrs later (12th
August). Retrieved nets
with (still alive) mosquitoes were placed in a sealed bag, labelled, and placed in an equally labelled
cardboard box for protection (one net per box). These labelled boxes were kept in the car for later
mosquito species identification. In week 33, the same procedure was followed: traps were switched on, on
the 24th of August, and retrieved subsequently 24 and 48 hrs later (25th
and 26th
August).
Within a maximum of three hours after collection from the trap, the nets with mosquitoes were removed
from the box and placed by -20 °C for approximately 3-10 minutes to kill them. Subsequently, they were
gently taken from the nets, separated from other insects, and identified. Mosquitoes were morphologically
identified using a Culicidae key specifically designed for rapid field-identification of Dutch Culicidae
adult female mosquitoes (Scholte, 2009a modified after Schaffner et al., 2001, Becker et al., 2003,
Verdonschot, 2002). Males were not identified. Differentiation of members of the Anopheles maculipennis
complex (An. messeae, An. maculipennis senso stricto, and An. melanoon), and between the biotypes of
Cx. pipiens (pipiens and molestus forms), was not carried out. The species identification was confirmed by
a specialist on European mosquito species.
Trap deciduous
forest a)
open
grassland
b)
Marshes c)
Canals
d)
Ponds e)
estimated presence of
hosts (large mammals)
estimated
presence of hosts
(birds)
A no yes no yes no regular always
B yes no no no no occasionally always
C yes no yes no no rarely always
D no yes no no yes very often always
E no yes no no yes very often always
F yes no no no no occasionally always
G yes no no no yes occasionally always
H no yes no no yes rarely always
I yes no yes no no occasionally always

6. 74
Monitoring for presence of West Nile virus
Upon identification, the collected mosquitoes were immediately flash frozen using dry-ice and stored at -
80°C until analysis. Mosquitoes were pooled by sampling site, sampling date and species. Mosquitoes
were ground using liquid N2 and pestles, and RNA was extracted using the RNeasy ® RNA Isolation
Minikit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions and including a
Qiashredder step for homogenization and DNaseI treatment. RNA was isolated from pools of no more
than five mosquitoes. As a quality control for the isolation step, each RNA isolate was checked by an actin
reverse transcriptasepolymerase chain reaction (RT-PCR) (Koopmans et al., 2008). Although these
primers were selected based on alignments of known actin sequences from Aedes spp. available in
GenBank, we have successfully used them on Cx. pipiens s.l., Cx. modestus, Cx. subochrea, Cx.
torrentium, Cx. territans, Culiseta annulata, Aedes cinereus, An. maculipennis s.l., An. plumbeus, An.
claviger, Oc. punctor, Oc. cantans, and Coquillettidia richiardii that are all endemic in the Netherlands
(data not shown).
The presence of WNV was analysed using a multiplex real-time RT-PCR assay, allowing detection and
discrimination of lineages 1 and 2 (Chao et al., 2007). An additional lineage 2 probe was designed and
added to the reaction, viz. WNV-2: 5’-FAM-tgaaggaagttggaacaaagcctgg-BHQ1. To avoid false negative
results arising from inhibitory factors known to be present in mosquito total RNA preparations, each pool
was also analysed in the presence of a ‘spike-mix’ consisting of viral RNA from both lineage 1 and 2 of
WNV at an experimentally pre-determined detectable concentration. Only the results from pools that were
positive for both lineages in the spiked RT-PCR were used in this study.
Results
Entomological survey
In total 36 trap collections were carried out. Four times a trap had failed (traps B, D, and I for the
collections on 11th
of August, and trap E for the collection on the 26th
of August), resulting in a total of 32
effective collections. In these 32 samples, a total of 410 mosquitoes were collected. Mosquito
identification up to species level was carried out for 396 specimens (Table 2): two specimens were too
damaged (of which one could be identified only as Culicidae, and one only up to genus level), and 12
were males. The identified species are An. algeriensis, An. claviger, An. maculipennis s.l., An. plumbeus,
Cq. richiardii, Cx. modestus, Cx. pipiens, Cx. torrentium, Cs. annulata, Cs. morsitans, Cs. subochrea, Oc.
cantans and Dahliana geniculata. Very interesting is the finding of An. algeriensis, a Mediterranean
mosquito species that is rare in northern regions, here newly identified for the Netherlands.
