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Contents lists available at ScienceDirect

Preventive Veterinary Medicine

j ourna l h om epa ge: www.elsev ier .com/ locate /prevetmed

he emergence of Schmallenberg virus across Culicoidesommunities and ecosystems in Europe

homas Balenghiena,b,∗, Nonito Pagèsc, Maria Goffredod, Simon Carpentere,enis Augot f, Elisabeth Jacquiera,b, Sandra Talaverac, Federica Monacod,

érôme Depaquit f, Colette Grilleta,b, Joan Pujolsc, Giuseppe Sattag,ohamed Kasbari f, Marie-Laure Setier-Rioh, Francesca Izzog, Cigdem Alkani,j,

ean-Claude Delécollek, Michela Quagliag, Rémi Charrel i,j, Andrea Polcig,mmanuel Bréard l, Valentina Federici g, Catherine Cêtre-Sossaha,b,laire Garrosa,b

Cirad, UMR15 CMAEE, F-34398 Montpellier, FranceINRA, UMR1309 CMAEE, F-34398 Montpellier, FranceCentre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra

Cerdanyola del Vallès), SpainIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100 Teramo, ItalyThe Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF, United KingdomUniversité de Reims Champagne-Ardenne, ANSES, SFR Cap Santé, EA4688 - USC « VECPAR », Reims, FranceIstituto Zooprofilattico della Sardegna, Via Duca degli Abruzzi 8, Sardinia, ItalyEID Méditerranée, 34184 Montpellier, FranceUMR D 190 “Emergence des Pathologies Virales”, Aix Marseille Univ, IRD French Institute of Research for Development, EHESP Frenchchool of Public Health, 13005 Marseille, FranceIHU Mediterranee Infection, APHM Public Hospitals of Marseille, 13005 Marseille, FranceIPPTS, 67000 Strasbourg, FranceANSES, UMR Virologie, Maisons-Alfort, France

r t i c l e i n f o

rticle history:eceived 23 September 2013eceived in revised form 24 January 2014ccepted 8 March 2014

eywords:rbovirusector competence

a b s t r a c t

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread acrossEurope since 2011 inflicting congenital deformities in the offspring of infected adult rumi-nants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have beenimplicated in the transmission of SBV through studies conducted in northern Europe. Inthis study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) werescreened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletusand C. scoticus) in transmission of SBV were confirmed in France and SBV was also discov-ered in a pool of C. nubeculosus for the first time, implicating this species as a potential

Please cite this article in press as: Balenghien, T., et al., The emergence of Schmallenberg virus across Culicoides commu-nities and ecosystems in Europe. PREVET (2014), http://dx.doi.org/10.1016/j.prevetmed.2014.03.007

eratopogonidaerthobunyavirusunyaviridae

vector. While collections in Sardinia were dominated by C. imicola, only relatively smallquantities of SBV RNA were detected in pools of this species and conclusive evidence of itspotential role in transmission is required.

In addition to these field-based studies, infection rates in colony-derived individuals ofC. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Ratesof infection in C. nubeculosus were low, confirming previous studies, while preliminary

∗ Corresponding author at: Cirad, TA A-15/G Campus international de Baillarguet, 34398 Montpellier Cedex 5, France. Tel.: +33 4 67 59 37 53;ax: +33 4 67 59 37 95.

E-mail address: thomas.balenghien@cirad.fr (T. Balenghien).

http://dx.doi.org/10.1016/j.prevetmed.2014.03.007167-5877/© 2014 Elsevier B.V. All rights reserved.

ARTICLE IN PRESSG ModelPREVET-3539; No. of Pages 10

2 T. Balenghien et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx

examination of C. scoticus demonstrated that while this species can replicate SBV to a poten-tially transmissible level, further work is required to fully define comparative competencebetween species in the region. Finally, the oral competence for SBV of two abundant andwidespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictusnor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appearunlikely to play a major role in transmission. Other vector competence data produced fromstudies across Europe to date is then comprehensively reviewed and compared with thatgenerated previously for bluetongue virus.

1. Introduction

Schmallenberg virus (SBV) is a member of the Orthobun-yavirus genus first identified in North Rhine-Westphalia,Germany, during summer 2011 (Hoffmann et al., 2012).Infection with SBV can lead to mild clinical signs in adultruminants, including pyrexia, decreased milk productionand diarrhea (Davies et al., 2012). The primary economicimpact of SBV, however, lies in severe congenital malfor-mations following transplacental transmission in cattle andsheep (van den Brom et al., 2012). Initial phylogenetic stud-ies placed SBV in the Simbu serogroup, sharing a closerelationship to Sathuperi and Douglas viruses and secon-darily to Shamonda virus and included in the same lineagethan the Akabane virus (Saeed et al., 2001; Goller et al.,2012). These viruses have been primarily isolated fromCulicoides (Doherty et al., 1972; St George et al., 1978; Lee,1979; Cybinski, 1984; Blackburn and Searle, 1985; Kurogiet al., 1987; Yanase et al., 2005) and more rarely frommosquitoes (Dandawate et al., 1969; Metselaar and Robin,1976). The recent emergence of the similarly Culicoides-borne bluetongue virus (BTV) in western and northernEurope (Carpenter et al., 2009), therefore led to immedi-ate suspicion that SBV was transmitted by female midgesfrom the Culicoides genus.

