Transboundary and Emerging Diseases
RAPID COMMUNICATION
Rapid Spread of Schmallenberg Virus-infected Biting Midges (Culicoides spp.) across Denmark in 2012 L. D. Rasmussen1, C. Kirkeby2, R. Bødker2, B. Kristensen2, T. B. Rasmussen1, G. J. Belsham1 and A. Bøtner1 1 2
National Veterinary Institute, Technical University of Denmark, Kalvehave, Denmark National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark
Keywords: Schmallenberg virus; Culicoides spp.; insect vector; biting midges Correspondence: A. Bøtner. National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark. Tel.: +45 3588 7858; Fax: +45 3588 7901; E-mail: [email protected]
Summary Detection of Schmallenberg virus RNA, using real-time RT-PCR, in biting midges (Culicoides spp.) caught at 48 locations in 2011 and four well-separated farms during 2012 in Denmark, revealed a remarkably rapid spread of virus-infected midges across the country. During 2012, some 213 pools of obsoletus group midges (10 specimens per pool) were examined, and of these, 35 of the 174 parous pools were Schmallenberg virus RNA positive and 11 of them were positive in the heads. Culicoides species-specific PCRs identified both C. obsoletus and C. dewulfi as vectors of Schmallenberg virus.
Received for publication August 26, 2013 doi:10.1111/tbed.12189
Introduction Schmallenberg virus (SBV) was discovered and identified as a novel orthobunya virus in the autumn of 2011 in Germany (Hoffmann et al., 2012). It caused relatively mild clinical symptoms within infected cattle, including diarrhoea and loss of milk production, but, more seriously, it induced abortions and malformations in the offspring of pregnant cattle and sheep. Subsequently, several European countries have reported the presence of SBV [e.g. The Netherlands, France, U.K. and Denmark (Beer et al., 2013)]. In Denmark, the virus was first detected within biting midges (Culicoides spp.) that had been captured in October 2011 close to the Danish border and a reported SBV outbreak within Germany (Rasmussen et al., 2012). This was before any clinical signs were observed or virus was detected within domestic animals in Denmark. The role of Culicoides midges, within the obsoletus group, as vectors for SBV has now been confirmed (De Regge et al., 2012; Elbers et al., 2013a,b; Larska et al., 2013a,b). These initial results prompted a thorough investigation of additional midges caught in 2011 and also of midges caught in 2012 from across Denmark to determine the © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
spatial spread of the SBV vector and to identify the important vector species groups. Materials and Methods In total, 1169 Culicoides, in 182 pools containing between 1 and 37 unsorted biting midges, were captured in the summer and autumn of 2011 from 47 other collection sites (20% of which were at cattle farms) scattered throughout the country (see Fig. 1). These midges were caught, as a byproduct of mosquito collection, using a Mosquito Magnet Independence trap (Mosquito Magnet, Lititz, PA, USA) baited with carbon dioxide and octenol. Biting midges were sorted manually, using a dissection microscope, into specimens of the obsoletus group, the pulicaris group and ‘others’ (unclassified midges not belonging to the two specific groups) and preserved at 20°C. In 2012, capture of midges occurred at four cattle farms (labelled I, II, III and IV) in different regions of Denmark (Fig. 1) using Onderstepoort Veterinary Institute traps with 8-w UV tubes. Midges were collected weekly from July 30 until September 17. Preliminary investigations of these midges used 23 pools of 50 unsorted (but non-engorged) 1
Spread of Schmallenberg Virus across Denmark in 2012
L. D. Rasmussen et al.
Fig. 1. Location of trap sites in 2011 (red dots) and 2012 (blue circles). The red star marks the site where Schmallenberg virus-positive midges were trapped in 2011, as described previously (Rasmussen et al., 2012).
individuals, collected as one pool per week per sampling per site. For more detailed analysis, a portion of the remaining midges was sorted into species groups, as described above; the obsoletus group midges were also segregated into groups based on their parous or nulliparous status as described by Dyce (1969). In total, 260 pools of female midges were analysed. The obsoletus group pools [as 213 heads and 213 abdomens separately (10 specimens/ pool)] included 174 parous and 39 nulliparous midges, while 27 pools (8–50 specimens/pool with a total of 1030 individuals) contained the pulicaris group and 20 pools (2– 50 specimens/pool with a total of 442 individuals) had unclassified midges (‘others’). Only midges in the obsoletus group, already identified as a vector group for SBV (Rasmussen et al., 2012), were dissected into heads and abdomens and stored in ethanol until required. The pools (whole insects or separated heads and abdomens) were homogenized in nuclease-free water (100 ll), using a 3-mm stainless steel bead (Dejay Distribution, 2
Launceston, UK) in a TissueLyser II (Qiagen, Hilden, Germany), for 1 min at 25 Hz (Veronesi et al., 2008). After homogenization, additional nuclease-free water (100 ll) was added to the samples which were then centrifuged at 3000 g for 5 min. Nucleic acids were extracted from the supernatant (100 ll) using a MagNA pure LC Total Nucleic Acid Isolation Kit on a MagNA pure LC system (Roche Diagnostics, Basel, Switzerland) and eluted in water (50 ll) (Veronesi et al., 2008). Two separate one-step RTqPCR assays, targeting the L-segment and the S-segment of SBV RNA, were performed on the extracted nucleic acids using a M93005p qPCR system (Agilent Technologies, Palo Alto, CA, USA) as described previously (Hoffmann et al., 2012; Rasmussen et al., 2012). Only pools positive in both reactions were scored as positive for SBV RNA. Another RT-qPCR targeting ruminant beta-actin mRNA was performed to test midges for the presence of bovine/ ovine beta-actin as a marker for the recent intake of a blood meal (Toussaint et al., 2007). © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
