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Due to the severe outbreaks of bluetongue disease in the years 2006/2007 in Germany in the absence of the main African vector Culicoides imicola, the role of authochtonous midges as possible vectors of the bluetongue disease were discussed. Only few local studies regarding the midge fauna of Germany existed therefore an entomological monitoring project covering a great part of Germany was launched. At 89 dairy farms located within the restriction zone of the bluetongue epidemic of 2006 UV-light traps for catching target insects as well as weather stations for collecting climate data were installed. The catches were performed during March 2006 and May 2007 and were analysed regarding the numbers of caught midges, their sex ratio, their belonging to the C. obsoletus group, C. pulicaris group or other, as well as their status of blood engorgement.
Furthermore, a certain number of midges were analysed down to species level as well as tested for BTV-8 genome by cooperating institutes. The results of the monitoring project confirmed the abscence of the main vector C. imicola in the region of the epidemic of 2006/2007. Midges of the C. obsoletus group were caught most numerous, followed by midges of the C. pulicaris group. BTV-8 genome was mostly found in midges of the C. obsoletus group but also in some of the C. pulicaris group affirming their vector potential.
Because midges were still caught in the months of winter a “vector-free period” could not be defined.
In order to identify caught midges down to species level, a rapid and easy applicable method for identification of autochthonous Culicoides spp. had to be developed.
Since morphological differentiation is time-consuming and demands specialised knowledge of preparation and identification, a polymerase chain reaction (PCR)-based procedure in connection with species-specific primers provided a solution. The region of internal transcribed spacer 1 (ITS1) of the ribosomal DNA was chosen as target sequence for the development of speciesspecific primers. The DNA used for the development of primers originated from Culicoides obsoletus s.s., C. dewulfi, C. scoticus, C. chiopterus, C. punctatus and C. pulicaris midges captured during the entomological monitoring of March/April 2007 – May 2008. In order to determine a gold standard body parts of the chosen midges (wings, head, abdomen with genitals) were mounted on slides for morphological identification. The DNA was extracted from the remaining parts (thorax with legs and upper abdomen) and the ITS1-region amplified using the conservative primers PanCulF/PanCulR (Cêtre-Sossah et al., 2004). Potential forward-primer regions were determined with the help of DNA-software; PanCulR was kept as reverse primer.
The qualification of the potential primers was tested using DNAsoftware and the synthezisation of the primers CulObsF2, CulDewF, CulChioF, CulPunctF and CulPulF was carried out by a spezialised laboratory. For each primer an optimized PCR protocol was developed by determining the ideal MgCl2 concentration and annealing temperatur; for some primers a touchdown method was applied. Using different procedures the primers’s analytical specificity and sensitivity was defined. The deployment of the primers was tested through different methods. Suggestions of improvement for subsequent entomological investigations were mentioned and the utilization of species-specific primers in future studies discussed.