Following detection of the SBV incursion, virus RNA wasrapidly identified in field-collected Culicoides from farms inthe affected regions (De Regge et al., 2012; Rasmussen et al.,2012; Elbers et al., 2013a,b; Goffredo et al., 2013; Larskaet al., 2013). Taken in their entirety, these studies con-vincingly implicated a range of widespread and abundantfarm-associated Culicoides species in the transmission ofSBV including Culicoides obsoletus, Culicoides scoticus, Culi-coides dewulfi and Culicoides chiopterus. A detailed studyof SBV replication and dissemination in the model speciesCulicoides sonorensis also allowed confirmation that lev-els of viral RNA in studies carried out in the Netherlands(Elbers et al., 2013b) were likely to represent transmissibleinfections (Veronesi et al., 2013).

From 2011 to 2013, SBV has spread across a huge geo-graphic area in Europe at a rate substantially exceeding thatof the BTV-8 epidemic which occurred in the same regionfrom 2006 to 2010. Current distribution of SBV rangesfrom the Mediterranean to Scandinavian regions, and fromIreland to Estonia (European Food Safety Authority, 2013).The rate of seroconversion recorded in many farms was

Please cite this article in press as: Balenghien, T., et al., The emenities and ecosystems in Europe. PREVET (2014), http://dx.doi.o

rapid (Elbers et al., 2012; Meroc et al., 2013) with a highproportion of ruminants apparently becoming infectedin a short time frame. A partial explanation for this

© 2014 Elsevier B.V. All rights reserved.

phenomenon could be the absence of animal movementrestrictions that were employed for BTV but not for SBVin the vast majority of countries reporting incursions.Additionally, however, it is suspected that the vector com-petence of Culicoides for SBV may exceed rates recordedfor BTV either in the number of species capable of trans-mitting the virus or in the proportion of individuals withina species able to act as vectors. This hypothesis receivessupport from the fact that the related Akabane virus isisolated at a far higher frequency than BTV from Culicoidesin Australia (St George et al., 1978), although comparativelaboratory-based investigations of susceptibility rates invector species have not been performed.

In this study we present the first data on detectionof SBV RNA in Culicoides from mainland France and Italy(Sardinia), examine infection rates in colony-derived indi-viduals of Culicoides nubeculosus (Meigen) and additionallypreliminary results for experimental infections of field-collected individuals. A significant advantage in carryingout such detections across a wide geographic range is thatit enables an understanding of SBV transmission acrossdisparate ecosystems and potential vector species. Thisincludes the examination of the role of Culicoides imicola,a major afrotropical vector of BTV with a distribution thatcould facilitate the spread of SBV into new areas, includ-ing Asia. We also examine two abundant and widespreadmosquito vector species in the laboratory to assess whetheralternative vectors could be involved in transmission of SBVacross Europe. Finally, we also review vector competencedata produced from studies across Europe to date com-prehensively and compare with that generated previouslyfor BTV. By contrasting the diverse ecosystems and poten-tial vectors present across these countries we subsequentlydraw conclusions regarding the vulnerability of Europe tofurther incursions.

2. Material and methods

2.1. Laboratory infection of mosquitoes and Culicoides

Experimental infection with SBV was carried out at theCentre de Recerca en Sanitat Animal (CReSA) using lines ofCulex pipiens Linnaeus and Aedes albopictus Skuse that hadbeen maintained for two and four years, respectively. Inaddition, a line of C. nubeculosus was also infected at CReSAwhich had originated from that originally established at

rgence of Schmallenberg virus across Culicoides commu-rg/10.1016/j.prevetmed.2014.03.007

The Pirbright Institute. All studies using both intrathoracicinoculation and artificial membrane-based blood-feedingwere conducted at the CReSA Biosafety level 3 facilities.