L. D. Rasmussen et al.
Spread of Schmallenberg Virus across Denmark in 2012
The PCR amplicon of 7 SBV-positive pools from the L-segment-specific RT-qPCR assay (145 bp) was sequenced using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer, resulting in 125-bp sequences, excluding primer regions. Results and Discussion Except for the midges caught close to the border with Germany (as described by Rasmussen et al., 2012), none of the pools of midges (containing in total 1169 specimens) caught in Denmark during 2011 were found positive for SBV RNA. In contrast, in 2012, the initial screen showed that 9 of 23 unsorted pools (50 midges each) collected from the four sampling sites contained SBV RNA, with at least one positive sample from each farm; these SBV-positive pools were collected between August 13 and September 17 (Table 1). For the more detailed analysis, the 260 midge pools were divided into species groups and the obsoletus group were further divided into parous and nulliparous groups. The 39 nulliparous obsoletus group pools were all negative for SBV RNA (data not shown). Of the remaining 221 pools, a total of 41 were positive for SBV RNA; from the 174 obsoletus group pools, 36 (21%) were positive, while 2 (7%) of 27 pulicaris pools and 3 (15%) of 20 of the ‘others’ contained SBV RNA (Table 2). It should be noted that the highest prevalence of SBV RNA was found within obsoletus group pools although the pools of the pulicaris group and ‘other’ Culicoides consisted, on average, of four and two times more individuals, respectively. The highest prevalence of SBV RNA was detected at site III (eastern Jutland); it was found in 23 of 60 pools in the obsoletus group, two of seven in the pulicaris group and one of six in the ‘others’ group. At site IV, on Zeeland, all SBV-positive pools (10 of 54) belonged to the obsoletus group (Table 2), while at site II, the most western site (Fig. 1), two of 6 pools of the ‘others’ group were positive Table 1. Results of the preliminary analysis of pools of 50 unsorted biting midges for the presence of Schmallenberg virus RNA at four separate sites within Denmark in 2012. The na indicates catch days when either no or a limited number of midges were captured, and these were reserved for the detailed investigation (see Table 2) Sampling date (2012)
Sampling area I II III IV
July 30 na na na
August 6
13
September 20
27
3
10
17
+ + +
na +
na
+ na +
+ na na
+
na
+
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
and at site I, the most northern site (Fig. 1), just three of the 49 pools of the obsoletus group were identified as being positive and only in the abdomens (Table 2). Overall, the frequency of SBV-positive abdomens was 2–5 times higher than positive heads. All 11 pools with SBV-positive heads, except one, also showed positive abdomens. The Ct values (S-Segment assay) were between 24 and 31 with no apparent difference between the groups of heads and abdomens. All specimens were examined under the microscope and judged visually not to have had a recent blood meal. SBV replication can be expected to start in the abdomen following the uptake of infected blood and then spread towards the head as for bluetongue virus (Carpenter et al., 2008). Finding virus in the head implies that the infection has spread to the salivary glands situated in the anterior part of the thorax. To identify the individual species within the obsoletus group acting as SBV vectors, all 36 pools containing SBV RNA were analysed using species-specific PCRs, as described previously (Nolan et al., 2007), distinguishing 4 separate species within the obsoletus group (these are C. obsoletus, C. chiopterus, C. dewulfi and C. scoticus). Only C. obsoletus and C. dewulfi were detected in the SBVpositive pools, both individually and in mixtures. Results were consistent for the pools of heads and abdomens originating from the same pool of midges. The presence of ruminant beta-actin mRNA, a marker for recent intake of a blood meal (Toussaint et al., 2007), was assayed in all samples, and it was undetected in most SBV RNA-positive pools of heads (8 of 11) and abdomens (20 of 35) derived from the obsoletus group; similarly, this marker was absent from most SBV RNA-positive pools that contained C. obsoletus and C. dewulfi both individually and in mixtures. This makes it very likely that both C. obsoletus and C. dewulfi are vectors for SBV in Denmark. These results complement the studies reported by De Regge et al. (2012) who showed that heads of C. obsoletus, C. dewulfi and C. chiopterus could be shown to contain SBV RNA. Most of the SBV-positive pools in Denmark contained obsoletus group midges, but positive pools of pulicaris group and ‘other’ Culicoides were detected at lower frequency. However, despite visual inspection for blood in the abdomens to allow the removal of newly fed specimens, ruminant beta-actin mRNA was detected in all positive pools in these latter groups indicative of a recent blood meal. For these species groups, heads and abdomens were not tested separately and it therefore remains unknown to what degree these species were functional vectors (i.e. in which replication occurred) or whether they just carried the virus from a blood meal. PCR amplicons (145 bp), derived from seven different pools of midges (as indicated in Table 2) collected at different sites and sampling times and using both heads and 3
