In August 2006, Bluetongue virus disease (BTD) was detected for the first time in the Netherlands, Belgium, Germany and Northern France. Serological tests as well as reverse transcriptase polymerase chain reaction (RT-PCR) proved the occurrence of Bluetongue virus (BTV) in diseased sheep and cattle, and the virus was identified as serotype 8. Therefore, the search for possible vectors was immediately initiated in the outbreak region in Germany. Traps with automatically regulated ultraviolet light lamps were placed at two different farms with sero-positive cattle, and insect monitoring was done from August 2006 until January 2007. The caught arthropods were weekly determined, and it could be observed that midges of the dipteran family Ceratopogonidae occurred in large numbers, sometimes representing up to 40% of all individuals. The microscopical analysis of the wing morphology showed that the species (complex) Culicoides obsoletus was most abundant covering about 97% of the analysed midges. On the second place ranged C. pulicaris, while C. nubeculosus and C. festivipennis were found only as single individuals. Fed and unfed females were separated, sent to the National Reference Laboratory for Bluetongue disease (Friedrich-Loeffler-Institut, Isle of Riems, Germany) and investigated with a BTV-8-specific real-time RT-PCR. It could be demonstrated that at both farms both fed and unfed C. obsoletus were tested positive for BTV-8 genomes, while none of the other species scored positive. This finding strongly supports that the BTD-epidemic, which reached in the meantime wide regions of North Rhine-Westphalia in Germany and of the neighbouring countries with several hundreds of affected farms, is initiated by virus transmission during the blood meal of midges of the C. obsoletus complex. Since they were captured still at the 21st of December close to cattle with clinical signs, it must be feared that BTV-8 is now established in Central Europe, where it had been absent until now.

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In the summer of 2006, a bluetongue epidemic started in the border area of Belgium, The Netherlands, and Germany, spread within 2 years over large areas of Western and Central Europe, and caused substantial losses in farm ruminants. Especially sheep and cattle were severely affected, leading to a case-fatality ratio of nearly 40% in sheep (Conraths et al., Emerg Inf Dis 15(3):433-435, 2009). The German federal ministry of food, agriculture, and consumer protection (BMELV) established a countrywide monitoring on the occurrence of the vectors of this virus, i.e., midges (family Ceratopogonidae) of the genus Culicoides. The monitoring was done on 91 sites, most of which were localized in the 150-km restriction zone that existed in December 2006. A grid consisting of 45 x 45 km(2) cells was formed that covered the monitoring area. As a rule, one trap was placed into each grid cell. The monitoring program started at the end of March 2007 and lasted until May 2008. It included the catching of midges by ultraviolet light traps-done each month from days 1 until 8, the selection of midges of the Culicoides obsoletus, Culicoides pulicaris group, and other Culicoides spp., the testing of midges for bluetongue virus (BTV) by polymerase chain reaction (PCR), and the daily registration of weather data at each trap site for the whole monitoring period. The following main results were obtained: (1) Members of the C. obsoletus group were most commonly found in the traps, reaching often 3/4 of the catches. The African and South European vector of BTV-the species Culicoides imicola-was never found. (2) Members of the C. obsoletus group were most frequently found infected with BTV besides a few cases in the C. pulicaris group and other species. (3) Members of the C. obsoletus group were also found in winter. Their numbers were reduced, however, and they were caught mostly close to stables. Therefore, a true midge-free period does not exist during the year in Germany. (4) The amounts of midges caught daily depended on the weather conditions. If it was cold and/or windy, the traps contained only a few specimens. Since the months from January to May 2008 were considerably colder (at all farms) than their correspondents in 2007, the growing of the population of midges started 2-3 months later in 2008 than in 2007. (5) The highest populations of midges occurred in both years (2007 and 2008) during the months September and October. This corresponded significantly to the finding of highest numbers of infected midges and to the number of diseased cattle and sheep during these 2 months. (6) It is noteworthy that in general, the first virus-positive midges of the species C. obsoletus were found about 1 1/2 months later than the first clinical cases had occurred or later than the first PCR-proven virus-positive sentinel animals had been documented. In 2007, the first BTV-positive cattle were detected in May in North Rhine-Westphalia, while the first positive Culicoides specimens were only found in August on the same farm. Evaluating these main...