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For intrathoracic (IT) inoculation, females of eachpecies (of ages 2–4 days) were anesthetised using carbonioxide and placed under a stereomicroscope. A volumef 0.2–0.4 �l (Culicoides) and 1–2 �l (mosquitoes) of a.38 TCID50/ml SBV viral suspension, produced on a babyamster kidney (BHK-21) cell line was inoculated intratho-acically into each individual using a glass micro-needle.nsertion of the needle was made between the epimeronnd episterum using a manual micro-injector (Sutternstruments, California, USA). Inoculated mosquitoes and

idges were placed in groups of 15–20 individuals insideardboard and plastic primary containers for Culicoides andosquitoes respectively. Females were fed with 5% sucrose

d libitum and maintained at 24 ± 2 ◦C and 80% RH, with 14:10 (light:dark) photoperiod for eight to nine daysrior to processing. Females of the same age range werelso exposed to oral infection using an artificial membraneystem fitted with a one day old chick skin membraneHemotek, UK). This study used a 1:1 mixture of bovinelood and 4.38 TCID50/ml SBV suspension, to obtain anal concentration of 4.08 TCID50/ml, that during the trialas heated to 38 ◦C during a 45–60 min exposure. Fully

ngorged females of all three species were subsequentlyelected and placed inside primary containers for an extrin-ic incubation period of 10 days using same maintenanceonditions as for IT inoculations.

Experimental infection trials with SBV were also carriedut with field collected Culicoides to provide preliminarystimates of their vector competence for SBV at the Uni-ersity Champagne-Ardenne Biosafety level 2 facilities.ulicoides were collected in a farm located in north-astern France (Louvois: 49◦06′06′′N, 4◦07′00′′E) usingight/suction traps (UV CDC trap, John W. Hock Compagny,ainesvile, FL, USA) or after emergence from manure or

Please cite this article in press as: Balenghien, T., et al., The emenities and ecosystems in Europe. PREVET (2014), http://dx.doi.o

he margins of ponds. A total of 60 females were allowedo feed on 10 ml of sucrose solution containing a final con-entration of 103 TCID50/ml SBV (produced on Vero3 celline) using a cotton pledglet for four hours. Females were

Fig. 1. Localisation of traps and Schmallenberg vi

PRESSry Medicine xxx (2014) xxx–xxx 3

fed with 10% sucrose ad libitum and maintained at 25 ± 2 ◦Cand 80% RH with a 15:9 (light:dark) photoperiod for fourto eight days prior to processing. Identification of orallyexposed Culicoides was confirmed by cytochrome oxydaseI sequencing, as barcode sequence reference (Hebert andGregory, 2005).

2.2. Screening of field-collected Culicoides for SBVpresence

Collection sites for detection of SBV transmission in Culi-coides were selected in temperate Europe (north-easternFrance) and in the Mediterranean island of Sardinia (Italy)(Fig. 1). In France, wide-scale monitoring of the activity ofCulicoides populations was already in place prior to the SBVoutbreak and was subsequently carried out from 2009 to2012 across both mainland areas and Corsica (Balenghienet al., 2011; Venail et al., 2012). This consisted of approxi-mately 160 light-suction UV traps (either one or two basedin each department), run at weekly intervals during mid-February to April and during November and December andat monthly intervals for the rest of the year (Balenghienet al., 2011; Venail et al., 2012). SBV detection was targetedat traps located in departments where SBV surveillancehad highlighted significant incidence in 2011, i.e. >1.5%as defined by the French national surveillance program(Dominguez et al., 2013). In total, Culicoides collected from53 traps in 28 distinct departments were selected (includ-ing 6 traps selected prior to the final results of the SBVsurveillance which subsequently were found to have anincidence comprised between 0 and 0.5%). Screening forSBV RNA was conducted on Culicoides collected from the3rd to the 6th October 2011 as late summer/autumn repre-sented the likely time of transmission of the virus described

rgence of Schmallenberg virus across Culicoides commu-rg/10.1016/j.prevetmed.2014.03.007

in other studies.In Italy, clinical outbreaks of SBV infection were

reported in Sardinia from October to December 2012, char-acterised by abortions that occurred in small ruminants

rus reports in France and Sardinia (Italy).

Please cite this article in press as: Balenghien, T., et al., The emenities and ecosystems in Europe. PREVET (2014), http://dx.doi.o

ARTICLE ING ModelPREVET-3539; No. of Pages 10

4 T. Balenghien et al. / Preventive Veterina

Tab

le

1Ex

per

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tal i

nfe

ctio

ns

of

mos

quit

o

and

Culic

oide

s

labo

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h

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mal

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Spec

ies

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sus

Intr

ath

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tion

Day

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pos

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on

0

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0

8–9

0

8–9

No.

inoc

ula

ted

/No.

test

ed5

51

4

89

9

130

No.

pos

itiv

e

(%)

5

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.0)

51

(100

.0)

4

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.0)

62

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7)9

(100

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129

(99.

2)M

ean

C tva

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[min

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28.7

[28.

0–29

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20.0

[16.

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28.8

[28.

3–29

.2]

23.4

[18.

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.8]

34.1

[32.

2–36

.5]

24.2

[15.

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No.

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8

149

2

27

12

102

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8

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2

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2

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0

12

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4

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C tva

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[28.

0–30

.8]

30.4

[29.

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.3]

28.8

[28.