Spread of Schmallenberg Virus across Denmark in 2012
L. D. Rasmussen et al.
Table 2. Detailed investigation of Schmallenberg virus (SBV) within sorted midge pools. Pools of midges were prepared following sorting. The obsoletus group midges (selected as parous) were divided into heads and abdomens prior to RNA extraction. The numbers of pools and the number which contained SBV RNA are indicated. The na indicates catch days when no midges were captured Sampling date (2012)
Catch site
Midge group
I
Obsoletus Heads Abdomens
Number
Pools Positive Pools Positive
Pulicarisa ‘Others’a II
Obsoletus Heads Abdomens
Pools Positive Pools Positive
Pulicarisa ‘Others’a III
Obsoletus Heads Abdomens
Obsoletus Heads Abdomens Pulicarisa ‘Others’a
a
August
September
6
13
20
27
0 0 0 0 na na
10 0 10 0
10 0 10 0
10 0 10 1b
5 0 5 0
0 0 0 0
0 0 0 0
Pools Positive Pools Positive
Pools Positive Pools Positive
3
8 0 8 0
10
17
0 0 0 0
6 0 6 2 na
1 0 1 0
10 0 10 0
0 0 0 0
0 0 0 0 na
0 0 0 0
0 0 0 0
+
na
10 1 10 5b + na
10 2 10 4
10 0 10 4b
10 1b 10 3
8 2b 8 2b
6 0 6 1
na
na
na
+
na
Pulicarisa ‘Others’a IV
July 30
0 0 0 0 na na
10 2 10 3
0 0 0 0
10 0 10 0 na
0 0 0 0
10 0 10 1
na
na
One pool was tested per sampling date in these groups, and thus, Indicates sample used for sequencing.
10 0 10 0
0 0 0 0 na na
10 3 10 6 +
10 0 10 3
+b
Total positive pools (%)
Number of pools examined
49 0 3 (6%) 0 0
7 6
11 0 0 0 2 (33%)
7 6
60 8 (13%) 22 (37%) 2 (29%) 1 (17%)
7 6
54 3 (6%) 10 (19%) 0/6 0/2
and + indicate negative and positive for SBV RNA.
b
abdomens, that were generated in the L-segment-specific RT-qPCR assays were sequenced and the 125-bp sequences, excluding primer regions, had 100% identity to the targeted region of the SBV segment L as described previously (Hoffmann et al., 2012). Conclusions Schmallenberg virus was identified within midges collected at four well-separated sites within Denmark during 2012 although it had only been identified in samples collected, in summer and autumn 2011, at 1 of 48 sampling sites (Fig. 1); thus, the virus had been able to spread across the country in about a year. It is noteworthy that during this time, in Denmark, only a single malformed calf was born and found to be infected with SBV although no systematic 4
survey of sheep and cattle for the presence of the virus or for anti-SBV antibodies was undertaken which clearly could have been helpful in evaluating the spread of SBV infection. In contrast to the situation in Denmark, Elbers et al. (2013b) have reported a large decrease in the proportion of SBV-containing midges in the Netherlands during 2012; however, a widespread infection of ruminant host animals had occurred in 2011 in the Netherlands and this had left only a minor proportion of the animal population being susceptible to SBV in 2012. It should be noted that Norway and Sweden, both to the north of Denmark, also reported the presence of SBV in 2012. Thus, it appears that having reached southern Denmark late in 2011 (Rasmussen et al., 2012), the virus was then spread to each of the four randomly selected Danish farms and further to other parts of Scandinavia during 2012. © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