By far the most abundant species was Cs. annulata (207 specimens), followed by Cq. richiardii (96
specimens) and An. claviger (55 specimens). Together, these three species made up 90% of the collected
mosquitoes. Of four species, only one specimen was collected: Cx. modestus, Cs. morsitans, Cs.
subochrea, and Da. geniculata. Nine specimens were collected of Cx. pipiens. Anopheles claviger, An.
maculipennis s.l., Cq. richiardii, and Cs. annulata were found at most locations. From the traps located at
D and E most mosquitoes were collected, with 96 and 119 specimens respectively. The lowest number of
mosquitoes was collected from the trap located at location I, where only two mosquitoes were collected.
The number of different species ranged between 2 and 8 species per trap location. The highest number of
species was collected from the traps at location B and E, with eight different species each.

7. 75
Table 2. Overview of collected mosquito species per trap
mosquito speciestrap # A B C D E F G H I Total
Anopheles algeriensis 1 4 1 6
Anopheles claviger 12 3 1 17 15 1 5 1 55
Anopheles maculipennis sl 1 1 1 1 1 1 6
Anopheles plumbeus 4 4
Coquillettidia richiardii 17 16 3 12 30 5 8 5 96
Culex modestus 1 1
Culex pipiens 1 2 1 5 9
Culex torrentium 1 1 2
Culiseta annulata 5 26 1 61 66 21 26 1 207
Culiseta morsitans 1 1
Culiseta subochrea 1 1
Dahliana geniculata 1 1
Ochlerotatus cantans 2 1 2 2 7
Total 36 51 7 96 119 32 42 11 2 396
Ochlerotatus sp. 1 1
Male 2 10 12
Culicidae heavily damaged 1 1
Total 36 53 7 96 119 34 52 11 2 410
West Nile virus survey
In total 314 mosquitoes, belonging to five different genera and 13 different mosquito species, were
analysed for the presence of WNV lineages 1 and 2 (Table 3). None of the analysed specimens were
found positive for WNV.
Table 3. Overview of mosquitoes collected in the nature reserve “De Oostvaardersplassen”
Mosquito species No.
captured
No. tested
for WNV
Anopheles algeriensis 6 3
Anopheles claviger 55 40
Anopheles maculipennis sl 6 6
Anopheles plumbeus 4 2
Coquillettidia richiardii 96 79
Culex modestus 1 0
Culex pipiens 9 9
Culex torrentium 2 2
Culiseta annulata 207 165
Culiseta morsitans 1 0
Culiseta subochrea 1 1
Dahliana geniculata 1 1
Ochlerotatus cantans 7 5
Ochlerotatus spp. 1 1
Total 397 314

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Discussion
Mosquito species
For the Netherlands the presence of 36 different mosquito species has been documented, including the
recently discovered Ochlerotatus atropalpus (Scholte et al., 2009). In addition, Stegomyia albopicta is
occasionally introduced into the Netherlands from South-East China with the import of Lucky Bamboo
plants. However this species has not established in the Netherlands (Scholte et al., 2007, 2008). Thirteen
different mosquito species, covering five different genera were collected during the survey. This
represents 37% of the total number of species known to be present in the Netherlands, and confirms the
presence of high mosquito species richness in the Oostvaardersplassen.
Remarkable is the presence of An. algeriensis in the nature reserve. Anopheles algeriensis is a
mediterranean mosquito species. The species is rare in southern Europe (only two populations are known
from southern France (Poncon et al., 2007), and isolated populations have been described for northern
Europe, viz. Britain (Rees & Rees, 1989; Snow et al., 1998), Estonia (Remm, 1957), Germany (Mohrig,
1969), western France (Ramsdale & Snow, 2000) and Ireland (Ashe et al., 1991). In terms of spatial
species composition; the collected species per location match with the mosquito fauna that could be
expected at these types of locations. However one exception was observed: Four adults of the forest
species An. plumbeus were collected at location E, a relatively open area, approximately 200 metres from
the nearest forest. This species was not collected at any of the forested locations (B, C, I, F, G). In terms
of temporal species composition; only eight specimens, belonging to two species of the genus
Ochlerotatus were collected, suggesting an under-representation of the genus. This can most likely be
explained by the timing of the study in ‘the season’: many Ochlerotatus species have only one generation,
and are abundant in (early) spring, after the overwintering eggs have hatched. Examples are Oc.
annulipes, Oc. cantans, Oc. communis, Oc. excrucians, Oc. riparius, Oc. leucomelas, and Oc. rusticus (all
endemic species). The absence of these species is therefore easily explained. Ochlerotatus species that,
based on their known ecology, could be expected in this study but were not collected, include Oc.
sticticus, Oc. nigrinus, Oc. flavescens, and Oc. caspius. A relatively low number (nine) of specimens were
collected of Cx. pipiens, a species that is generally an abundant species in Culicidae surveys in different
habitats in the Netherlands (Takken et al., 2007). As the type of trap used in entomological surveys is one
of the factors determining the relative abundance and diversity of the mosquitoes collected, one should
keep in mind that the mosquito magnet used in this study might not be the optimal trap for all species
present in the nature reserve.
