Belsham, Graham

Bødker, Rene

Bøtner, Anette

Kirkeby, Carsten

Kristensen, Birgit

Rasmussen, Lasse Dam

Rasmussen, Thomas Bruun

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 20, 2017Occurrence of Schmallenberg virus in Danish biting midges (Culicoides spp.)Rasmussen, Lasse Dam; Kirkeby, Carsten Thure; Kristensen, Birgit; Rasmussen, Thomas Bruun;Belsham, Graham; Bødker, Rene; Bøtner, AnettePublication date:2013Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Rasmussen, L. D., Kirkeby, C., Kristensen, B., Rasmussen, T. B., Belsham, G., Bødker, R., & Bøtner, A. (2013).Occurrence of Schmallenberg virus in Danish biting midges (Culicoides spp.). Poster session presented atEDENext Annual Meeting, Barcelona, Spain.Occurrence of Schmallenberg virus in Danish biting midges (Culicoides spp.) Lasse Dam Rasmussen1, Carsten Kirkeby2, Birgit Kristensen2, Thomas Bruun Rasmussen1, Graham J. Belsham1, René Bødker2, Anette Bøtner1 1)DTU National Veterinary Institute, Lindholm, DK-4771 Kalvehave, Denmark   2)DTU National Veterinary Institute, DK-1870 Copenhagen, Denmark In autumn 2011, an unidentified disease of livestock was reported on both sides of the Dutch-German border. Using Metagenomics the etiological agent of this disease was identified as a novel orthobunyavirus and named Schmallenberg virus (SBV) (1). Other members of this virus genus (e.g. Akabane virus) are widespread in Africa and Asia; both biting midges (Culicoides spp.) and mosquitoes are responsible for transmission of these viruses. Hence it was a reasonable assumption that European Culicoides species may be responsible for the transmission of SBV within Europe.  The first find of SBV in Danish Culicoides were caught in October 2011 close (6 km) to the German border, and less than 10 km to an SBV- infected sheep farm in Germany (2) (     on the map) . After this intial find a thorough investigation of midge pools were initiated. Pools of midges caught in 2011 at sites scattered across the entire country (represented by     on the map) were tested for the presents of SBV and Bluetongue virus (BTV). In 2012 investigations were focused on just 4 different areas (I, II, III, IV) where midges were collected on a weekly basis from the end of July until the middle of September. After a preliminary “rough and dirty” investigation, the midges were divided into Obsoletus group, Pulicaris group and others. Furthermore, were the heads and abdomen of the midges in the Obsoletus group tested separately. 1. Hoffmann B, et al.  Novel orthobunyavirus in cattle, europe, 2011. Emerg Infect Dis. 2012;18:469-72. 2. Rasmussen LD, et al. Culicoids as Vectors of Schmallenberg Virus. Emerg Infect Dis. 2012 18:1204–1206 3. Nolan DV, et al. Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris     species complexes,implicated vectors of bluetongue virus in Europe. Vet Microbiol. 2007 124:82-94  “Heads” or “Tails” After the initial finding of two SBV positive pools close to the German border, all other midge pools (approx.250) tested negative for both SBV and BTV Results 2011 Results 2012 Samplingarea 30/7 6/8 13/8 20/8 27/8 3/9 10/9 17/9I na negative negative positive negative negative negative positiveII na na negative negative positive na na naIII na negative negative negative positive positive negative positiveIV negative na positive na positive negative positive negativeCollection date“Rough and dirty” One pool pr. Sampling area pr. date containing 50 unsorted Culicoides spp. “Up close and personal” Date Pools positive % positive % mean Ct positive % mean Ct30/76/813/8 3 0 0 0 0 0 020/8 11 0 0 0 0 0 027/83/910/917/9Over all6 2 33 29,2unidentifiedPools positive frequency % Mean CtPools positive frequency % Mean Ct7 0 0IIObsoletus groupHead AbdomenPulicaris groupDate Pools positive % positive % mean Ct positive % mean Ct30/7 12 0 0 0 0 0 06/813/8 12 1 8 0 0 1 8 29,720/827/8 12 3 25 0 0 3 25 27,83/9 12 3 25 1 8 24,3 3 25 28,510/9 10 2 20 2 20 28,7 2 20 24,317/9 8 1 13 0 0 1 13 30,6Over allPools positive frequency % Mean Ct2 0 06 0 0unidentifiedObsoletus groupIVPulicaris groupPools positive frequency % Mean CtHead AbdomenDate Pools