L. D. Rasmussen et al.
Within the obsoletus group midges, both C. obsoletus and C. dewulfi species have now been shown to contain SBV RNA within both the heads and the abdomens. Schmallenberg virus RNA was detected in the heads in the absence of residual ruminant blood, strongly suggesting that both species act as vectors for SBV. It is not yet established whether the pulicaris group and ‘other’ Culicoides are true vectors because all SBV-positive midges of these groups also showed evidence of a recent blood meal. Larska et al. (2013b) have recently reported the isolation of SBV RNA from nulliparous obsoletus group midges and therefore suggested transovarial transmission of the virus. However, no evidence for the presence of SBV RNA was obtained in 39 nulliparous pools of unfed obsoletus group midges examined here which were collected during an 8-week warm period with a high prevalence of SBV in parous individuals. The indirect assessment of parity based on abdominal pigmentation (Dyce, 1969) can be uncertain, for example newly hatched specimens in the obsoletus group may emerge as apparently parous (Harrup et al., 2013). The mechanism behind this rapid spread of SBV in the obsoletus group population is unknown and needs further investigation in order to be prepared for future incursions of pathogens carried by Culicoides vectors. Acknowledgements We thank Helle Rasmussen and Astrid Blok van Witteloostuijn for excellent technical assistance. Also thanks to Claire Garros (CIRAD) for providing control species for the obsoletus-specific PCR. This work was supported by EU Grant GOCE-2003-010284 EDENext and is catalogued by the EDENext Steering Committee as EDENext 137 (http://www.edenext.eu) and OrbiNet a Green Development and Demonstration Programme and the European Research Area Network Emerging and Major Infectious Diseases of Livestock – funded project by The Ministry of Food, Agriculture and Fisheries. References Beer, M., F.J. Conraths, and W.H. van der Poel, 2013: ‘Schmallenberg virus’ – a novel orthobunyavirus emerging in Europe. Epidemiol. Infect. 141, 1–8. Carpenter, S., C. McArthur, R. Selby, R. Ward, D. V. Nolan, A. J. Mordue Luntz, J. F. Dallas, F. Tripet, and P. S. Mellor, 2008: Experimental infection studies of UK Culicoides species midges with bluetongue virus serotypes 8 and 9. Vet. Rec. 163, 589–592.
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
Spread of Schmallenberg Virus across Denmark in 2012
De Regge, N., I. Deblauwe, R. De Deken, P. Vantieghem, M. Madder, D. Geysen, F. Smeets, B. Losson, T. van den Berg, and A. B. Cay, 2012: Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR. Transbound. Emerg. Dis. 59, 471–475. Dyce, A. L., 1969: The recognition of nulliparous and parous Culicoides (Diptera: Ceratopogonidae) without dissection. Aust. J. Entomol. 8, 11–15. Elbers, A. R., R. Meiswinkel, E. van Weezep, M. M. van Oldruitenborgh-Oosterbaan, and E. A. Kooi, 2013a: Schmallenberg virus in Culicoides spp. biting midges, The Netherlands, 2011. Emerg. Infect. Dis. 19, 106–109. Elbers, A. R. W., R. Meiswinkel, E. van Weezep, E. A. Kooi, and W. H. M. van der Poel, 2013b: Schmallenberg virus in Culicoides biting midges in the Netherlands in 2012. Transbound. Emerg. Dis. Epub doi: 10.1111/tbed.12128. Harrup, L. E., B. V. Purse, N. Golding, P. S. Mellor, and S. Carpenter, 2013: Larval development and emergence sites of farm-associated Culicoides in the United Kingdom. Med. Vet. Entomol. Epub doi: 10.1111/mve.12006. Hoffmann, B., M. Scheuch, D. Hoper, R. Jungblut, M. Holsteg, H. Schirrmeier, M. Eschbaumer, K. V. Goller, K. Wernike, M. Fischer, A. Breithaupt, T. C. Mettenleiter, and M. Beer, 2012: Novel orthobunyavirus in cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469–472. Larska, M., M. P. Polak, M. Grochowska, L. Lechowski, J. S. Zwiaz zek, and J. F. Zmudzi nski, 2013a: First report of Schmallenberg virus infection in cattle and midges in Poland. Transbound. Emerg. Dis. 60, 97–101. Larska, M., L. Lechowski, M. Grochowska, and J. F. Zmudzinski, 2013b: Detection of Schmallenberg virus in nulliparous Culicoides obsoletus/scoticus complex and C. punctatus- the possibility of transovarial virus transmission in the midge population and of a new vector. Vet. Microbiol. 166, 467–473. Nolan, D. V., S. Carpenter, J. Barber, P. S. Mellor, J. F. Dallas, A. J. Mordue, and S. B. Piertney, 2007: Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe. Vet. Microbiol. 124, 82–94. Rasmussen, L. D., B. Kristensen, C. Kirkeby, T. B. Rasmussen, G. J. Belsham, R. Bødker, and A. Bøtner, 2012: Culicoids as vectors of Schmallenberg virus. Emerg. Infect. Dis. 18, 1204–1206. Toussaint, J. F., C. Sailleau, E. Breard, S. Zientara, and K. De Clercq, 2007: Bluetongue virus detection by two real-time RTqPCRs targeting two different genomic segments. J. Virol. Methods 140, 115–123. Veronesi, E., P. P. Mertens, A. E. Shaw, J. Brownlie, P. S. Mellor, and S. T. Carpenter, 2008: Quantifying bluetongue virus in adult Culicoides biting midges (Diptera: Ceratopogonidae). J. Med. Entomol. 45, 129–132.