The following notes give some information on the five most commonly encountered species in this
survey:
Anopheles claviger. This species was encountered at all locations except H. This species occurs mostly in
permanent pools, shallow canals and ditches, with a preference for shaded sites. This species hibernates as
a larva.
Coquillettidia richiardii. An interesting feature of the larvae of this species, is that they obtain their
oxygen not directly from the air (as other mosquito larvae), but from plant tissues by penetrating the roots
of certain aquatic plants.
Culiseta annulata. This species was by far the most abundant species in the Oosvaardersveld at the time
of the study. This species is a relatively large mosquito and an aggressive biter, also to humans. Larvae
are known to be present almost throughout the year, although the species is known to overwinter as
adults.

9. 77
Anopheles maculipennis s.l. Anopheles maculipennis consists of a complex, of which seven sibling
species occur in Europe and three in the Netherlands: An. messeae, An. atroparvus, and An. maculipennis
sensu stricto. Females are often present in barns and stables, but bite humans as well. Adults hibernate in
cool rooms such as cellars.
Culex pipiens complex. The Cx. pipiens complex consists of two biotypes: the pipiens and molestus
forms. The first is ornithophilic (not attracted to humans), whereas the latter is mainly zoophilic but
occasionally bites birds as well. In the case of West Nile virus circulation, biotype pipiens can play a role
in maintaining the virus circulating among birds only, whereas biotype molestus may form the bridge
species, transmitting the virus to horses and humans. Biotype pipiens breeds in a large variety of
waterbodies, including shallow ponds and artificial containers. The species overwinters as adults.
Disease vectors
Ten species endemic to the Netherlands are reported as field vectors for WNV in foreign studies of which
five have been collected in this study, viz. An. maculipennis s.l., Cx. modestus, Cx. pipiens s.l., Oc.
cantans and Cq. richiardii (Table 4). WNV has been isolated from all five species in Europe (Hannoun et
al., 1964, Filipe, 1972, Labuda et al., 1974, Katsarov et al., 1980, Fyodorova et al., 2006, Koopmans et
al., 2008). Two species are reported laboratory vectors for WNV, viz. An. plumbeus and Da. geniculata
(Table 4). Although seven potential WNV vector species were collected in this nature reserve, no
evidence for WNV circulation in mosquitoes was found in this study.
This could mean that although there is WNV circulation in the nature reserve, it cannot be detected with
the current survey set-up. Enzootic WNV transmission may occur only at a low intensity in certain birds
and mosquito species. The number of mosquitoes collected in this study is most likely not enough to
detect a low endemic WNV circulation in the area. In total 314 mosquitoes were analysed for the presence
of WNV of which only 9 specimens of the Cx. pipiens complex which, together with Cx. modestus, are
considered as the vector species with the highest vectorial capacity for WNV. Furthermore there might be
WNV circulation in the reserve but not in the area of study. De Oostvaardersveld may not completely
reflect the total of the other areas of the nature reserve. Even though the Oostvaardersveld has several
waterbodies, these are not comparable in size to the lakes in the other areas. The number of birds visiting
the Oostvaardersveld, is relatively low compared to the other areas of the nature reserve. However these
areas are not open to the public and researchers.
On the other hand the failure to detect WNV in the collected mosquitoes could reflect the absence of
WNV circulation in De Oostvaardersplassen. Although the virus might be introduced (repeatedly) in the
reserve by migratory birds, the local basic reproduction rate, R0, might be too low for WNV
establishment in a sylvatic cycle. Further research into factors of influence like the vector competence of
the local mosquito population, the susceptibility of the indigenous bird population and ecological factors
like temperature, precipitation and occurrence of floods is necessary.
