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The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe Thomas Balenghien a,b,∗ , Nonito Pagès c , Maria Goffredo d , Simon Carpenter e , Denis Augot f , Elisabeth Jacquier a,b , Sandra Talavera c , Federica Monaco d , Jérôme Depaquit f , Colette Grillet a,b , Joan Pujols c , Giuseppe Satta g , Mohamed Kasbari f , Marie-Laure Setier-Rio h , Francesca Izzo g , Cigdem Alkan i,j , Jean-Claude Delécolle k , Michela Quaglia g , Rémi Charrel i,j , Andrea Polci g , Emmanuel Bréard l , Valentina Federici g , Catherine Cêtre-Sossah a,b , Claire Garros a,b a

Cirad, UMR15 CMAEE, F-34398 Montpellier, France INRA, UMR1309 CMAEE, F-34398 Montpellier, France c Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain d Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100 Teramo, Italy e The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF, United Kingdom f Université de Reims Champagne-Ardenne, ANSES, SFR Cap Santé, EA4688 - USC « VECPAR », Reims, France g Istituto Zooprofilattico della Sardegna, Via Duca degli Abruzzi 8, Sardinia, Italy h EID Méditerranée, 34184 Montpellier, France i UMR D 190 “Emergence des Pathologies Virales”, Aix Marseille Univ, IRD French Institute of Research for Development, EHESP French School of Public Health, 13005 Marseille, France j IHU Mediterranee Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France k IPPTS, 67000 Strasbourg, France l ANSES, UMR Virologie, Maisons-Alfort, France b

a r t i c l e

i n f o

Article history: Received 23 September 2013 Received in revised form 24 January 2014 Accepted 8 March 2014

Keywords: Arbovirus Vector competence Ceratopogonidae Orthobunyavirus Bunyaviridae

a b s t r a c t Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of 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; fax: +33 4 67 59 37 95. E-mail address: [email protected] (T. Balenghien). http://dx.doi.org/10.1016/j.prevetmed.2014.03.007 0167-5877/© 2014 Elsevier B.V. All rights reserved.

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examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Schmallenberg virus (SBV) is a member of the Orthobunyavirus 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 adult ruminants, including pyrexia, decreased milk production and diarrhea (Davies et al., 2012). The primary economic impact of SBV, however, lies in severe congenital malformations following transplacental transmission in cattle and sheep (van den Brom et al., 2012). Initial phylogenetic studies placed SBV in the Simbu serogroup, sharing a close relationship to Sathuperi and Douglas viruses and secondarily to Shamonda virus and included in the same lineage than the Akabane virus (Saeed et al., 2001; Goller et al., 2012). These viruses have been primarily isolated from Culicoides (Doherty et al., 1972; St George et al., 1978; Lee, 1979; Cybinski, 1984; Blackburn and Searle, 1985; Kurogi et al., 1987; Yanase et al., 2005) and more rarely from mosquitoes (Dandawate et al., 1969; Metselaar and Robin, 1976). The recent emergence of the similarly Culicoidesborne bluetongue virus (BTV) in western and northern Europe (Carpenter et al., 2009), therefore led to immediate suspicion that SBV was transmitted by female midges from the Culicoides genus. Following detection of the SBV incursion, virus RNA was rapidly identified in field-collected Culicoides from farms in the affected regions (De Regge et al., 2012; Rasmussen et al., 2012; Elbers et al., 2013a,b; Goffredo et al., 2013; Larska et al., 2013). Taken in their entirety, these studies convincingly implicated a range of widespread and abundant farm-associated Culicoides species in the transmission of SBV including Culicoides obsoletus, Culicoides scoticus, Culicoides dewulfi and Culicoides chiopterus. A detailed study of SBV replication and dissemination in the model species Culicoides sonorensis also allowed confirmation that levels of viral RNA in studies carried out in the Netherlands (Elbers et al., 2013b) were likely to represent transmissible infections (Veronesi et al., 2013). From 2011 to 2013, SBV has spread across a huge geographic area in Europe at a rate substantially exceeding that of the BTV-8 epidemic which occurred in the same region from 2006 to 2010. Current distribution of SBV ranges from the Mediterranean to Scandinavian regions, and from Ireland to Estonia (European Food Safety Authority, 2013). The rate of seroconversion recorded in many farms was rapid (Elbers et al., 2012; Meroc et al., 2013) with a high proportion of ruminants apparently becoming infected in a short time frame. A partial explanation for this