positive % positive % mean Ct positive % mean Ct30/7 12 0 0 0 0 0 06/8 24 4 17 2 8 34,6 3 13 34,613/8 27 1 4 0 0 1 4 29,720/8 35 1 3 0 0 1 3 24,327/8 31 9 29 3 10 30,3 9 29 27,73/9 34 8 24 2 6 26,9 8 24 30,010/9 22 6 27 4 18 28,5 6 27 25,817/9 28 7 25 0 0 7 25 29,7Over allPools positive frequency % Mean Ct20 3 15 29,527 2 7 31,5unidentifiedObsoletus groupHead AbdomenPulicaris groupPools positive frequency % Mean CtDenmark 2012Date Pools positive % positive % mean Ct positive % mean Ct30/76/8 12 4 33 2 17 34,6 3 25 34,613/820/8 12 0 0 0 0 0 027/8 12 6 50 3 25 30,3 6 50 27,63/9 12 5 42 1 8 29,6 5 42 31,510/9 12 4 33 2 17 28,4 4 33 27,417/9 12 4 33 0 0 4 33 27,5Over all6 1 17 31unidentifiedPools positive frequency % Mean CtPools positive frequency % Mean Ct7 2 29 31,5IIIObsoletus groupHead AbdomenPulicaris groupDate Pools positive % positive % mean Ct positive % mean Ct30/76/8 12 0 0 0 0 0 013/8 1220/8 12 1 8 0 0 1 8 24,327/8 7 0 0 0 0 0 03/9 10 0 0 0 0 0 010/917/9 8 2 25 0 0 2 25 30,9Over all0 0unidentifiedPools positive frequency % Mean Ct6 0 0Head AbdomenPulicaris groupPools positive frequency % Mean Ct7IObsoletus groupbloodDate Pools positive % C. obsoletus C. dewulfi both30/7 12 06/8 24 2 10013/8 27 020/8 35 027/8 31 3 33 2 13/9 34 2 0 1 110/9 22 4 0 2 217/9 28 0Overall 213 11 27 4 3 2HeadsSpecies specific PCRTwo unidentified poolsbloodDate Pools positive % C. obsoletus C. dewulfi both30/7 12 06/8 24 3 100 113/8 27 1 0 120/8 35 1 100 127/8 31 9 56 5 1 33/9 34 8 38 6 1 110/9 22 6 17 3 2 117/9 28 7 29 2 1 4Overall 213 35 43 17 6 10two unidentifiedAbdomenSpecies specific PCRbloodDate Pools positive % C. obsoletus C. dewulfi both30/7 12 06/8 24 4 100 113/8 27 1 0 120/8 35 1 100 127/8 31 9 56 5 1 33/9 34 8 38 6 1 110/9 22 6 17 3 2 117/9 28 7 29 2 1 4Overall 213 36 44 17 6 10overallSpecies specific PCRthree unidentifiedFor Culicoides belonging to the Obsoletus group, heads and abdomen were tested separately for SBV (10/pool). The positive pools were subject to species specific multiplex PCR, detecting C. obsoletus, C. dewulfi, C. chiopterus and C. scoticus (3). In 2012 collection of Culicoides was focused on four areas. Midges were collected on a weekly basis from July 30th to September 17th and divided into the Obsoletus group, the Pulicaris group and “others”. Furthermore, were the heads and abdomen of the midges in the Obsoletus group tested separately in pools of 10. The samples were homogenized using A Tissue Lyser II and RNA was purified by MagNa Pure robot. RT-qPCR was performed using both S3 and L1 primers and probes. Only pools positive in both were considered positive for SBV. The mean Ct values in the tables are from the S3 PCR.  In 2011, Culicoides containing Schmallenberg virus (SBV) was only found at the most southern location, of 44 catch sites located across the entire country, only 10 km from an infected German farm. In 2012 infected biting midges were found on all of the four catch sites which were all located in large distances from the infected site from 2011, and representing most of Denmark. In other words, the virus has managed to spread to midges in the entire country in just under a year and that without extensive reports of outbreaks. Only a single lamb was found infected with SBV. The Obsoletus group were the most frequent caught and is also responsible for 36 of the 41 positive pools. In total 17% of the Obsoletus group pools were positive for SBV. Only C. obsoletus and C. dewulfi were found in the positive pools. Both were found as the only species in the pool and in a mixture. This was the case for both head and abdomen and without the presents of ruminant blood which indicates that both species can act as vector for SBV. It is still a puzzle how SBV manages to infect so many animals in so little time but these results more than indicate that Culicoides play a crucial role. Discussion and Conclusions 

Occurrence of Schmallenberg virus in Danish biting midges (Culicoides spp.)

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Occurrence of Schmallenberg virus in Danish biting midges (Culicoides spp.)

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