In total 11 of the 13 different mosquito species collected in the Oostvaardersplassen are reported field or
laboratory vectors for infectious pathogens, including Tahyna virus (TAHV), Sindbis virus (SINV), Usutu
virus (USUV), Batai virus (BATV), Lednice virus (LEDV), Francisella tularensis and Dirofilaria immitis
(Table 4). Especially SINV, USUV, LEDV and BATV are likely other candidates for circulation in the
nature reserve. Like WNV, SINV, USUV and LEDV are maintained in an avian-mosquito cycle. The
vertebrate reservoirs are largely passeriform (WNV, USUV) and anseriform (LEDV) birds, with
migratory members being responsible for wide geographic distribution (Hubálek, 2008, Weissenbock et
al., 2009). Natural foci of SINV and LEDV infections occur mainly in wetland ecosystems like the
Oostvaardersplassen (Hubálek 2008). Although BATV has principally a domestic animal – mosquito

10. 78
cycle it can persistently infect birds and the virus is thought to spread across Europe through migratory
birds (Hubálek & Halouzka, 1996).
Sindbis virus circulates in Eurasia, Africa and Oceania (Taylor et al., 1955, Hubálek, 2008). Outbreaks in
Europe of Pogosta disease (human Sindbis virus infections) have thus far emerged in Fennoscandia every
seven years since the 1st outbreak was noted in 1974 with hundreds to thousands of clinical cases
(Hubálek, 2008). However in 2009 this seven-year cycle did not recur (Sane et al., 2010). Human disease
is characterized by fever, rash and (chronic) arthritis (Hubálek, 2008).
In Europe evidence for USUV circulation in birds exists for Austria, Hungary, Italy, Switzerland,
England, Poland and the Czech Republic (Weissenböck et al., 2008). In 2009 the first European human
cases of USUV-related neurological disease occurred in Italy (Pecorari et al., 2009; Cavrini et al., 2009).
Lednice virus circulates in birds in the Czech Republic and Romania. No evidence for disease in
mammals, including man, exists sofar (Hubálek, 2008).
Bataivirus is present in Europe, Asia and Africa. In Europe evidence for circulation exists for Norway,
Sweden, Finland, Slovakia, the Czech Republic, Croatia, Serbia, Bosnia, Montenegro, Italy, Hungary,
Romania, Portugal, Germany and Belarus (Hubálek, 2008). Vertebrate hosts are pigs, horses and
ruminants. Closely related viruses cause stillbirths and congenital abnormalities in ruminants in the USA
(Chung et al., 1990). However, European veterinary data are lacking. Human BATV infections are
associated with influenza-like illness in Europe and febrile illness in Asia and Africa (Juricová et al.,
2009, Hubálek, 2008).
Considering the wide variety of wildlife, richness in mosquito species and frequent avian visitors from
arbovirus endemic areas in Europe and Africa, De Oostvaardersplassen is considered a high risk ecosytem
for arbovirus circulation in the Netherlands. The establishment of a sylvatic cycle of WNV in the
Oostvaardersplassen creates an opportunity for WNV introduction into the urban cycle through peri-
domestic birds and subsequent WNV transmission to humans through bridge vectors. The city of Almere
with 190,000 inhabitants borders directly on the nature reserve while the city of Lelystad with 75,000
inhabitants is less then 3 km away (Figure 1). Further research into arbovirus circulation in the nature
reserve is essential for assessing the risks for public health by emerging arboviruses endemic elsewhere in
Europe or even Africa.
Acknowledgements
The authors are indebted to Francis Schaffner for checking and confirmation of the species identifications
in this study. The authors thank Joke van der Giessen for critical reading of the manuscript and
Staatsbosbeheer for the kind cooperation in the nature reserve.

11. 79
Table 4. Overview diseases putatively transmitted by mosquito species collected in De Oostvaardersplassen.
mosquito species reported field vector reported laboratory vector probable vector for
Anopheles algeriensis malaria.
An. claviger malaria Tahyna, Batai, myxomatosis (virus), canine
filariasis, Setaria labiatopapillosa (nematode),
anaplasmosis, Lyme, Tularaemia (bacterium).
An. maculipennis s.l.
- sensu stricto malaria (minor role) Batai, Tahyna, West Nile, Dirofilaria immitis,
myxomatosis, Tularaemia.
