Insect bite hypersensitivity (IBH) is an
IgE-mediated (
Type I) hypersensitivity reaction induced by
allergens from biting midges of the Culicoides spp. The aim of the present study was to identify, clone and express recombinant
allergens from C. obsoletus, the main species found feeding on horses in the Netherlands, by sequence homology searches on the C. obsoletus specific RNA database, with previously described
allergens from C. nubeculosus and C. sonorensis. BLAST searches with these described
allergens resulted in similarity hits with 7 genes coding for C. obsoletus
allergens. These
allergens were expressed as
hexahistidine tagged
recombinant proteins in E. coli.
Allergens were termed Cul o 1-Cul o 7. A
maltase (Cul o 1) plus Cul s 1 (
maltase of C. sonorensis) were additionally expressed in insect cells using the baculovirus expression system to compare homologous
allergens from different species produced with different expression systems in diagnostic in vitro and in vivo tests. We demonstrate that IBH affected horses in the Netherlands show higher
IgE levels to Cul o 1 than to Cul s 1, as determined by an
IgE ELISA. Furthermore, we show that Cul o 1 produced in E. coli is at least as suitable for in vitro diagnosis of IBH affected horses as Cul o 1 produced in the baculovirus/insect cell expression system. The resulting
proteins were evaluated for their ability to discriminate IBH affected and healthy horses by ELISA and intradermal testing. The frequency of positive test results by ELISA within IBH affected horses ranged from 38% to 67% for the different
allergens. When results of
IgE-binding to Cul o 1-Cul o 7 were combined the test had a sensitivity of 92% and specificity of 85%. The capability of the
allergens to induce
Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test. The results show that E. coli expressed recombinant
allergens from C. obsoletus are valuable tools to determine the
allergen specific sensitisation profile (component resolved diagnosis) in horses with IBH in countries were C. obsoletus is the most abundant species and may facilitate in the development of future
immunotherapy.