Doreen Werner

Monitoring of putative vectors of bluetongue virus serotype 8, Germany.

To identify the vectors of bluetongue virus (BTV) in Germany, we monitored Culicoides spp. biting midges during April 2007–May 2008. Molecular characterization of batches of midges that tested positive for BTV suggests C. obsoletus sensu stricto as a relevant vector of bluetongue disease in central Europe.

European Surveillance for West Nile Virus in Mosquito Populations

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany

BackgroundAs a result of globalization and climate change, Dirofilaria immitis and Dirofilaria repens, the causative agents of dirofilariosis in Europe, continue to spread from endemic areas in the Mediterranean to northern and northeastern regions of Europe where autochthonous cases of dirofilarial infections have increasingly been observed in dogs and humans. Whilst D. repens was recently reported from mosquitoes in putatively non-endemic areas, D. immitis has never been demonstrated in mosquitoes from Europe outside the Mediterranean.MethodsFrom 2011 to 2013, mosquitoes collected within the framework of a German national mosquito monitoring programme were screened for filarial nematodes using a newly designed filarioid-specific real-time PCR assay. Positive samples were further processed by conventional PCR amplification of the cytochrome c oxidase subunit I (COI) gene, amplicons were sequenced and sequences blasted against GenBank.ResultsApproximately 17,000 female mosquitoes were subjected to filarial screening. Out of 955 pools examined, nine tested positive for filariae. Two of the COI sequences indicated D. immitis, one D. repens and four Setaria tundra. Two sequences could not be assigned to a known species due to a lack of similar GenBank entries. Whilst D. immitis and the unknown parasites were detected in Culex pipiens/torrentium, D. repens was found in a single Anopheles daciae and all S. tundra were demonstrated in Aedes vexans. All positive mosquitoes were collected between mid-June and early September.ConclusionThe finding of dirofilariae in German mosquitoes implies the possibility of a local natural transmission cycle. While the routes of introduction to Germany and the origin of the filariae cannot be determined retrospectively, potential culicid vectors and reservoir hosts must prospectively be identified and awareness among physicians, veterinarians and public health personnel be created. The health impact of S. tundra on the indigenous cervid fauna needs further investigation.

Out of the bush: the Asian bush mosquito Aedes japonicus japonicus (Theobald, 1901) (Diptera, Culicidae) becomes invasive

The Asian bush or rock pool mosquito Aedes japonicus japonicus is one of the most expansive culicid species of the world. Being native to East Asia, this species was detected out of its original distribution range for the first time in the early 1990s in New Zealand where it could not establish, though. In 1998, established populations were reported from the eastern US, most likely as a result of introductions several years earlier. After a massive spread the mosquito is now widely distributed in eastern North America including Canada and two US states on the western coast. In the year 2000, it was demonstrated for the first time in Europe, continental France, but could be eliminated. A population that had appeared in Belgium in 2002 was not controlled until 2012 as it did not propagate. In 2008, immature developmental stages were discovered in a large area in northern Switzerland and bordering parts of Germany. Subsequent studies in Germany showed a wide distribution and several populations of the mosquito in various federal states. Also in 2011, the species was found in southeastern Austria (Styria) and neighbouring Slovenia. In 2013, a population was detected in the Central Netherlands, specimens were collected in southern Alsace, France, and the complete northeastern part of Slovenia was found colonized, with specimens also present across borders in adjacent Croatia. Apparently, at the end of 2013 a total of six populations occurred in Europe although it is not clear whether all of them are completely isolated. Similarly, it is not known whether these populations go back to the same number of introductions. While entry ports and long-distance continental migration routes are also obscure, it is likely that the international used tyre trade is the most important mode of intercontinental transportation of the mosquito. Aedes j. japonicus does not only display an aggressive biting behaviour but is suspected to be a vector of various disease agents and to displace indigenous culicid species. Therefore, Aedes j. japonicus might both cause public health problems in the future and have a significant impact on the biodiversity of the invaded territories.

Approaches to passive mosquito surveillance in the EU

The recent emergence in Europe of invasive mosquitoes and mosquito-borne disease associated with both invasive and native mosquito species has prompted intensified mosquito vector research in most European countries. Central to the efforts are mosquito monitoring and surveillance activities in order to assess the current species occurrence, distribution and, when possible, abundance, in order to permit the early detection of invasive species and the spread of competent vectors. As active mosquito collection, e.g. by trapping adults, dipping preimaginal developmental stages or ovitrapping, is usually cost-, time- and labour-intensive and can cover only small parts of a country, passive data collection approaches are gradually being integrated into monitoring programmes. Thus, scientists in several EU member states have recently initiated programmes for mosquito data collection and analysis that make use of sources other than targeted mosquito collection. While some of them extract mosquito distribution data from zoological databases established in other contexts, community-based approaches built upon the recognition, reporting, collection and submission of mosquito specimens by citizens are becoming more and more popular and increasingly support scientific research. Based on such reports and submissions, new populations, extended or new distribution areas and temporal activity patterns of invasive and native mosquito species were found. In all cases, extensive media work and communication with the participating individuals or groups was fundamental for success. The presented projects demonstrate that passive approaches are powerful tools to survey the mosquito fauna in order to supplement active mosquito surveillance strategies and render them more focused. Their ability to continuously produce biological data permits the early recognition of changes in the mosquito fauna that may have an impact on biting nuisance and the risk of pathogen transmission associated with mosquitoes. International coordination to explore synergies and increase efficiency of passive surveillance programmes across borders needs to be established.