- atroparvus malaria, West Nile, myxomatosis Tahyna, Dirofilaria immitis, Tularaemia
- messeae malaria (minor role) Batai, Tahyna, West Nile, canine filarias,
myxomatosis, Tularaemia
An. plumbeus malaria, West Nile, filariasis
Coquillettidia richiardii Batai, West Nile, Tahyna
Culex modestus West Nile, Tahyna, myxomatosis,
Sindbis, Lednice, Dirofilaria immitis
Tularaemia
Cx. pipiens West Nile, Sindbis, Batai, Usutu, Tahyna, Dirofilaria immitis
Cx. torrentium Sindbis
Culiseta annulata Usutu, myxomatosis Tahyna
Cs. morsitans Sindbis (minor role)
Da. geniculata Tularaemia West Nile, yellow fever
Ochlerotatus cantans Tahyna, West Nile, myxomatosis
Taken from (Schaffner et al., 2001) and adapted based on (Poncon et al., 2007, Weissenböck et al., 2008)

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References
Almeida, A.P., Galao, R.P., Sousa, C.A., Novo, M.T., Parreira, R., Pinto, J., Piedade, J. & Esteves, A.
(2008) Potential mosquito vectors of arboviruses in Portugal: species, distribution, abundance and
West Nile infection. Transactions of the Royal Society of Tropical Medicine and Hygiene 102,
823-32.
Ashe P., O'Connor, J.P. & Casey, R.J. (1991) Irish mosquitoes (Diptera: Culicidae): a checklist of the
species and their known distribution. Proceedings of the Royal Irish Academy 91B, 21-36.
Bakonyi, T., Ivanics, E., Erdelyi, K., Ursu, K., Ferenczi, E., Weissenbock, H. & Nowotny, N. (2006)
Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerging Infectious
Diseases 12, 618-23.
Barzon, L., Squarzon, L., Cattai, M., Franchin, E., Pagni, S., Cusinato, R. & Palu, G. (2009) West Nile
virus infection in Veneto region, Italy, 2008-2009. Eurosurveillance 14.
Becker, N., Petric, D., Zgomba, M., Boase, C., Dahl, C., Lane, J. & Kaiser, A. (2003) Mosquitoes and
their control. Kluwer Academic/Plenum Publishers, NY, NY U.S.A.
Burke, D.S. & Monath, T.P. (2001) Flaviviruses. In: Virology, Fields, B.N. 4th edition. Eds: Knipe, D.M.,
Lippincott, Williams and Wilkins, USA.
Cavrini, F., Gaibani, P., Longo, G., Pierro, A.M., Rossini, G., Bonilauri, P., Gerundi, G.E., Di Benedetto,
F., Pasetto, A., Girardis, M., Dottori, M., Landini, M.P. & Sambri, V. (2009) Usutu virus
infection in a patient who underwent orthotropic liver transplantation, Italy, August-September
2009. Eurosurveillance 14.
Ceianu, C.S., Ungureanu, A., Nicolescu, G., Cernescu, C., Nitescu, L., Tardei, G., Petrescu, A., Pitigoi,
D., Martin, D., Ciulacu-Purcarea, V., Vladimirescu, A. & Savage, H.M. (2001) West Nile virus
surveillance in Romania: 1997-2000. Viral Immunology 14, 251-62.
Chao, D.Y., Davis, B.S. & Chang, G.J. (2007) Development of multiplex real-time reverse transcriptase
PCR assays for detecting eight medically important flaviviruses in mosquitoes. Journal of
Clinical Microbiology 45, 584-9.
Chung, S.I., Livingston, C.W., Edwards, J.F., Crandell, R.W., Shope, R.E., Shelton, M.J. & Collisson,
E.W. (1990) Evidence that Cache Valley virus induces congenital malformations in sheep.
Veterinary Microbiology 21, 297-307.
Connell, J., McKeown, P., Garvey, P., Cotter, S., Conway, A., O'Flanagan, D.P., O'Herlihy, B.,
Morgan, D., Nicoll, A. & Lloyd, G. (2004) Two linked cases of West Nile virus (WNV) acquired
by tourists in the Algarve, Portugal. Eurosurveillance 8.
Dauphin, G., Zientara, S., Zeller, H. & Murgue B. (2004) West Nile: worldwide current situation in
animals and humans. Comparative Immunology, Microbiology and Infectious Diseases 27, 343-
55.
Del Giudice, P., Schuffenecker, I., Vandenbos, F., Counillon, E. & Zeller, H. (2004) Human West Nile
virus, France. Emerging Infectious Diseases 10, 1885-1886.
Esteves, A., Almeida, A.P., Galao, R.P., Parreira, R., Piedade, J., Rodrigues, J.C., Sousa, C.A. & Novo,
M.T. (2005) West Nile virus in Southern Portugal, 2004. Vector Borne and Zoonotic Diseases 5,
410-3.