5
RAPID COMMUNICATION
Rapid Spread of Schmallenberg Virus-infected Biting Midges (Culicoides spp.) across Denmark in 2012 L. D. Rasmussen1, C. Kirkeby2, R. Bødker2, B. Kristensen2, T. B. Rasmussen1, G. J. Belsham1 and A. Bøtner1 1 2
National Veterinary Institute, Technical University of Denmark, Kalvehave, Denmark National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark
Keywords: Schmallenberg virus; Culicoides spp.; insect vector; biting midges Correspondence: A. Bøtner. National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark. Tel.: +45 3588 7858; Fax: +45 3588 7901; E-mail: [email protected]
Summary Detection of Schmallenberg virus RNA, using real-time RT-PCR, in biting midges (Culicoides spp.) caught at 48 locations in 2011 and four well-separated farms during 2012 in Denmark, revealed a remarkably rapid spread of virus-infected midges across the country. During 2012, some 213 pools of obsoletus group midges (10 specimens per pool) were examined, and of these, 35 of the 174 parous pools were Schmallenberg virus RNA positive and 11 of them were positive in the heads. Culicoides species-specific PCRs identified both C. obsoletus and C. dewulfi as vectors of Schmallenberg virus.
Received for publication August 26, 2013 doi:10.1111/tbed.12189
Introduction Schmallenberg virus (SBV) was discovered and identified as a novel orthobunya virus in the autumn of 2011 in Germany (Hoffmann et al., 2012). It caused relatively mild clinical symptoms within infected cattle, including diarrhoea and loss of milk production, but, more seriously, it induced abortions and malformations in the offspring of pregnant cattle and sheep. Subsequently, several European countries have reported the presence of SBV [e.g. The Netherlands, France, U.K. and Denmark (Beer et al., 2013)]. In Denmark, the virus was first detected within biting midges (Culicoides spp.) that had been captured in October 2011 close to the Danish border and a reported SBV outbreak within Germany (Rasmussen et al., 2012). This was before any clinical signs were observed or virus was detected within domestic animals in Denmark. The role of Culicoides midges, within the obsoletus group, as vectors for SBV has now been confirmed (De Regge et al., 2012; Elbers et al., 2013a,b; Larska et al., 2013a,b). These initial results prompted a thorough investigation of additional midges caught in 2011 and also of midges caught in 2012 from across Denmark to determine the © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
spatial spread of the SBV vector and to identify the important vector species groups. Materials and Methods In total, 1169 Culicoides, in 182 pools containing between 1 and 37 unsorted biting midges, were captured in the summer and autumn of 2011 from 47 other collection sites (20% of which were at cattle farms) scattered throughout the country (see Fig. 1). These midges were caught, as a byproduct of mosquito collection, using a Mosquito Magnet Independence trap (Mosquito Magnet, Lititz, PA, USA) baited with carbon dioxide and octenol. Biting midges were sorted manually, using a dissection microscope, into specimens of the obsoletus group, the pulicaris group and ‘others’ (unclassified midges not belonging to the two specific groups) and preserved at 20°C. In 2012, capture of midges occurred at four cattle farms (labelled I, II, III and IV) in different regions of Denmark (Fig. 1) using Onderstepoort Veterinary Institute traps with 8-w UV tubes. Midges were collected weekly from July 30 until September 17. Preliminary investigations of these midges used 23 pools of 50 unsorted (but non-engorged) 1
Spread of Schmallenberg Virus across Denmark in 2012
L. D. Rasmussen et al.
Fig. 1. Location of trap sites in 2011 (red dots) and 2012 (blue circles). The red star marks the site where Schmallenberg virus-positive midges were trapped in 2011, as described previously (Rasmussen et al., 2012).
individuals, collected as one pool per week per sampling per site. For more detailed analysis, a portion of the remaining midges was sorted into species groups, as described above; the obsoletus group midges were also segregated into groups based on their parous or nulliparous status as described by Dyce (1969). In total, 260 pools of female midges were analysed. The obsoletus group pools [as 213 heads and 213 abdomens separately (10 specimens/ pool)] included 174 parous and 39 nulliparous midges, while 27 pools (8–50 specimens/pool with a total of 1030 individuals) contained the pulicaris group and 20 pools (2– 50 specimens/pool with a total of 442 individuals) had unclassified midges (‘others’). Only midges in the obsoletus group, already identified as a vector group for SBV (Rasmussen et al., 2012), were dissected into heads and abdomens and stored in ethanol until required. The pools (whole insects or separated heads and abdomens) were homogenized in nuclease-free water (100 ll), using a 3-mm stainless steel bead (Dejay Distribution, 2
Launceston, UK) in a TissueLyser II (Qiagen, Hilden, Germany), for 1 min at 25 Hz (Veronesi et al., 2008). After homogenization, additional nuclease-free water (100 ll) was added to the samples which were then centrifuged at 3000 g for 5 min. Nucleic acids were extracted from the supernatant (100 ll) using a MagNA pure LC Total Nucleic Acid Isolation Kit on a MagNA pure LC system (Roche Diagnostics, Basel, Switzerland) and eluted in water (50 ll) (Veronesi et al., 2008). Two separate one-step RTqPCR assays, targeting the L-segment and the S-segment of SBV RNA, were performed on the extracted nucleic acids using a M93005p qPCR system (Agilent Technologies, Palo Alto, CA, USA) as described previously (Hoffmann et al., 2012; Rasmussen et al., 2012). Only pools positive in both reactions were scored as positive for SBV RNA. Another RT-qPCR targeting ruminant beta-actin mRNA was performed to test midges for the presence of bovine/ ovine beta-actin as a marker for the recent intake of a blood meal (Toussaint et al., 2007). © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
