PCR identification of Culicoides dewulfi midges (Diptera: Ceratopogonidae), potential vectors of bluetongue in Germany.

Abstract

Due to the severe outbreaks of bluetongue disease (BTD) in the years 2006/2007 in Germany in the absence of the main African vector Culicoides imicola, a rapid and easy applicable method for identification of autochthonous Culicoides spp. had to be developed. Morphological identification is time-consuming, rendering impossible the identification of large numbers of midges in a short period of time. A polymerase chain reaction (PCR)-based procedure in connection with a species-specific primer greatly simplifies the identification process. The region of internal transcribed spacer 1 (ITS-1) of the ribosomal DNA has shown great potential for developing a reliable PCR-based procedure. Culicoides midges were caught with ultraviolet-light traps installed on different farms in Germany during 2007 and 2008. The midges were mounted on slides and morphologically characterised. Midge DNA was extracted and the ITS-1 region amplified using conservative primers. Potential primer regions within ITS-1 were determined and a species-specific Culicoides dewulfi primer was developed to correctly identify autochthonous C. dewulfi, one of the suspected BTV vectors in northwestern Europe. The developed primer was used to identify C. dewulfi in a pool of Culicoides midges from a farm in the state of Brandenburg.