A new focus of Aedes japonicus japonicus (Theobald, 1901) (Diptera, Culicidae) distribution in Western Germany: rapid spread or a further introduction event?

BackgroundThe Asian bush mosquito, Aedes japonicus japonicus, a potential vector of several viruses, was first detected in Germany in 2008 on the Swiss-German border. In the following years, this invasive species apparently succeeded in establishing populations in southern Germany and in spreading northwards. In 2011, its distribution area already covered large areas of the federal state of Baden-Wurttemberg, and its northernmost German collection point was reported to be close to Stuttgart. Several independent submissions to our laboratories of Ae. j. japonicus specimens in July 2012, originating from the same area in the federal state of North Rhine-Westphalia, western Germany, prompted us to carry out an immediate surveillance in this region in the expectation of finding a further distribution focus of Ae. j. japonicus in Germany.MethodsAfter inspecting the places of residence of the collectors of the submitted mosquito specimens, all kinds of water containers in 123 cemeteries in surrounding towns and villages were checked for mosquito developmental stages. These were collected and kept to produce adults for morphological species identification. One specimen per collection site was identified genetically by COI sequence analysis.ResultsAedes j. japonicus adults and immature stages were found in 36 towns/villages that were checked (29%) over an area of approximately 2,000 km2 in southern North Rhine-Westphalia and northern Rhineland Palatinate. The species could not be demonstrated further south when monitoring towards the northernmost previous collection sites in southern Germany. It therefore remains to be elucidated whether the species has entered western Germany from the south, from Belgium in the west where it has been demonstrated to occur locally since 2002, or through a new introduction.ConclusionsAedes j. japonicus is obviously much more widely distributed in Germany than previously thought. It appears to be well adapted, to have a strong expansion tendency and to replace indigenous mosquito species. Thus, a further spread is anticipated and elimination seems hardly possible anymore. The vector potency of the species should be reason enough to thoroughly monitor its future development in Germany.

Further specimens of the Asian tiger mosquito Aedes albopictus (Diptera, Culicidae) trapped in southwest Germany

After two previous demonstrations of introductions of the Asian tiger mosquito, Aedes albopictus, into southern Germany in 2007 and 2011, another three specimens were trapped in the city of Freiburg in the Upper Rhine Valley. The females were caught in early September 2011 (n = 2) and mid-July 2012 (n = 1). The trap was located at a railway container station where cargo is transferred to trains from trucks predominantly coming from southern Europe where A. albopictus is widely distributed. The reported findings confirm vehicle transport of A. albopictus to be an important and probably frequent mode of importation, and suggest that more regular and intense monitoring for invasive mosquito species in the Upper Rhine Valley should be undertaken in order to detect an establishment and implement adequate control measures in good time.