7–28

.9]

30.0

[26.

9–33

.8]

27.5

[21.

7–38

.7]

PRESSry Medicine xxx (2014) xxx–xxx

held at farms in Sassari, Ogliastra and Cagliari provinces(Fig. 1). Following the confirmation of SBV, an entomo-logical survey was performed in the island. Between the30th October and 4th December 2012, a total of 26 Culi-coides collections were performed on 15 farms in sevenmunicipalities of Sardinia, located in the provinces ofCarbonia-Iglesias (San Giovanni Suergiu and Sant’AnnaArresi), Cagliari (Muravera), Ogliastra (Barisardo, Girasoleand Tertenia) and Sassari (Mores).

In both France and Italy, Culicoides were stored in70% ethanol and subsequently categorised according totheir physiological status (nulliparous, parous and bloodengorged). All Culicoides were identified morphologicallyusing a stereomicroscope (Delécolle, 1985) and females ofC. obsoletus and C. scoticus were grouped as the Obsole-tus complex. In each site in France, up to 50 unengorgedfemales of the Obsoletus complex were pooled (maximum1 pool) and additional pools of up to 50 unengorged femalesof other species (maximum 3 pools) were assayed for SBVpresence. In each site in Italy, parous and engorged femaleswere assayed separately.

2.3. Detection of SBV genome

In experimental infections conducted in Spain and infield-collected Culicoides screenings conducted in France,viral RNA was extracted using NucleoSpin RNA Virus(Macherey Nagel, Germany) following the manufac-turer’s instructions. In Italy, Culicoides nucleic acids wereextracted and purified using a BioSprint 96 One-For-All VetKit procedure based on the BioSprint 96 instrument (Qia-gen, California, USA). In experimental infections conductedin France, RNA was extracted using EZ1 virus mini kit v2.0

rgence of Schmallenberg virus across Culicoides commu-rg/10.1016/j.prevetmed.2014.03.007

(Qiagen, California, USA) following the manufacturer’sinstructions. RNA was extracted individually from entireinsects for both inoculated and orally exposed femalesin Spain or France, and for females from the Obsoletus

Fig. 2. Observed Ct values for Schmallenberg virus in mosquitoes andCulicoides 8–9 days after intrathoracic inoculation.

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omplex collected in France and in Italy, and from pools ofaximum 50 entire midges for all other species collected

n the field. The S gene of the Schmallenberg virus wasranscribed and amplified according to the one step real-ime RT-PCR protocol published by Hoffmann et al. (2012).ollowing extraction, SBV RNA was quantified using thene step real-time reverse transcription PCR (RT-PCR) fol-owing the protocol developed by Friedrich Loeffler InstitutFLI) targeting the S3 genomic fragment (Bilk et al., 2012),xcept for Cirad where RT-PCR was performed accordingo Taq Vet Schmallenberg virus S gene 50 kit (Laboratoireervice International, LSI, France). Assessments of infec-ion status were made using Ct values generated fromhe samples as a semi-quantitative measure of viral RNAuantity. In France, pools screened for SBV presence andith a 37 < Ct < 45 values were tested twice and statedositive when both tests gave the same 37 < Ct < 45 valuesith appropriate shape curves. Positive females of thebsoletus complex were identified to species level usingolecular species identification assay (Nolan et al., 2007).

. Results

.1. Experimental infections using laboratory colonies

.1.1. Intrathoracic inoculationsPresence of SBV RNA was detected by real-time RT-

CR in a subsample of the inoculated mosquitoes andidges on the day of inoculation (Table 1): the mean

t values were equivalent between the mosquito speciesssessed (28.7 ± 0.5 [confidence interval (CI) 28.3–29.2] fore. albopictus and 28.8 ± 0.4 [CI 28.4–29.2] for Cx. pipiens)ut higher in C. nubeculosus (34.1 ± 1.5 [CI 33.1–35.1]), dueo a smaller inoculation volume used. The presence of SBVNA was detected after 8–9 days post-inoculation (dpi)

n all Ae. albopictus, in 69.7% of Cx. pipiens females and in9.2% of C. nubeculosus females (Table 1). The mean Ct val-es decreased in positive females between the inoculationay and 8–9 dpi by 8.0 Ct in Ae. albopictus (20.7 ± 5.1 [CI9.7–21.7]), by 5.4 in Cx. pipiens (23.4 ± 4.2 [CI 22.6–24.3])nd by 10.0 in C. nubeculosus (24.2 ± 4.9 [CI 23.3–25.0])Fig. 2).