phenomenon could be the absence of animal movement restrictions that were employed for BTV but not for SBV in the vast majority of countries reporting incursions. Additionally, however, it is suspected that the vector competence of Culicoides for SBV may exceed rates recorded for BTV either in the number of species capable of transmitting the virus or in the proportion of individuals within a species able to act as vectors. This hypothesis receives support from the fact that the related Akabane virus is isolated at a far higher frequency than BTV from Culicoides in Australia (St George et al., 1978), although comparative laboratory-based investigations of susceptibility rates in vector species have not been performed. In this study we present the first data on detection of SBV RNA in Culicoides from mainland France and Italy (Sardinia), examine infection rates in colony-derived individuals of Culicoides nubeculosus (Meigen) and additionally preliminary results for experimental infections of fieldcollected individuals. A significant advantage in carrying out such detections across a wide geographic range is that it enables an understanding of SBV transmission across disparate ecosystems and potential vector species. This includes the examination of the role of Culicoides imicola, a major afrotropical vector of BTV with a distribution that could facilitate the spread of SBV into new areas, including Asia. We also examine two abundant and widespread mosquito vector species in the laboratory to assess whether alternative vectors could be involved in transmission of SBV across Europe. Finally, we also review vector competence data produced from studies across Europe to date comprehensively and compare with that generated previously for BTV. By contrasting the diverse ecosystems and potential vectors present across these countries we subsequently draw conclusions regarding the vulnerability of Europe to further incursions. 2. Material and methods 2.1. Laboratory infection of mosquitoes and Culicoides Experimental infection with SBV was carried out at the Centre de Recerca en Sanitat Animal (CReSA) using lines of Culex pipiens Linnaeus and Aedes albopictus Skuse that had been maintained for two and four years, respectively. In addition, a line of C. nubeculosus was also infected at CReSA which had originated from that originally established at The Pirbright Institute. All studies using both intrathoracic inoculation and artificial membrane-based blood-feeding were conducted at the CReSA Biosafety level 3 facilities.

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

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For intrathoracic (IT) inoculation, females of each species (of ages 2–4 days) were anesthetised using carbon dioxide and placed under a stereomicroscope. A volume of 0.2–0.4 ␮l (Culicoides) and 1–2 ␮l (mosquitoes) of a 4.38 TCID50/ml SBV viral suspension, produced on a baby hamster kidney (BHK-21) cell line was inoculated intrathoracically into each individual using a glass micro-needle. Insertion of the needle was made between the epimeron and episterum using a manual micro-injector (Sutter instruments, California, USA). Inoculated mosquitoes and midges were placed in groups of 15–20 individuals inside cardboard and plastic primary containers for Culicoides and mosquitoes respectively. Females were fed with 5% sucrose ad libitum and maintained at 24 ± 2 ◦ C and 80% RH, with a 14:10 (light:dark) photoperiod for eight to nine days prior to processing. Females of the same age range were also exposed to oral infection using an artificial membrane system fitted with a one day old chick skin membrane (Hemotek, UK). This study used a 1:1 mixture of bovine blood and 4.38 TCID50/ml SBV suspension, to obtain a final concentration of 4.08 TCID50/ml, that during the trial was heated to 38 ◦ C during a 45–60 min exposure. Fully engorged females of all three species were subsequently selected and placed inside primary containers for an extrinsic incubation period of 10 days using same maintenance conditions as for IT inoculations. Experimental infection trials with SBV were also carried out with field collected Culicoides to provide preliminary estimates of their vector competence for SBV at the University Champagne-Ardenne Biosafety level 2 facilities. Culicoides were collected in a farm located in northeastern France (Louvois: 49◦ 06 06 N, 4◦ 07 00 E) using light/suction traps (UV CDC trap, John W. Hock Compagny, Gainesvile, FL, USA) or after emergence from manure or the margins of ponds. A total of 60 females were allowed to feed on 10 ml of sucrose solution containing a final concentration of 103 TCID50 /ml SBV (produced on Vero3 cell line) using a cotton pledglet for four hours. Females were