In August 2006, the blue tongue virus (BTV-type South Africa serotype 8) was detected for the first time in cattle blood probes in the Netherlands, immediately followed by cases in Belgium and in cattle on German farms, which were situated close to Aachen at the border to those countries. Within less than 2 months the disease spread eastwards crossing the Rhine, southwards to Luxemburg and to Northern France. At the end of the year 2006, nearly 1,000 farms were affected in Germany. Catches on two German cattle farms proved that the ceratopogonid species Culicoides obsoletus was obviously the vector, since many females-fed and unfed ones-were found to be infected with this virus. This sudden outbreak of bluetongue disease (BTD) is surely not a primary result of global warming, but rather an effect of globalization-i.e. the intensive worldwide import and export of animals; but a hot summer, as in 2006, and a warm winter like that of the years 2006/2007 supported the new spread starting again in masses in August 2007 leading to 596 PCR-confirmed cases until then with more than 200,000 animals infected. Thus, new agents coming from elsewhere have only a chance to spread if appropriate vectors are available and the conditions remain favourable during a reasonably long period. Effects of global warming-of course-will support persistence of such outbreaks of diseases due to offering of spreading of imported viruses, bacteria and/or parasites.

The entomological monitoring programs done on 19 farms all over Northrhine-Westfalia (Germany) in the years 2007 and 2008 showed that the species Culicoides obsoletus and C. pulicaris are the most common ones and that both act as vectors of the bluetongue-virus of the serotype 8. Especially the species C. obsoletus was found all over the year and also inside or close to stables during the winter months. Therefore, there exists no midge-free period that would interrupt the transmission cycle of bluetongue virus. This makes it necessary that vaccination programs and insecticidal treatment have to consider this and must be preceded even in winter months. From the fact that there was no northward migration of southern Culicoides species (e.g., Culicoides imicola), apparently, globalization and its intense transportation of animals, plants, and other goods is the reason of this BTD outbreak in Central Europe. This conclusion needs urgently special attention on other luring epidemics in the near future.

When studying the vectorship of Culicoides species during the outbreak of Bluetongue disease (BTD) in Central Europe, the question arose whether the most common species and additionally proven vectors of BTV (C. obsoletus and C. pulicaris) are definitive species or do they belong to so-called complexes, since the determination based on morphological criteria is not very significant and knowledge on the life cycles is poor or even absent. Therefore, the present molecular biological study on their ITS-1, ITS-2 and 18SrDNA characteristics was initiated to investigate specimens, which had been determined by their wing morphology during an entomological monitoring in the years 2007 and 2008 at 91 farms in Germany (Mehlhorn et al. 2009). This study revealed novel types respectively different forms, which appeared very similar to Culicoides obsoletus, but showed slightly varying wing patterns. The molecular biological data were compared to those in data banks and combined to provisional dendrograms. The ITS-1 and ITS-2 analysis showed that the specimens determined in the monitoring as C. obsoletus inclusive those with different wing pattern correlate significantly with the data of C. obsoletus in the data banks and surrounded the data bank specifications of C. montanus and C. scoticus so closely that the latter might be only hardly separate species. A similar interpretation can also be drawn when looking at the 18S rDNA dendrogram. Thus, C. scoticus and C. montanus might be races of C. obsoletus rather than separate species. With respect to the ITS-1 and ITS-2 characteristics of C. pulicaris females, which morphologically and by size can be significantly differentiated from C. obsoletus, it was seen that this species is significantly situated on another rame of the dendrograms and in very close relationship to C. punctatus and C. lupicaris, so that the latter might also be only races of C. pulicaris. One of the two other most common species found in Northrhine-Westfalia-C. festivipennis-belongs to the rame of the dendrogram, where C. pulicaris is situated close to C. circumscriptus, while the other common species (C. nubeculosus) has its place close to C. puncticollis and C. variipennis on the rame, where C. obsoletus is found. Thus, this paper again clearly points out that the question "what is a definite species" is far from being solved, if the life cycle is not defined and morphology misleading. However, it also became clear that C. obsoletus and C. pulicaris are Europe-wide occurring species and that several other clearly described separate species are probably only races.

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