Fernandes, T., Clode, M.H.H., Simões, M.J., Ribeiro, H. & Anselmo, M.L. (1998) Isolation of virus West
Nile from a pool of unfed Anopheles atroparvus females in the Tejo River estuary, Portugal. Acta
Parasitologica Portugal 5, 7.
Filipe, A. R. (1972) Isolation in Portugal of West Nile virus from Anopheles maculipennis mosquitoes.
Acta Virologica 16, 361.
Fyodorova, M. V., Savage, H.M., Lopatina, J.V., Bulgakova, T.A., Ivanitsky, A.V., Platonova, O.V. &
Platonov, A.E. (2006) Evaluation of potential West Nile virus vectors in Volgograd region,
Russia, 2003 (Diptera: Culicidae): species composition, bloodmeal host utilization, and virus
infection rates of mosquitoes. Journal of Medical Entomology 43, 552-63.

13. 81
Hannoun, C., Panthier, R., Mouchet, J. & Ezouan, J. (1964) Isolation in France of the WNV from patients
and from the vector Culex modestus Ficalbi. Comptes Rendus Hebdomadaires des Séances de
l'Académie des Sciences 259, 4170-4172.
Higgs, S., Snow, K. & Gould, E.A. (2004) The potential for West Nile virus to establish outside of its
natural range: a consideration of potential mosquito vectors in the United Kingdom. Transactions
of the Royal Society of Tropical Medicine and Hygiene 98, 82-7.
Hubálek, Z. (2008) Mosquito-borne viruses in Europe. Parasitology Research 103 Suppl 1, S29-43.
Hubálek, Z. & Halouzka, J. (1996) Arthropod-borne viruses of vertebrates in Europe. Acta Scientarum
Naturalium Academiae Scientiarum Bohemicae Brno 30, 1-95.
Hubálek, Z. & Halouzka, J. (1999) West Nile fever-a reemerging mosquito-borne viral disease in Europe.
Emerging Infectious Diseases 5, 643-50.
Hubálek, Z., Savage, H.M., Halouzka, J., Juricova, Z., Sanogo, Y.O. & Lusk, S. (2000) West Nile virus
investigations in South Moravia, Czechland. Viral Immunology 13, 427-33.
Hurlbut, H.S., Rizk, F., Taylor, R.M. & Work, T.H. (1956) A study of the ecology of West Nile virus in
Egypt. American Journal of Tropical Medicine and Hygiene 5, 579-620.
Juriková, Z., Hubálek, Z., Halouzka, J. & Sikutová, S. (2009) Serological examination of songbirds
(passeriformes) for mosquito-borne viruses Sindbis, Tahyna, and Batai in a South Moravian
wetland (Czech Republic). Vector-borne and Zoonotic Diseases 9, 295-299.
Kaptoul, D., Viladrich, P., Domingo, C., Niubó, J., Martínez-Yélamos, S., De Ory, F. & Tenorio, A.
(2007) West Nile virus in Spain: report of the first diagnosed case (in Spain) in a human with
aseptic meningitis. Scandinavian Journal of Infectious Diseases 39, 70-71.
Katsarov, G., Vasilenko, S., Vargin, V., Burtenko, S. & Tkachenko, E. (1980) Serological studies on the
distribution of some arboviruses in Bulgaria. Problems Infectious Parasite Diseases 8, 32-35.
Kooijman, G. & Vulink, Th. J. (2007) De Oostvaardersplassen natuurlijk! Evaluatie van ontwikkeling en
beheer van het ecosysteem 1996-2005. Ministerie van Verkeer en Waterstaat. Internal report (in
Dutch), 59pp.
Koopmans, M., Martina, B., Reusken, C.B.E.M. & Van Maanen, K. (2008) West Nile virus in Europe.
Chapter 8. In: Takken, W. & Knols, B. (Eds.) Emerging pests and vector-borne diseases in
Europe. Wageningen Academic publishers.
Kramer, L.D., Li, J. & Shi, P.Y. (2007) West Nile virus. Lancet Neurology 6, 171-81.
Krisztalovics, K., Ferenczi, E., Molnar, Z., Csohan, A., Ban, E., Zoldi, V. & Kaszas, K. (2008) West Nile
virus infections in Hungary, August-September 2008. Eurosurveillance 13.