.1.2. Oral exposure

Please cite this article in press as: Balenghien, T., et al., The emenities and ecosystems in Europe. PREVET (2014), http://dx.doi.o

Presence of SBV RNA was detected by real time RT-PCRn a subsample of the orally exposed mosquitoes and Culi-oides on the day of exposure (Table 1): the mean Ct valuesere similar between mosquitoes and Culicoides (29.0 ± 1.1

able 2xperimental infections (orally) of field-collected midges with Schmallenberg vir

Species No. exposed Days post-exposure

C. scoticus 45 0

4

5

8

Forcipomyia sp. 6 4

C. obsoletus 1 0

C. newsteadi 3 8

Other species 5 –

PRESSry Medicine xxx (2014) xxx–xxx 5

[CI 28.2–29.7] for Ae. albopictus, 28.8 ± 0.1 [CI 28.6–29.0]for Cx. pipiens and 30.0 ± 2.1 [CI 28.9–31.2] for C. nubecu-losus) despite a smaller blood-meal in Culicoides. At 10days post-exposure, SBV RNA was detected in 1.3% of Ae.albopictus (2/149), in none of the 27 blood-fed Cx. pipiensfemales and in 3.9% of the blood-fed C. nubeculosus (4/102).The infection rate was significantly greater for C. nubeculo-sus (95% binomial confidence interval of 1.08–9.73%) thanfor Ae. albopictus (0.16–4.76%): p = 0.049 under bionomialassumptions. It was not possible to distinguish the infec-tion rates between Cx. pipiens (0–12.77%) and the two otherspecies due to too few mosquitoes being tested (N = 27). TheCt values were similar ten days post-infection than the dayof exposure for the two positive Ae. albopictus (29.53 and31.25 versus 29.0), whereas they were much lower for 3 ofthe 4 positive C. nubeculosus (21.67, 24.21 and 25.3 versus30.0) strongly suggesting SBV replication.

Culicoides scoticus was the dominant species of field-collected females exposed orally to SBV (45 of the 60individuals). For this species, mean Ct values were 32.2 atday 0, 18.5 at day 4, 33.8 at day 5 and 29.0 at day 8 (Table 2)suggesting SBV replication in some C. scoticus individuals.Variation in Ct values may be due to differences in the quan-tity of ingested blood and in individual susceptibility withinC. scoticus population, and was amplified by the limitednumber of individuals.

It was not possible to interpret results for other speciesdue to too few individuals. Single viral RNA identificationin Forcipomyia or C. newsteadi individuals, especially witha high Ct value, was not sufficient to incriminate thesespecies as potential vectors without further investigations.

3.2. Screening of field-collected individuals for SBVpresence

In France, a total of 224,870 Culicoides, belonging toat least 20 species, were collected in the studied period.The species diversity was dominated by C. dewulfi (58.4%of the total catch), the Obsoletus complex (35.9%) and C.chiopterus (4.5%). The parity rates (No. parous females/No.females) of C. dewulfi and of the Obsoletus complex wereabout 32.0% and 40%. In Italy, a total of 53,531 Culicoideswere collected (Table 3). Culicoides imicola was the domi-nant species in these collections, (74.5% of the total catch),followed by Culicoides newsteadi Austen (21.7%), species

rgence of Schmallenberg virus across Culicoides commu-rg/10.1016/j.prevetmed.2014.03.007

of the Obsoletus complex (0.8%), Culicoides pulicaris (Lin-neaus) (0.4%) and Culicoides punctatus (Meigen) (0.01%)(Table 3). The parity rate of C. imicola was approximately55.0%.

us.

No. positive Mean Ct value [min–max]

4 32.2 [31.4–34.2]1 18.53 33.8 [33.1–34.2]2 29.0 [28.9–29.1]

1 31.51 33.91 35.20

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Table 3Culicoides collected in Sardinia (Italy) from the 30th October to the 5th December 2012 and assessed for the presence of Schmallenberg virus.

Species Province Ogliastra Sassari Cagliari Carbonia-Iglesias Total

Municipality (No. collections) Barisardo (7) Girasole (1) Tertenia (5) Mores (4) Muravera (1) San Giovanni Suergiu (3) Sant’Anna Arresi (5)

C. imicola Total No. midges 19,576 11 769 3778 99 15,589 44 39,866 (74.47%)No. PFa (No. pools) 10,909 (221) 7 (1) 603 (14) 395 (10) 50 (1) 10,132 (204) 30 (5) 22,126 (456)No. EFa (No. pools) 75 (3) 4 (1) 13 (3) 1 (1) 106 (4) 199 (12)

C. newsteadi Total No. midges 695 3 108 9248 151 1326 103 11,634 (21.73%)No. PF (No. pools) 303 (10) 45 (4) 4299 (87) 107 (3) 684 (15) 65 (5) 5503 (124)No. EF (No. pools) 12 (2) 2 (2) 104 (5) 2 (1) 8 (1) 1 (1) 129 (12)

Obsoletus complex Total No. midges 48 0 81 137 7 130 9 421 (0.77%)No. PF 7 41 59 6 14 4 131No. EF 4 4 2 10