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fed with 10% sucrose ad libitum and maintained at 25 ± 2 ◦ C and 80% RH with a 15:9 (light:dark) photoperiod for four to eight days prior to processing. Identification of orally exposed Culicoides was confirmed by cytochrome oxydase I sequencing, as barcode sequence reference (Hebert and Gregory, 2005). 2.2. Screening of field-collected Culicoides for SBV presence Collection sites for detection of SBV transmission in Culicoides were selected in temperate Europe (north-eastern France) and in the Mediterranean island of Sardinia (Italy) (Fig. 1). In France, wide-scale monitoring of the activity of Culicoides populations was already in place prior to the SBV outbreak and was subsequently carried out from 2009 to 2012 across both mainland areas and Corsica (Balenghien et al., 2011; Venail et al., 2012). This consisted of approximately 160 light-suction UV traps (either one or two based in each department), run at weekly intervals during midFebruary to April and during November and December and at monthly intervals for the rest of the year (Balenghien et al., 2011; Venail et al., 2012). SBV detection was targeted at traps located in departments where SBV surveillance had 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 from 53 traps in 28 distinct departments were selected (including 6 traps selected prior to the final results of the SBV surveillance which subsequently were found to have an incidence comprised between 0 and 0.5%). Screening for SBV RNA was conducted on Culicoides collected from the 3rd to the 6th October 2011 as late summer/autumn represented the likely time of transmission of the virus described in other studies. In Italy, clinical outbreaks of SBV infection were reported in Sardinia from October to December 2012, characterised by abortions that occurred in small ruminants

Fig. 1. Localisation of traps and Schmallenberg virus reports in France and Sardinia (Italy).

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0 12 12 (100.0) 30.0 [26.9–33.8]

held at farms in Sassari, Ogliastra and Cagliari provinces (Fig. 1). Following the confirmation of SBV, an entomological survey was performed in the island. Between the 30th October and 4th December 2012, a total of 26 Culicoides collections were performed on 15 farms in seven municipalities of Sardinia, located in the provinces of Carbonia-Iglesias (San Giovanni Suergiu and Sant’Anna Arresi), Cagliari (Muravera), Ogliastra (Barisardo, Girasole and Tertenia) and Sassari (Mores). In both France and Italy, Culicoides were stored in 70% ethanol and subsequently categorised according to their physiological status (nulliparous, parous and blood engorged). All Culicoides were identified morphologically using a stereomicroscope (Delécolle, 1985) and females of C. obsoletus and C. scoticus were grouped as the Obsoletus complex. In each site in France, up to 50 unengorged females of the Obsoletus complex were pooled (maximum 1 pool) and additional pools of up to 50 unengorged females of other species (maximum 3 pools) were assayed for SBV presence. In each site in Italy, parous and engorged females were assayed separately. 2.3. Detection of SBV genome In experimental infections conducted in Spain and in field-collected Culicoides screenings conducted in France, viral RNA was extracted using NucleoSpin RNA Virus (Macherey Nagel, Germany) following the manufacturer’s instructions. In Italy, Culicoides nucleic acids were extracted and purified using a BioSprint 96 One-For-All Vet Kit procedure based on the BioSprint 96 instrument (Qiagen, California, USA). In experimental infections conducted in France, RNA was extracted using EZ1 virus mini kit v2.0 (Qiagen, California, USA) following the manufacturer’s instructions. RNA was extracted individually from entire insects for both inoculated and orally exposed females in Spain or France, and for females from the Obsoletus

Oral exposure

Days post-exposure No. exposed No. positive (%) Mean Ct value [min–max]

0 8 8 (100.0) 29.0 [28.0–30.8]

10 149 2 (1.3) 30.4 [29.5–31.3]

0 2 2 (100.0) 28.8 [28.7–28.9]

10 27 0

130 129 (99.2) 24.2 [15.2–38.6] 9 9 (100.0) 34.1 [32.2–36.5]

8–9 0

89 62 (69.7) 23.4 [18.2–37.8] 5 5 (100.0) 28.7 [28.0–29.2] No. inoculated/No. tested No. positive (%) Mean Ct value [min–max]

51 51 (100.0) 20.0 [16.3–40.6]

4 4 (100.0) 28.8 [28.3–29.2]

8–9

Culicoides nubeculosus Culex pipiens

0 0 Intrathoracic inoculation

8–9 Aedes albopictus

Days post-inoculation Species

Table 1 Experimental infections of mosquito and Culicoides laboratory colonies with Schmallenberg virus.