Labuda, M., Kozuch, O. & Gresikova, M. (1974) Isolation of West Nile virus from Aedes cantans
mosquitoes in West Slovakia. Acta Virologica 18, 429-433.
Lvov, D.K., Butenko, A.M., Gromashevsky, V.L., Larichev, V.P., Gaidamovich, S.Y., Vyshemirsky,
O.I., Zhukov, A.N., Lazorenko, V.V., Salko, V.N., Kovtunov, A.I., Galimzyanov, K.M.,
Platonov, A.E., Morozova, T.N., Khutoretskaya, V., Shishkina, E.O. & Skvortsova, T.M. (2000)
Isolation of two strains of West Nile virus during an outbreak in southern Russia, 1999. Emerging
Infectious Diseases 6, 373-6.
Mailles, A., Dellamonica, P., Zeller, H., Durand, J.P., Zientara, S., Goffette, R., Gloaguen, C.,
Armengaud, A., Schaffner, F., Hars, J., Chodorge, E. & Barbat, J. (2003) Human and equine
West Nile virus infections in France, August-September 2003. Eurosurveillance 7.
Mohrig, W. (1969) Die Culiciden Deutschlands. In: Eichler, W., Sprehn, C.E., Stamme, H.J. (Eds.)
Untersuchungen zur Taxonomie, Biologie und Ökologie der einheimischen stechmücken. Gustav
Fischer Verlag.
Murgue, B., Murri, S., Triki, H., Deubel, V. & Zeller, H.G. (2001) West Nile in the Mediterranean basin:
1950-2000. Annals of the New York Academy of Sciences 951, 117-26.
Pecorari, M., Longo, G., Gennari, W., Grottola, A., Sabbatini, A.M., Tagliazucchi, S., Savini, G.,
Monaco, F., Simone, M.L., Lelli, R., Rumpianesi, F. (2009) First human case of Usutu virus
neuroinvasive infection, Italy, August-September 2009. Eurosurveillance 14.

14. 82
Platonov, A.E., Shipulin, G.A., Shipulina, O.Y., Tyutyunnik, E.N., Frolochkina, T.Y., Lanciotti, R.S.,
Yazyshina, S., Platonova, O.V., Obukhov, I.L., Zhukov, A.N., Vengerov, Y.Y. & Pokrovskii,
V.I. (2001) Outbreak of West Nile virus infection, Volgograd Region, Russia, 1999. Emerging
Infectious Diseases 7, 128-32.
Poncon, N., Toty, C., L'Ambert, G., Le Goff, G., Brengues, C., Schaffner, F. & Fontenille, D. (2007)
Biology and dynamics of potential malaria vectors in Southern France. Malaria Journal 6, 18.
Popovici, F., Sarbu, A., Nicolae, O., Pistol, A., Cucuiu, R., Stolica, B., Furtunescu, F., Manuc, M. &
Popa, M.I. (2008) West Nile fever in a patient in Romania, August 2008: case report.
Eurosurveillance 13.
Ramsdale, C. & Snow, K. (2000) Distribution of Anopheles species in the British Isles. European
Mosquito Bulletin 7, 1-26.
Rappole, J.H. & Hubálek, Z. (2003) Migratory birds and West Nile virus. Journal of Applied
Microbiology 94 Suppl 47S-58S.
Rappole, J.H., Derrickson, S.R. & Hubálek, Z. (2000) Migratory birds and spread of West Nile virus in
the Western Hemisphere. Emerging Infectious Diseases 6, 319-28.
Rees, A.T. & Rees, A.E. (1989) Anopheles algeriensis on Anglesey: the story so far. British
Mosquito Group Newsletter 6, 1-5.
Remm, K.Y. (1957) Data on the fauna and ecology of mosquitoes in Estonia. Entomologicheskoe
Obozrenie 36, 148-160.
Rizzo, C., Vescio, F., Declich, S., Finarelli, A.C., Macini, P., Mattivi, A., Rossini, G., Piovesan, C.,
Barzon, L., Palu, G., Gobbi, F., Macchi, L., Pavan, A., Magurano, F., Ciufolini, M.G.,
Nicoletti, L., Salmaso, S. & Rezza, G. (2009) West Nile virus transmission with human cases in
Italy, August - September 2009. Eurosurveillance 14.
Sane, J., Guedes, S., Kurkela, S., Lyytikäinen, O. & Vapalahti, O. (2010) Epidemiological analysis of
mosquito-borne Pogosta disease in Finland 2009. Eurosurveillance 15.