C. pulicaris Total No. midges 81 3 74 33 1 0 0 192 (0.36%)No. PF (No. pools) 25 (3) 2 (1) 28 (4) 16 (4) 1 (1) 72 (13)No. EF (No. pools) 2 (2) 2 (2)

C. punctatus Total No. midges 1 0 0 0 0 0 5 6 (0.01%)No. PF (No. pools) 1 (1) 4 (2) 5 (3)

Other species Total No. midges 740 5 74 149 5 228 220 1421 (2.65%)

Total Total No. midges 21,141 22 1106 13,345 263 17,273 381 53,531No. PF (No. pools) 11,245 (242) 9 (2) 717 (63) 4769 (160) 164 (11) 10,830 (233) 103 (16) 27,837 (727)No. EF (No. pools) 87 (5) 10 (5) 123 (13) 3 (2) 116 (6) 1 (1) 340 (32)

a PF: parous females; EF: blood-fed females.

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Table 4Retrospective detection of Schmallenberg virus from Culicoides collected in France in October 2011.

Species No. Culicoides(pools) tested

No. positivepools

Mean Ct value[min–max]

Overall minimuminfection rate(MIR)

Mean MIR in positivesites [min–max]

Obsoletus complexa 1734 (347) 10 32.9 [23.4–38.2]a 0.58%a 4.14% [2.00–6.00]C. obsoletus 8 34.4 [28.3–38.2]a

C. dewulfi 1729 (47) 0C. chiopterus 1224 (40) 2 32.0 [30.6–33.4] 0.16% 1.17% [1.12–1.22]C. pulicaris 271 (27) 1 38.3 0.37% 1.25%C. newsteadi 65 (12) 0C. nubeculosus 43 (7) 1 28.8 2.33% 3.70%C. lupicaris 24 (9) 0C. punctatus 17 (7) 0C. festivipennis 9 (3) 0C. clastrieri 8 (1) 0C. circumscriptus 4 (2) 0C. fascipennis 2 (1) 0C. alazanicus 1 (1) 0C. cataneii/gejgelensis 1 (1) 0C. parroti 1 (1) 0C. puncticollis 1 (1) 0C. subfasciipennis 1 (1) 0

e identis duals cos rate sam

flatc0pgwiaI3eiawpp

4

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a Eight of the 10 positive individuals from the Obsoletus complex werpecies level. The Ct values associated with the Obsoletus complex indivipecies) leading to minimum infection rates being the effective infection

For collections made in France 5135 females were testedor SBV RNA presence. Ten pools of females from the Obso-etus complex contained detectable quantities of SBV RNAnd all females processed individually that tested posi-ive were identified as C. obsoletus. Pools of the Obsoletusomplex had an overall minimum infection rate (MIR) of.58%, with C. chiopterus giving an MIR of 0.16%, C. pulicarisossessing an overall MIR of 0.37% and of C. nubeculosusiving an overall MIR of 2.33% (Table 4). Excluding siteshere SBV was not recovered increased the MIR to approx-

mately 4% for the Obsoletus complex and C. nubeculosusnd around 1% for C. chiopterus and C. pulicaris (Table 4). Fortalian collections, 27,837 parous females (727 pools) and40 engorged females (32 pools) were tested for SBV pres-nce. Detectable SBV RNA was identified in three pools of C.micola of which two were pools of 50 pigmented femalesnd one was a pool of 33 engorged females. All these poolsere collected at San Giovanni Suergiu (Carbonia-Iglesiasrovince) leading to a MIR = 0.04% within the pigmentedopulation of C. imicola in this site (Table 5).

Please cite this article in press as: Balenghien, T., et al., The emenities and ecosystems in Europe. PREVET (2014), http://dx.doi.o

. Discussion

This study, together with previous work in theetherlands (Elbers et al., 2013b), Belgium (De Regge et al.,

able 5etection of Schmallenberg virus from Culicoides collected at San Giovanni Suergi

Species No. midgescollected

Physiologicalstatus

No. midg(pools) t

C. imicola 7704 Parous 5050 (10Engorged 66 (2)

C. newsteadi 846 Parous 500 (10Engorged 8 (1)

Obsoletus complex 27 Parous 6 (6)

fied as C. obsoletus; it was not possible to identify the two others at therrespond to assay on single individuals (compared to pools for the otherpled populations of the Obsoletus complex.