L. D. Rasmussen et al.
Spread of Schmallenberg Virus across Denmark in 2012
The PCR amplicon of 7 SBV-positive pools from the L-segment-specific RT-qPCR assay (145 bp) was sequenced using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer, resulting in 125-bp sequences, excluding primer regions. Results and Discussion Except for the midges caught close to the border with Germany (as described by Rasmussen et al., 2012), none of the pools of midges (containing in total 1169 specimens) caught in Denmark during 2011 were found positive for SBV RNA. In contrast, in 2012, the initial screen showed that 9 of 23 unsorted pools (50 midges each) collected from the four sampling sites contained SBV RNA, with at least one positive sample from each farm; these SBV-positive pools were collected between August 13 and September 17 (Table 1). For the more detailed analysis, the 260 midge pools were divided into species groups and the obsoletus group were further divided into parous and nulliparous groups. The 39 nulliparous obsoletus group pools were all negative for SBV RNA (data not shown). Of the remaining 221 pools, a total of 41 were positive for SBV RNA; from the 174 obsoletus group pools, 36 (21%) were positive, while 2 (7%) of 27 pulicaris pools and 3 (15%) of 20 of the ‘others’ contained SBV RNA (Table 2). It should be noted that the highest prevalence of SBV RNA was found within obsoletus group pools although the pools of the pulicaris group and ‘other’ Culicoides consisted, on average, of four and two times more individuals, respectively. The highest prevalence of SBV RNA was detected at site III (eastern Jutland); it was found in 23 of 60 pools in the obsoletus group, two of seven in the pulicaris group and one of six in the ‘others’ group. At site IV, on Zeeland, all SBV-positive pools (10 of 54) belonged to the obsoletus group (Table 2), while at site II, the most western site (Fig. 1), two of 6 pools of the ‘others’ group were positive Table 1. Results of the preliminary analysis of pools of 50 unsorted biting midges for the presence of Schmallenberg virus RNA at four separate sites within Denmark in 2012. The na indicates catch days when either no or a limited number of midges were captured, and these were reserved for the detailed investigation (see Table 2) Sampling date (2012)
Sampling area I II III IV
July 30 na na na
August 6
13
September 20
27
3
10
17
+ + +
na +
na
+ na +
+ na na
+
na
+
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
and at site I, the most northern site (Fig. 1), just three of the 49 pools of the obsoletus group were identified as being positive and only in the abdomens (Table 2). Overall, the frequency of SBV-positive abdomens was 2–5 times higher than positive heads. All 11 pools with SBV-positive heads, except one, also showed positive abdomens. The Ct values (S-Segment assay) were between 24 and 31 with no apparent difference between the groups of heads and abdomens. All specimens were examined under the microscope and judged visually not to have had a recent blood meal. SBV replication can be expected to start in the abdomen following the uptake of infected blood and then spread towards the head as for bluetongue virus (Carpenter et al., 2008). Finding virus in the head implies that the infection has spread to the salivary glands situated in the anterior part of the thorax. To identify the individual species within the obsoletus group acting as SBV vectors, all 36 pools containing SBV RNA were analysed using species-specific PCRs, as described previously (Nolan et al., 2007), distinguishing 4 separate species within the obsoletus group (these are C. obsoletus, C. chiopterus, C. dewulfi and C. scoticus). Only C. obsoletus and C. dewulfi were detected in the SBVpositive pools, both individually and in mixtures. Results were consistent for the pools of heads and abdomens originating from the same pool of midges. The presence of ruminant beta-actin mRNA, a marker for recent intake of a blood meal (Toussaint et al., 2007), was assayed in all samples, and it was undetected in most SBV RNA-positive pools of heads (8 of 11) and abdomens (20 of 35) derived from the obsoletus group; similarly, this marker was absent from most SBV RNA-positive pools that contained C. obsoletus and C. dewulfi both individually and in mixtures. This makes it very likely that both C. obsoletus and C. dewulfi are vectors for SBV in Denmark. These results complement the studies reported by De Regge et al. (2012) who showed that heads of C. obsoletus, C. dewulfi and C. chiopterus could be shown to contain SBV RNA. Most of the SBV-positive pools in Denmark contained obsoletus group midges, but positive pools of pulicaris group and ‘other’ Culicoides were detected at lower frequency. However, despite visual inspection for blood in the abdomens to allow the removal of newly fed specimens, ruminant beta-actin mRNA was detected in all positive pools in these latter groups indicative of a recent blood meal. For these species groups, heads and abdomens were not tested separately and it therefore remains unknown to what degree these species were functional vectors (i.e. in which replication occurred) or whether they just carried the virus from a blood meal. PCR amplicons (145 bp), derived from seven different pools of midges (as indicated in Table 2) collected at different sites and sampling times and using both heads and 3
