Vet. Res. 35 (2004) 325-337
DOI: 10.1051/vetres:2004015

Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe

Catherine Cêtre-Sossah^a, Thierry Baldet^a, Jean-Claude Delécolle^b, Bruno Mathieu^c, Aurélie Perrin^a, Colette Grillet^a and Emmanuel Albina<sup>a

a</sup>  CIRAD-EMVT, Campus international de Baillarguet, TA30/G, 34398 Montpellier Cedex 5, France
^b  Université Louis Pasteur de Strasbourg, Musée zoologique, 29 bd de la Victoire, 67000 Strasbourg, France
^c  Entente Interdépartementale pour la Démoustication, 165 avenue Paul Rimbaud, 34184 Montpellier Cedex 4, France

(Received 12 November 2003; accepted 9 February 2004)

Abstract - Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap network for the collection of Culicoides, was implemented in 2002 in southern mainland France. The morphological identification of Culicoides can be both tedious and time-consuming because its size ranges from 1.5 to 3 mm. Therefore, an ITS₁ rDNA polymerase chain reaction (PCR)-based diagnostic assay was developed to rapidly and reliably identify Culicoides spp. and C. imicola. The aim of this work was to set up a rapid test for the detection of C. imicola amongst a pool of insects collected in areas at risk for BT. The sequence similarity of the rDNA (nuclear ribosomal DNA), which is greater within species than between species, is the foundation of its utilisation in species-diagnostic assays. The alignment of the 11 ITS1 sequences of Culicoides obtained from Genbank and EMBL databases helped us to identify one region in the 5' end and one in the 3' end that appear highly conserved. PCR primers were designed within these regions to amplify genus-specific fragments. In order to set up a C. imicola-specific PCR, another forward primer was designed and used in combination with the previously designed reverse primer. These primers proved to be highly specific and sensitive and permitted a rapid diagnostic separation of C. imicola from Culicoides spp.

Key words: molecular detection / Culicoides imicola / ITS1 / PCR / bluetongue

Corresponding author: Catherine Cêtre-Sossah catherine.cetre-sossah@cirad.fr

© INRA, EDP Sciences 2004