Insect bite hypersensitivity (IBH) is an
IgE-mediated seasonal
dermatitis of the horses associated with
bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the
allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous
allergen, present in the salivary glands of both insects, shows extended
IgE cross-reactivity in vitro and in vivo. The
cDNA sequences coding for both
antigen 5 like
allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [
His](6)-tagged
proteins in Escherichia coli. The highly pure
recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum
IgE levels to both
allergens in 24 unaffected horses did not show any value above background. Both
proteins strongly bound serum
IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the
recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum
IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the
allergens share common
IgE-binding
epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the
allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant
immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides
crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed
recombinant proteins. In conclusion our data demonstrate that
IgE-mediated cross-reactivity to homologous
allergens, a well-known clinically relevant phenomenon in human
allergy, also occurs in veterinary
allergy.