Spread of Schmallenberg Virus across Denmark in 2012
L. D. Rasmussen et al.
Table 2. Detailed investigation of Schmallenberg virus (SBV) within sorted midge pools. Pools of midges were prepared following sorting. The obsoletus group midges (selected as parous) were divided into heads and abdomens prior to RNA extraction. The numbers of pools and the number which contained SBV RNA are indicated. The na indicates catch days when no midges were captured Sampling date (2012)
Catch site
Midge group
I
Obsoletus Heads Abdomens
Number
Pools Positive Pools Positive
Pulicarisa ‘Others’a II
Obsoletus Heads Abdomens
Pools Positive Pools Positive
Pulicarisa ‘Others’a III
Obsoletus Heads Abdomens
Obsoletus Heads Abdomens Pulicarisa ‘Others’a
a
August
September
6
13
20
27
0 0 0 0 na na
10 0 10 0
10 0 10 0
10 0 10 1b
5 0 5 0
0 0 0 0
0 0 0 0
Pools Positive Pools Positive
Pools Positive Pools Positive
3
8 0 8 0
10
17
0 0 0 0
6 0 6 2 na
1 0 1 0
10 0 10 0
0 0 0 0
0 0 0 0 na
0 0 0 0
0 0 0 0
+
na
10 1 10 5b + na
10 2 10 4
10 0 10 4b
10 1b 10 3
8 2b 8 2b
6 0 6 1
na
na
na
+
na
Pulicarisa ‘Others’a IV
July 30
0 0 0 0 na na
10 2 10 3
0 0 0 0
10 0 10 0 na
0 0 0 0
10 0 10 1
na
na
One pool was tested per sampling date in these groups, and thus, Indicates sample used for sequencing.
10 0 10 0
0 0 0 0 na na
10 3 10 6 +
10 0 10 3
+b
Total positive pools (%)
Number of pools examined
49 0 3 (6%) 0 0
7 6
11 0 0 0 2 (33%)
7 6
60 8 (13%) 22 (37%) 2 (29%) 1 (17%)
7 6
54 3 (6%) 10 (19%) 0/6 0/2
and + indicate negative and positive for SBV RNA.
b
abdomens, that were generated in the L-segment-specific RT-qPCR assays were sequenced and the 125-bp sequences, excluding primer regions, had 100% identity to the targeted region of the SBV segment L as described previously (Hoffmann et al., 2012). Conclusions Schmallenberg virus was identified within midges collected at four well-separated sites within Denmark during 2012 although it had only been identified in samples collected, in summer and autumn 2011, at 1 of 48 sampling sites (Fig. 1); thus, the virus had been able to spread across the country in about a year. It is noteworthy that during this time, in Denmark, only a single malformed calf was born and found to be infected with SBV although no systematic 4
survey of sheep and cattle for the presence of the virus or for anti-SBV antibodies was undertaken which clearly could have been helpful in evaluating the spread of SBV infection. In contrast to the situation in Denmark, Elbers et al. (2013b) have reported a large decrease in the proportion of SBV-containing midges in the Netherlands during 2012; however, a widespread infection of ruminant host animals had occurred in 2011 in the Netherlands and this had left only a minor proportion of the animal population being susceptible to SBV in 2012. It should be noted that Norway and Sweden, both to the north of Denmark, also reported the presence of SBV in 2012. Thus, it appears that having reached southern Denmark late in 2011 (Rasmussen et al., 2012), the virus was then spread to each of the four randomly selected Danish farms and further to other parts of Scandinavia during 2012. © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
