Culicoides biting midges are vectors of internationally important arboviruses including bluetongue virus (BTV). The ecological constraints imposed by the small body size of these insects strongly influence the epidemiology of the diseases they can carry. Bluetongue virus recently emerged in northern Europe, and atmospheric dispersion models have subsequently been employed to simulate vector movement (and hence likely spread of BTV). The data underlying such models, however, have hitherto either been obtained from small-scale studies or from outside the north-western Palaearctic. The effects of seasonality and local meteorological conditions upon the daily presence and abundance of Culicoides vectors were examined using 2760 samples collected across a network of 12 different habitat types in England during 2008. Over 50 000 individuals were estimated to be in the samples with males constituting 62% of the total collection, allowing straightforward comparison between potential vector species in terms of their activity rates and seasonality. Culicoides abundance was linked to livestock density and land use. Farm-associated Culicoides species were recorded at all sites including species thought to be restricted to this ecosystem by larval habitat, suggesting a greater potential for dispersal over land than previously thought. Synthesis and applications. The model developed has already been applied in a functional dispersion model to predict disease risk from wind-borne infected Culicoides incursion into the UK and elsewhere. The study has expounded the long-distance dispersal potential of Culicoides, essential for future prediction of the incursion and spread of Culicoides-borne pathogens. It has additionally contributed to the understanding of the ecology of highly dispersive insect vectors.

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.