2012) and Italy (Goffredo et al., 2013) confirmed the role ofC. obsoletus as a highly probable vector of SBV in northernEurope (Table 6). This species is among the most abun-dant livestock-associated species in the region (Meiswinkelet al., 2008; Carpenter et al., 2009; Venail et al., 2012) andits apparently ubiquitous distribution on farms across thePalaearctic and Nearctic may facilitate spread of SBV to newregions. In addition, C. nubeculosus was implicated for thefirst time as a potential vector in France, although quan-tities of SBV RNA detected were equivocal in defining thelevel of dissemination that had occurred (Veronesi et al.,2013). Studies of vector competence for SBV in colony linesof this species, both in the current study and in previousstudies in the UK (Veronesi et al., 2013), have indicatedextremely low rates of competence of approximately 3%.It is important to note, however, that such infection rateshave been demonstrated to vary with vector populationfor other Culicoides-borne arboviruses (Tabachnick, 1996).Preliminary evidence was also provided that C. scoticus isable to replicate SBV to transmissible levels, albeit usinga technique (pledglet feeding with sugar) that is likely to

rgence of Schmallenberg virus across Culicoides commu-rg/10.1016/j.prevetmed.2014.03.007

result in virus being transported to the crop rather than thegut (Jennings and Mellor, 1988).

Studies of C. imicola in Sardinia (Italy) failed to con-vincingly implicate this species in transmission through

u, Carbonia-Iglesias province, in Sardinia (Italy), the 7th November 2012.

esested

No. positivepools

Mean Ct value[min–max]

Minimuminfection rate

1) 2 36 [34–38] 0.04%1 33

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Table 6Published reports of Schmallenberg virus detection from field-collected Culicoides in Belgium, Denmark, Netherlands, Italy and Poland using detection assays from Hoffmann et al. (2012) and Bilk et al. (2012).

Country Period Pool constitutiona Species No. midges(pools) tested

No. positivepools

Mean Ct value[min–max]

Minimuminfection rate

Reference

Belgium August to October 2011 25 heads (PF) Obsoletus complex 688 (34) 5 33.9 [30.7–36.0] 0.73% De Regge et al. (2012)C. obsoletus 283 (32) 3 35.9 [34.9–36.5] 1.06%C. scoticus 240 (27) 0C. dewulfi 181 (20) 2 35.2 [32.2–38.1] 1.10%C. chiopterus 227 (23) 1 28.7 0.44%C. pulicaris 89 (11) 1 37.9 1.12%

Denmark October 2011 5 entire females Obsoletus group 91 2 26.0 [25.0–27.6] 2.20% Rasmussen et al. (2012)

Netherlands August to September 2011 10 heads (NF or PF) Obsoletus complex 2300 (230) 12 24.6 [19.6–36.0] 0.52% Elbers et al. (2013b)C. obsoletus 1 24.6C. scoticus 10 25.0 [19.6–36.0]C. dewulfi 1300 (130) 0C. chiopterus 1440 (144) 2 31.6 [27.9–35.4] 0.14%C. punctatus 1050 (105) 0

May to September 2012 50 entire females(PF or GF)

Obsoletus complex 2100 (42) 2 36.3 [35.0–37.7] 0.10% Elbers et al. (2013a)

C. dewulfi 1300 (26) 0C. chiopterus 1050 (21) 0C. punctatus 1550 (31) 0C. pulicaris 500 (10) 0

Italy June 2011 to June 2012 <50 entire females(PF or GF)

Obsoletus complex 5146 6 28.7 [26.0–33.0] 0.12% Goffredo et al. (2013)

C. pulicaris 29 (17) 0C. punctatus 28 (14) 0C. dewulfi 1 (1) 0Nubeculosus complex 296 (34) 0C. flavipulicaris 1 (1) 0

September to November2011

Obsoletus complex 1104 5 29.0 [26.0–33.0] 0.45%

May 2012 Obsoletus complex 769 1 27.0 0.13%C. obsoletus 1 27.0

Poland September/October 2011and April to October 2012

∼20 entire females(NP, PF or GF)b

Obsoletus complex ∼3600 (181) 28 ∼29.8 [17.5–39.4] 0.78% Larska et al. (2013)

C. punctatus ∼2100 (108) 6 ∼31.4 [23.9–37.2] 0.29%

a PF: parous females; NF: nulliparous females; GF: gravid females.b The number of Culicoides per pools was not given precisely, it ranged from 9 to 60 (meanly 20). Blood-fed females were also tested in this study, but we did not report the results here.

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etection of SBV RNA, despite its well documented role inransmission of other Culicoides-borne arboviruses (Mellort al., 2009b) and association with BTV outbreaks in ItalyGoffredo et al., 2003; Goffredo et al., 2004). Indeed, allumerous pools of C. imicola collected in farms reportingbortions tested negative for SBV. On the contrary, rela-ively small quantities of SBV RNA were detected in pools of. imicola in Iglesias province where no case was reportedFig. 1). The fact that C. imicola dominated the Culicoidesauna in Sardinia and especially at outbreak sites wherenly very limited numbers of the Obsoletus complex wereresent, however, indicates its probable involvement inransmission of SBV. An absence of pools of C. imicola con-aining significant quantities of SBV RNA may have beenue to the time of sampling. Samples were made in farmsxperiencing SBV reports few months after the probablenfection date. On the contrary, SBV RNA was detectedn C. imicola populations late in the season when ani-

al infection may be unapparent. Thus further screeningf field-collected Culicoides within the distribution of thispecies would be desirable.