L. D. Rasmussen et al.
Within the obsoletus group midges, both C. obsoletus and C. dewulfi species have now been shown to contain SBV RNA within both the heads and the abdomens. Schmallenberg virus RNA was detected in the heads in the absence of residual ruminant blood, strongly suggesting that both species act as vectors for SBV. It is not yet established whether the pulicaris group and ‘other’ Culicoides are true vectors because all SBV-positive midges of these groups also showed evidence of a recent blood meal. Larska et al. (2013b) have recently reported the isolation of SBV RNA from nulliparous obsoletus group midges and therefore suggested transovarial transmission of the virus. However, no evidence for the presence of SBV RNA was obtained in 39 nulliparous pools of unfed obsoletus group midges examined here which were collected during an 8-week warm period with a high prevalence of SBV in parous individuals. The indirect assessment of parity based on abdominal pigmentation (Dyce, 1969) can be uncertain, for example newly hatched specimens in the obsoletus group may emerge as apparently parous (Harrup et al., 2013). The mechanism behind this rapid spread of SBV in the obsoletus group population is unknown and needs further investigation in order to be prepared for future incursions of pathogens carried by Culicoides vectors. Acknowledgements We thank Helle Rasmussen and Astrid Blok van Witteloostuijn for excellent technical assistance. Also thanks to Claire Garros (CIRAD) for providing control species for the obsoletus-specific PCR. This work was supported by EU Grant GOCE-2003-010284 EDENext and is catalogued by the EDENext Steering Committee as EDENext 137 (http://www.edenext.eu) and OrbiNet a Green Development and Demonstration Programme and the European Research Area Network Emerging and Major Infectious Diseases of Livestock – funded project by The Ministry of Food, Agriculture and Fisheries. References Beer, M., F.J. Conraths, and W.H. van der Poel, 2013: ‘Schmallenberg virus’ – a novel orthobunyavirus emerging in Europe. Epidemiol. Infect. 141, 1–8. Carpenter, S., C. McArthur, R. Selby, R. Ward, D. V. Nolan, A. J. Mordue Luntz, J. F. Dallas, F. Tripet, and P. S. Mellor, 2008: Experimental infection studies of UK Culicoides species midges with bluetongue virus serotypes 8 and 9. Vet. Rec. 163, 589–592.
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
Spread of Schmallenberg Virus across Denmark in 2012
De Regge, N., I. Deblauwe, R. De Deken, P. Vantieghem, M. Madder, D. Geysen, F. Smeets, B. Losson, T. van den Berg, and A. B. Cay, 2012: Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR. Transbound. Emerg. Dis. 59, 471–475. Dyce, A. L., 1969: The recognition of nulliparous and parous Culicoides (Diptera: Ceratopogonidae) without dissection. Aust. J. Entomol. 8, 11–15. Elbers, A. R., R. Meiswinkel, E. van Weezep, M. M. van Oldruitenborgh-Oosterbaan, and E. A. Kooi, 2013a: Schmallenberg virus in Culicoides spp. biting midges, The Netherlands, 2011. Emerg. Infect. Dis. 19, 106–109. Elbers, A. R. W., R. Meiswinkel, E. van Weezep, E. A. Kooi, and W. H. M. van der Poel, 2013b: Schmallenberg virus in Culicoides biting midges in the Netherlands in 2012. Transbound. Emerg. Dis. Epub doi: 10.1111/tbed.12128. Harrup, L. E., B. V. Purse, N. Golding, P. S. Mellor, and S. Carpenter, 2013: Larval development and emergence sites of farm-associated Culicoides in the United Kingdom. Med. Vet. Entomol. Epub doi: 10.1111/mve.12006. Hoffmann, B., M. Scheuch, D. Hoper, R. Jungblut, M. Holsteg, H. Schirrmeier, M. Eschbaumer, K. V. Goller, K. Wernike, M. Fischer, A. Breithaupt, T. C. Mettenleiter, and M. Beer, 2012: Novel orthobunyavirus in cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469–472. Larska, M., M. P. Polak, M. Grochowska, L. Lechowski, J. S. Zwiaz zek, and J. F. Zmudzi nski, 2013a: First report of Schmallenberg virus infection in cattle and midges in Poland. Transbound. Emerg. Dis. 60, 97–101. Larska, M., L. Lechowski, M. Grochowska, and J. F. Zmudzinski, 2013b: Detection of Schmallenberg virus in nulliparous Culicoides obsoletus/scoticus complex and C. punctatus- the possibility of transovarial virus transmission in the midge population and of a new vector. Vet. Microbiol. 166, 467–473. Nolan, D. V., S. Carpenter, J. Barber, P. S. Mellor, J. F. Dallas, A. J. Mordue, and S. B. Piertney, 2007: Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe. Vet. Microbiol. 124, 82–94. Rasmussen, L. D., B. Kristensen, C. Kirkeby, T. B. Rasmussen, G. J. Belsham, R. Bødker, and A. Bøtner, 2012: Culicoids as vectors of Schmallenberg virus. Emerg. Infect. Dis. 18, 1204–1206. Toussaint, J. F., C. Sailleau, E. Breard, S. Zientara, and K. De Clercq, 2007: Bluetongue virus detection by two real-time RTqPCRs targeting two different genomic segments. J. Virol. Methods 140, 115–123. Veronesi, E., P. P. Mertens, A. E. Shaw, J. Brownlie, P. S. Mellor, and S. T. Carpenter, 2008: Quantifying bluetongue virus in adult Culicoides biting midges (Diptera: Ceratopogonidae). J. Med. Entomol. 45, 129–132.
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