10 102 4 (3.9) 27.5 [21.7–38.7]

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

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complex collected in France and in Italy, and from pools of maximum 50 entire midges for all other species collected in the field. The S gene of the Schmallenberg virus was transcribed and amplified according to the one step realtime RT-PCR protocol published by Hoffmann et al. (2012). Following extraction, SBV RNA was quantified using the one step real-time reverse transcription PCR (RT-PCR) following the protocol developed by Friedrich Loeffler Institut (FLI) targeting the S3 genomic fragment (Bilk et al., 2012), except for Cirad where RT-PCR was performed according to Taq Vet Schmallenberg virus S gene 50 kit (Laboratoire Service International, LSI, France). Assessments of infection status were made using Ct values generated from the samples as a semi-quantitative measure of viral RNA quantity. In France, pools screened for SBV presence and with a 37 < Ct < 45 values were tested twice and stated positive when both tests gave the same 37 < Ct < 45 values with appropriate shape curves. Positive females of the Obsoletus complex were identified to species level using molecular species identification assay (Nolan et al., 2007). 3. Results 3.1. Experimental infections using laboratory colonies 3.1.1. Intrathoracic inoculations Presence of SBV RNA was detected by real-time RTPCR in a subsample of the inoculated mosquitoes and midges on the day of inoculation (Table 1): the mean Ct values were equivalent between the mosquito species assessed (28.7 ± 0.5 [confidence interval (CI) 28.3–29.2] for Ae. albopictus and 28.8 ± 0.4 [CI 28.4–29.2] for Cx. pipiens) but higher in C. nubeculosus (34.1 ± 1.5 [CI 33.1–35.1]), due to a smaller inoculation volume used. The presence of SBV RNA was detected after 8–9 days post-inoculation (dpi) in all Ae. albopictus, in 69.7% of Cx. pipiens females and in 99.2% of C. nubeculosus females (Table 1). The mean Ct values decreased in positive females between the inoculation day and 8–9 dpi by 8.0 Ct in Ae. albopictus (20.7 ± 5.1 [CI 19.7–21.7]), by 5.4 in Cx. pipiens (23.4 ± 4.2 [CI 22.6–24.3]) and by 10.0 in C. nubeculosus (24.2 ± 4.9 [CI 23.3–25.0]) (Fig. 2). 3.1.2. Oral exposure Presence of SBV RNA was detected by real time RT-PCR in a subsample of the orally exposed mosquitoes and Culicoides on the day of exposure (Table 1): the mean Ct values were similar between mosquitoes and Culicoides (29.0 ± 1.1

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[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. nubeculosus) despite a smaller blood-meal in Culicoides. At 10 days post-exposure, SBV RNA was detected in 1.3% of Ae. albopictus (2/149), in none of the 27 blood-fed Cx. pipiens females and in 3.9% of the blood-fed C. nubeculosus (4/102). The infection rate was significantly greater for C. nubeculosus (95% binomial confidence interval of 1.08–9.73%) than for Ae. albopictus (0.16–4.76%): p = 0.049 under bionomial assumptions. It was not possible to distinguish the infection rates between Cx. pipiens (0–12.77%) and the two other species due to too few mosquitoes being tested (N = 27). The Ct values were similar ten days post-infection than the day of exposure for the two positive Ae. albopictus (29.53 and 31.25 versus 29.0), whereas they were much lower for 3 of the 4 positive C. nubeculosus (21.67, 24.21 and 25.3 versus 30.0) strongly suggesting SBV replication. Culicoides scoticus was the dominant species of fieldcollected females exposed orally to SBV (45 of the 60 individuals). For this species, mean Ct values were 32.2 at day 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 quantity of ingested blood and in individual susceptibility within C. scoticus population, and was amplified by the limited number of individuals. It was not possible to interpret results for other species due to too few individuals. Single viral RNA identification in Forcipomyia or C. newsteadi individuals, especially with a high Ct value, was not sufficient to incriminate these species as potential vectors without further investigations. 3.2. Screening of field-collected individuals for SBV presence In France, a total of 224,870 Culicoides, belonging to at 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 were about 32.0% and 40%. In Italy, a total of 53,531 Culicoides were collected (Table 3). Culicoides imicola was the dominant species in these collections, (74.5% of the total catch), followed by Culicoides newsteadi Austen (21.7%), species of the Obsoletus complex (0.8%), Culicoides pulicaris (Linneaus) (0.4%) and Culicoides punctatus (Meigen) (0.01%) (Table 3). The parity rate of C. imicola was approximately 55.0%.