Results from IT inoculation of mosquitoes stronglyuggested that SBV can replicate in individuals when intro-uced directly into the haemocoel, bypassing mid-gutarriers to arbovirus dissemination (Mellor, 2000). Oral

nfection, however, did not result in Ct values indicativef full SBV dissemination in either mosquito species. Whilehese results should be interpreted with caution as theytilised inbred colony lines, this study provides preliminaryvidence that the Culicidae may not play a substantial rolen transmission of SBV in the field. Previously it was demon-trated that Culicoides were efficient experimental vectorsor the closely related Akabane virus (Jennings and Mellor,989), whereas attempts to demonstrate replication of thisirus in mosquitoes have so far proved unsuccessful (Kayt al., 1975). Systematic studies to characterise biting ratesf mosquito species on livestock in Europe, however, woulde useful in understanding the role of this group to poten-ially transmit pathogens such as SBV.

The spread of SBV has been noted to have been quickernd its transmission more intense in comparison to BTVnd this has been hypothesised to be due to Culicoides pop-lations being more susceptible to infection (Elbers et al.,013b). A review of studies conducted to date found equiv-cal support for this hypothesis, as expected from the facthat the proportion of Culicoides exposed to viraemic hostsithin screened populations is unknown and in general

he numbers of individuals and sites investigated in initialtudies were low (De Regge et al., 2012; Rasmussen et al.,012; Elbers et al., 2013b). The most straightforward wayo assess the true competence of populations is to carryut infection studies of field collected Culicoides in the lab-ratory using either viraemic hosts or artificial means ofeeding, as conducted for BTV (Jennings and Mellor, 1988;arpenter et al., 2006; Carpenter et al., 2008). As the tim-

ng of animal based experiments in biosecure containmentith population peaks in Culicoides is logistically chal-

Please cite this article in press as: Balenghien, T., et al., The emenities and ecosystems in Europe. PREVET (2014), http://dx.doi.o

enging, pledglet-based blood feeding methods are mostommonly employed to assess infection rates. Membrane-ased methods, such as those employed to feed colonyulicoides and mosquitoes in this study currently result

PRESSry Medicine xxx (2014) xxx–xxx 9

in extremely poor rates of feeding in field collected Culi-coides from northern Europe (Jennings and Mellor, 1988;Venter et al., 2005). As it is known that pledglet feedingsignificantly underestimates the proportion of competentCulicoides in a population (Venter et al., 2005) it is there-fore vital that standard membrane-based techniques aredeveloped for northern European species.

A key observation during the SBV outbreak is therapid spread of the virus into new ecosystems in Europewhich are primarily dominated by different multitudes ofCulicoides species. This phenomenon has been observedpreviously during large-scale outbreaks of BTV in Europe,initially in the case of BTV-9, which was able to movefrom areas dominated by C. imicola into the Balkans, wherethe Culicoides fauna was dominated by Palaearctic species(Mellor and Wittmann, 2002). In later outbreaks, BTV-8was introduced by an as yet undefined route into north-ern Europe and then caused disease outbreaks in southernEurope on farms dominated by C. imicola. Similarly, BTV-1 moved from C. imicola dominated farms in Spain toareas dominated by the Obsoletus complex and C. dewulfiin France (Carpenter et al., 2009; Mellor et al., 2009a;Mellor et al., 2009b). The ease by which these arboviruseshave moved between disparate ecosystems implies thatany barriers presented by the process of adaptation ofCulicoides-borne arboviruses to novel vector groups are atbest only partially restrictive between these two primaryvector groups. In a period of significant globalisation oftrade and environmental change this has significant con-sequences for the worldwide spread of such pathogens.Conducting studies to understand factors limiting the cur-rent distribution of arboviruses is therefore likely to beuseful in determining the risk that emerging pathogenspose to other regions.

Conflict of interest

The authors declare that there are no conflicts ofinterest.

Acknowledgements

The study was mainly funded by the DirectorateGeneral for Health and Consumers (DG-SANCO), Euro-pean Commission (2012/349/UE). This study was partiallyfunded by EU grant FP7-261504 EDENext and is cata-logued by the EDENext Steering Committee as EDENext175(http://www.edenext.eu). The contents of this publicationare the sole responsibility of the authors and do not neces-sarily reflect the views of the European Commission. TheFrench authors would like to thank the national veteri-nary services, the farmers and the technicians for collectingthe biting midges; Xavier Allène, Ignace Rakotoarivony,Jonathan Lhoir (Cirad), Bethsabée Scheid (EID-Med) andDelphine Delécolle (IPPTS) for identifying the Culicoidessamples.

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