Savage, H.M., Ceianu, C., Nicolescu, G., Karabatsos, N., Lanciotti, R., Vladimirescu, A., Laiv, L.,
Ungureanu, A., Romanca, C. & Tsai, T.F. (1999) Entomologic and avian investigations of an
epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization
of a virus isolate from mosquitoes. American Journal of Tropical Medicine and Hygiene 61, 600-
11.
Schaffner, F., Angel, G., Geoffrey, B., Hervy, J.-P., Rhaiem, A. & Brunhes, J. (2001) The mosquitoes of
Europe. CD-ROM. Montpellier: Institut de Recherche pour le Développement/Entente
interdépartementale pour la démoustication du littoral (EID) Méditerrannée.
Scholte, E.J. (2009a). Diagnostic key for adult Culicidae endemic in the Netherlands. Dutch Centre for
Monitoring of Vectors, Ministry of Agriculture, Nature, and Food safety. Internal document, 4 pp.
Scholte, E., Den Hartog, W., Braks, M., Reusken, C., Dik, M. & Hessels, A. (2009) First report of a
North American invasive mosquito species Ochlerotatus atropalpus (Coquillett) in the
Netherlands, 2009. Eurosurveillance 14.
Scholte E.J., Dijkstra, E., Ruijs, H., Jacobs, F., Takken, W., Hofhuis, A., Reusken, C., Koopmans, M. &
Boer de, A. (2007) The Asian tiger mosquito (Aedes albopictus) in the Netherlands: should we
worry? Proceedings of the Netherlands Entomological Society, meeting 18, 131-136.
Scholte, E.J., Dijkstra, E., Blok, H., De Vries, A., Takken, W., Hofhuis, A., Koopmans, M., De Boer,
A. & Reusken, C. (2008) Accidental importation of the mosquito Aedes albopictus into the
Netherlands: a survey of mosquito distribution and the presence of dengue virus. Medical and
Veterinary Entomology 22, 352-8.
Snow, K.R. (1990) Mosquitoes. Naturalists’ Handbooks 14. The Richmond Publishing Co.Ltd.
Smithburn, K.C., Hughes, T.P., Burke, A.W. & Paul, J.H. (1940) A neurotropic virus isolated from the
blood of a native of Uganda. Journal of Tropical Medicine 20, 471-492.
Takken, W., Verhulst, N., Scholte, E.J., Jacobs, F., Jongema, Y., van Lammeren, R.J.A., Bergsma, A.R.,
Klok, T.C., van Roermund, H.J.W., de Koeijer, A.A. & Borgsteede, F.H.M. (2007) Distribution
and dynamics of arthropod vectors of zoonotic disease in the Netherlands in relation to risk of

15. 83
disease transmission. Internal Report Wageningen University and Research Centre for Dutch
Ministry of Agriculture, Nature and Food Quality.
Taylor, R.M., Hurlbut, H.S., Work, T.H., Kingston, J.R., Frothingham, T.E. (1955) Sindbis virus: a newly
recognized arthropodtransmitted virus. American Journal of Tropical Medicine and Hygiene 4,
844-62.
Tsai, T.F., Popovici, F., Cernescu, C., Campbell, G.L. & Nedelcu, N.I. (1998) West Nile encephalitis
epidemic in southeastern Romania. The Lancet 352, 767-71.
Verdonschot, P. (2002). Culicidae. pp. 98-100. In: Beuk, P.L.Th. (ed.). Checklist of the Diptera in the
Netherlands. KNNV publishers, Utrecht, the Netherlands
Weissenbock, H., Hubálek, Z., Bakonyi, T. & Nowotny, N. (2009) Zoonotic mosquito-borne flaviviruses:
Worldwide presence of agents with proven pathogenicity and potential candidates of future
emerging diseases. Veterinary Microbiology 140, 271-280.
Weissenböck, H., Chvala-Mannssberger, S., Bakonyi, T. & Nowotny, N. (2008) Emergence of Usutu
virus in Central Europe: diagnosis, surveillance and epizootology. In: Emerging pests and vector-
borne diseases in Europe. Takken, W. & Knols, B. (Eds.) Wageningen Academic publishers.
Zeller, H.G. & Schuffenecker, I. (2004) West Nile virus: an overview of its spread in Europe and the
Mediterranean basin in contrast to its spread in the Americas. European Journal of Clinical
Microbiology and Infectious Diseases 23, 147-56.