Table 2 Experimental infections (orally) of field-collected midges with Schmallenberg virus. Species

No. exposed

Days post-exposure

No. positive

Mean Ct value [min–max]

C. scoticus

45

0 4 5 8

4 1 3 2

32.2 [31.4–34.2] 18.5 33.8 [33.1–34.2] 29.0 [28.9–29.1]

6 1 3 5

4 0 8 –

1 1 1 0

31.5 33.9 35.2

Forcipomyia sp. C. obsoletus C. newsteadi Other species

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Species

Province

Ogliastra

Sassari

Cagliari

Carbonia-Iglesias

Total

Barisardo (7)

Girasole (1)

Tertenia (5)

Mores (4)

Muravera (1)

San Giovanni Suergiu (3)

Sant’Anna Arresi (5)

Total No. midges No. PFa (No. pools) No. EFa (No. pools)

19,576 10,909 (221) 75 (3)

11 7 (1)

769 603 (14) 4 (1)

3778 395 (10) 13 (3)

99 50 (1) 1 (1)

15,589 10,132 (204) 106 (4)

44 30 (5)

39,866 (74.47%) 22,126 (456) 199 (12)

C. newsteadi

Total No. midges No. PF (No. pools) No. EF (No. pools)

695 303 (10) 12 (2)

3

108 45 (4) 2 (2)

9248 4299 (87) 104 (5)

151 107 (3) 2 (1)

1326 684 (15) 8 (1)

103 65 (5) 1 (1)

11,634 (21.73%) 5503 (124) 129 (12)

Obsoletus complex

Total No. midges No. PF No. EF

48 7

0

81 41 4

137 59 4

7 6

130 14 2

9 4

421 (0.77%) 131 10

C. pulicaris

Total No. midges No. PF (No. pools) No. EF (No. pools)

81 25 (3)

3 2 (1)

74 28 (4)

33 16 (4) 2 (2)

1 1 (1)

0

0

192 (0.36%) 72 (13) 2 (2)

C. punctatus

Total No. midges No. PF (No. pools)

1 1 (1)

0

0

0

0

0

5 4 (2)

6 (0.01%) 5 (3)

Other species

Total No. midges

740

5

74

149

5

228

220

1421 (2.65%)

Total

Total No. midges No. PF (No. pools) No. EF (No. pools)

21,141 11,245 (242) 87 (5)

22 9 (2)

1106 717 (63) 10 (5)

13,345 4769 (160) 123 (13)

263 164 (11) 3 (2)

17,273 10,830 (233) 116 (6)

381 103 (16) 1 (1)

53,531 27,837 (727) 340 (32)

a

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

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Municipality (No. collections) C. imicola

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

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

No. Culicoides (pools) tested

No. positive pools

Mean Ct value [min–max]

Overall minimum infection rate (MIR)

Mean MIR in positive sites [min–max]

Obsoletus complexa C. obsoletus C. dewulfi C. chiopterus C. pulicaris C. newsteadi C. nubeculosus C. lupicaris C. punctatus C. festivipennis C. clastrieri C. circumscriptus C. fascipennis C. alazanicus C. cataneii/gejgelensis C. parroti C. puncticollis C. subfasciipennis

1734 (347)

10 8 0 2 1 0 1 0 0 0 0 0 0 0 0 0 0 0

32.9 [23.4–38.2]a 34.4 [28.3–38.2]a

0.58%a

4.14% [2.00–6.00]

32.0 [30.6–33.4] 38.3

0.16% 0.37%

1.17% [1.12–1.22] 1.25%

28.8

2.33%

3.70%

1729 (47) 1224 (40) 271 (27) 65 (12) 43 (7) 24 (9) 17 (7) 9 (3) 8 (1) 4 (2) 2 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1)

a Eight of the 10 positive individuals from the Obsoletus complex were identified as C. obsoletus; it was not possible to identify the two others at the species level. The Ct values associated with the Obsoletus complex individuals correspond to assay on single individuals (compared to pools for the other species) leading to minimum infection rates being the effective infection rate sampled populations of the Obsoletus complex.

For collections made in France 5135 females were tested for SBV RNA presence. Ten pools of females from the Obsoletus complex contained detectable quantities of SBV RNA and all females processed individually that tested positive were identified as C. obsoletus. Pools of the Obsoletus complex had an overall minimum infection rate (MIR) of 0.58%, with C. chiopterus giving an MIR of 0.16%, C. pulicaris possessing an overall MIR of 0.37% and of C. nubeculosus giving an overall MIR of 2.33% (Table 4). Excluding sites where SBV was not recovered increased the MIR to approximately 4% for the Obsoletus complex and C. nubeculosus and around 1% for C. chiopterus and C. pulicaris (Table 4). For Italian collections, 27,837 parous females (727 pools) and 340 engorged females (32 pools) were tested for SBV presence. Detectable SBV RNA was identified in three pools of C. imicola of which two were pools of 50 pigmented females and one was a pool of 33 engorged females. All these pools were collected at San Giovanni Suergiu (Carbonia-Iglesias province) leading to a MIR = 0.04% within the pigmented population of C. imicola in this site (Table 5).