Transmission of Schmallenberg Virus during Winter, Germany

To the Editor: Schmallenberg virus (SBV), an orthobunyavirus, emerged in northern Europe in 2011 (1). SBV infection causes transient fever, diarrhea, and a reduced milk yield in adult ruminants but, most notably, stillbirths and severe malformations in lambs and calves (2). Insect vectors play an essential role in transmission; the viral genome has been detected in various field-collected biting midges (Culicoides spp.) (3,4). During autumn 2012 and winter 2012–2013, blood samples were taken at several times from individual sheep on a farm located in the German federal state of Mecklenburg–Western Pomerania. The farm is surrounded by agricultural fields and meadows. Approximately 1,000 ewes and their lambs, a dog, and some cats were kept on the farm; most of the animals are outdoors year-round. Only dams with >2 lambs are housed in open stabling in December and January. The dung is regularly cleared away and stored ≈10 m from 1 of the stable entrances. Repellents or insecticides were not applied in the monitored period. Blood samples were taken in September 2012 and in January and February 2013 and analyzed by an SBV-specific real-time quantitative reverse transcription PCR (RT-qPCR) (5) and by an SBV antibody ELISA (ID Screen Schmallenberg virus Indirect; IDvet; Montpellier, France) by using the recommended cutoff of 50% relative optical density as compared with the positive control (sample-to-positive ratio [S/P]). In September 2012, blood samples from 60 sheep tested negative by the SBV antibody ELISA. Moreover, fetal malformations of the brain, spinal cord, or skeletal muscle, which might have suggested a previous SBV-infection of the dam, were not observed during the lambing season in December 2012. On January 10, 2013, blood samples were taken from 15 sheep that had not previously been tested; samples from all animals tested negative by ELISA. However, 4 sheep (S01–S04) tested positive by RT-qPCR (quantification cycle values: S01: 31.6, S02: 39.9, S03: 37.6, and S04: 34.9). Four weeks later, antibodies against SBV could be detected. Each of the PCR-positive blood samples was injected into 2 adult type I interferon receptor-knockout mice on a C57BL/6 genetic background. Both mice that had received blood samples of sheep S01 were seropositive after 3 weeks (S/P: 207.0 and 207.2), which demonstrates the presence of infectious virus in the inoculated blood. Assuming that viral RNA remains in the blood for just a few days, as reported after experimental infection with SBV (1,6), the sheep tested in this study had most likely been infected in early 2013. During this period, the lowest temperatures rose above 5°C for several consecutive days, with a maximum of ≈9°C (Figure, panel A). Within this brief interval, when the temperature was higher, some biting midges (Culicoides spp.) become active (7). Indeed, at the end of January, a single female biting midge (Obsoletus complex) was caught in a trap equipped with ultraviolet light; the midge tested negative by the SBV-specific RT-qPCR. Figure Results of analysis of samples from sheep and cattle for Schmallenberg virus (SBV), Germany, 2012–2013. A) Climate data and sampling. The maximum temperatures are shown with filled triangles and a solid line and the minimum temperatures with unfilled ... On January 23 and February 20, 2013, blood samples were taken from 90 sheep that had not previously been tested (Figure, panel A). A viral genome was not detected in any animal at any time. However, antibodies were detectable in 9 animals on the first sampling day. In 2 additional sheep, the S/P was in the inconclusive range; 1 of the animals tested positive after 4 weeks. In the remaining 79 sheep, no SBV antibodies could be detected; after 4 weeks, 76 sheep still tested negative by ELISA. However, the S/P of 1 sheep had increased to the inconclusive range, and 2 sheep were seropositive. Because antibodies may be detectable 10 days–3 weeks after experimental infection for the first time (8), the presumed period of infection was between mid-January and mid-February. At this time, the highest temperatures again rose above 6°C for a few days (Figure, panel A). Although the within-herd seroprevalence was >90% in ewes after confirmed or suspected SBV infection in 2011 (9), in this study, conducted during the cold season, only 12 (13%) of 90 tested sheep were positive by ELISA. Three animals seroconverted between mid-January and mid-February. Thus, SBV transmission appears to be possible at a low level, most likely because of the low activity of the involved insect vectors. In addition to the SBV cases found on the sheep holding in Mecklenburg–Western Pomerania, an additional 52 confirmed SBV cases (defined as virus detection by qRT-PCR or isolation in cell culture) in adult ruminants were reported to the German Animal Disease Reporting System from January 1 through February 20, 2013 (Figure, panel B). Most affected animal holdings were located in Bavaria, but cases were also reported from Thuringia, Saxony, Brandenburg, Mecklenburg–Western Pomerania, Hesse, and Lower Saxony. In conclusion, transmission of SBV by hematophagous insects seems possible, even during the winter in central Europe, if minimum temperatures rise above a certain threshold for several consecutive days.

Unexpected patterns of admixture in German populations of Aedes Japonicus Japonicus (Diptera: Culicidae) underscore the importance of human intervention

The mosquito Aedes japonicus japonicus, originally restricted to temperate East Asia, is now widespread in North America and more recently has become established in Europe. To ascertain the putative number of separate introductions to Europe and examine patterns of expansion we analyzed the genetic makeup of Ae. j. japonicus populations from five cemeteries in North Rhine-Westphalia and Rhineland-Palatinate, two western German federal states, as well as of specimens from populations in Belgium, Switzerland, and Austria/Slovenia. To do so, we genotyped individual specimens at seven pre-existing polymorphic microsatellite loci and sequenced part of the nad4 mitochondrial locus. We found evidence of two different genotypic signatures associated with different nad4 mitochondrial haplotypes, indicating at least two genetically differentiated populations of Ae. j. japonicus in Europe (i.e. two distinct genotypes). Belgian, Swiss, and Austrian/Slovenian populations all share the same genotypic signature although they have become differentiated since isolation. Contrary to expectations, the German Ae. j. japonicus are not closely related to those in Belgium which are geographically nearest but are also highly inbred. German populations have a unique genotype but also evidence of mixing between the two genotypes. Also unexpectedly, the populations closest to the center of the German infestation had the highest levels of admixture indicating that separate introductions did not expand and merge but instead their expansion was driven by punctuated human-mediated transport. Critically, the resulting admixed populations have higher genetic diversity and appear invasive as indicated by their increased abundance and recent spread across western Germany.

PCR identification of culicoid biting midges (Diptera, Ceratopogonidae) of the Obsoletus complex including putative vectors of bluetongue and Schmallenberg viruses

BackgroundBiting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV) in northern and central Europe during the 2006 outbreak of bluetongue disease (BT). Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV) in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI) gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus) plus the isomorphic species C. dewulfi.MethodsBiting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer.ResultsThe newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic band for each species, could be realized with high specificity and sensitivity.ConclusionTo elucidate the biological characteristics of potential vectors of disease agents, such as ecology, behaviour and vector competence, and the role of these haematophagous arthropods in the epidemiology of the diseases, simple, cost-effective and, most importantly, reliable identification techniques are necessary. The PCR assay presented will help to identify culicoid vector species and therefore add to bluetongue and Schmallenberg disease research including vector control and monitoring.