2012) and Italy (Goffredo et al., 2013) confirmed the role of C. obsoletus as a highly probable vector of SBV in northern Europe (Table 6). This species is among the most abundant livestock-associated species in the region (Meiswinkel et al., 2008; Carpenter et al., 2009; Venail et al., 2012) and its apparently ubiquitous distribution on farms across the Palaearctic and Nearctic may facilitate spread of SBV to new regions. In addition, C. nubeculosus was implicated for the first time as a potential vector in France, although quantities of SBV RNA detected were equivocal in defining the level of dissemination that had occurred (Veronesi et al., 2013). Studies of vector competence for SBV in colony lines of this species, both in the current study and in previous studies in the UK (Veronesi et al., 2013), have indicated extremely low rates of competence of approximately 3%. It is important to note, however, that such infection rates have been demonstrated to vary with vector population for other Culicoides-borne arboviruses (Tabachnick, 1996). Preliminary evidence was also provided that C. scoticus is able to replicate SBV to transmissible levels, albeit using a technique (pledglet feeding with sugar) that is likely to result in virus being transported to the crop rather than the gut (Jennings and Mellor, 1988). Studies of C. imicola in Sardinia (Italy) failed to convincingly implicate this species in transmission through

4. Discussion This study, together with previous work in the Netherlands (Elbers et al., 2013b), Belgium (De Regge et al.,

Table 5 Detection of Schmallenberg virus from Culicoides collected at San Giovanni Suergiu, Carbonia-Iglesias province, in Sardinia (Italy), the 7th November 2012. Species

No. midges collected

Physiological status

No. midges (pools) tested

No. positive pools

Mean Ct value [min–max]

Minimum infection rate

C. imicola

7704

Parous Engorged

5050 (101) 66 (2)

2 1

36 [34–38] 33

0.04%

846

Parous Engorged

500 (10) 8 (1)

0 0

6 (6)

0

C. newsteadi

Obsoletus complex

27

Parous

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

Pool constitutiona

Species

No. midges (pools) tested

Belgium

August to October 2011

25 heads (PF)

Obsoletus complex C. obsoletus C. scoticus C. dewulfi C. chiopterus C. pulicaris

688 (34) 283 (32) 240 (27) 181 (20) 227 (23) 89 (11)

5 3 0 2 1 1

No. positive pools

Reference

33.9 [30.7–36.0] 35.9 [34.9–36.5]

0.73% 1.06%

De Regge et al. (2012)

35.2 [32.2–38.1] 28.7 37.9

1.10% 0.44% 1.12%

2

26.0 [25.0–27.6]

2.20%

Rasmussen et al. (2012)

12 1 10 0 2 0 2

24.6 [19.6–36.0] 24.6 25.0 [19.6–36.0]

0.52%

Elbers et al. (2013b)

31.6 [27.9–35.4]

0.14%

36.3 [35.0–37.7]

0.10%

Elbers et al. (2013a)

Goffredo et al. (2013)

Denmark

October 2011

5 entire females

Obsoletus group

91

Netherlands

August to September 2011

10 heads (NF or PF)

Obsoletus complex C. obsoletus C. scoticus C. dewulfi C. chiopterus C. punctatus Obsoletus complex

2300 (230)

C. dewulfi C. chiopterus C. punctatus C. pulicaris

1300 (26) 1050 (21) 1550 (31) 500 (10)

0 0 0 0

Obsoletus complex

5146

6

28.7 [26.0–33.0]

0.12%

C. pulicaris C. punctatus C. dewulfi Nubeculosus complex C. flavipulicaris Obsoletus complex

29 (17) 28 (14) 1 (1) 296 (34) 1 (1) 1104

0 0 0 0 0 5

29.0 [26.0–33.0]

0.45%

Obsoletus complex C. obsoletus

769

1 1

27.0 27.0

0.13%

Obsoletus complex

∼3600 (181)

28

∼29.8 [17.5–39.4]

0.78%

C. punctatus

∼2100 (108)

6

∼31.4 [23.9–37.2]

0.29%

May to September 2012

Italy

June 2011 to June 2012

50 entire females (PF or GF)

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe.

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspr...
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