|Title||Culicoides obsoletus allergens for diagnosis of insect bite hypersensitivity in horses|
|Author(s)||Meide, N.M.A. van der|
|Source||University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - S.l. : s.n. - ISBN 9789461736697 - 228|
Cell Biology and Immunology
|Publication type||Dissertation, internally prepared|
|Keyword(s)||paarden - equus - culicoides obsoletus - insectenbeten - allergenen - overgevoeligheid - allergieën - cytokinen - diagnose - elisa - vectoren, ziekten - immunologie - horses - insect bites - allergens - hypersensitivity - allergies - cytokines - diagnosis - disease vectors - immunology|
|Categories||Equidae (Horses, Donkeys, Mules) / Immunology|
AInsect Bite Hypersensitivity (IBH) is the most common skin allergy in horses and involves a Type I (IgE mediated) hypersensitivity reaction against bites of insects, mainly of the Culicoides species. Welfare of affected horses is seriously reduced and no fully curative treatment is yet available. Furthermore, current diagnostic tests are unreliable because of their low sensitivity and specificity. Aim of our research was to increase the understanding of immunological aspects of IBH, with special attention to improving diagnosis by the characterization and production of recombinant allergens.
Whole body extracts (WBE) of three Culicoides species: C. obsoletus C. nubeculosus and C. sonorensis were evaluated for their applicability for diagnosis of IBH in horses in The Netherlands. They were tested for IgE binding by ELISA and Western blotting and for their capacity to degranulate basophils in a histamine release test. For all tests, best results were obtained with C. obsoletus. The ELISA was further evaluated using C. obsoletus extract on approximately 200 IBH affected and healthy horses, which demonstrated high test sensitivity and specificity. C. obsoletus-specific IgE serum levels were found to be the same in the IBH season and off season, suggesting that the test can be used to diagnose horses in winter when clincial symptoms are absent.
Since C. obsoletus was found to be the most important species for diagnosis of IBH in The Netherlands, mRNA of this Culicoides species was sequenced and assembled to create a transcriptome. Using the sequences from in literature described allergens from C. nubeculosus and C. sonorensis, similarity searches were performed on this transcriptome,. This resulted in the identification of seven allergens from C. obsoletus. These allergens were cloned and expressed as recombinant proteins in E. coli and named Cul o 1 – Cul o 7. The frequency of positive test results by ELISA within IBH affected horses ranged from 38 % to 67 %. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test.
The applicability of the 7 C. obsoletus derived recombinant allergens was further evaluated and compared with C. obsoletus WBE in an IgE ELISA using a large number of horses.The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISA’s with a combination of recombinant allergens, combi-1 (Cul o 3, 5 and 7) and combi-2 (Cul o 1, 2, 5 and 7) were additionally performed and both resulted in high test accuracies close to that obtained with WBE. Both combi-1 and combi-2 resulted in a lower test sensitivity with samples collected in winter compared to samples collected in IBH season, but most IBH affected horses could still also be correctly diagnosed in winter.
The association between several factors and IgE levels against C. obsoletus whole body extract and the 7 recombinant allergens was quantified. Furthermore, the relation between IgE levels and severity of symptoms was examined. Severity of symptoms and IgE levels against several C. obsoletus allergens were found to be related. Factors that were found to be associated with IgE levels were: breed, age, month of scoring, interaction between IBH status and month of scoring, degree of itchiness and number of seasons horses were affected with IBH.
The general discussion discussed the prospects to use the produced recombinant allergens for immunotherapy treatment of IBH affected horses. The panel of all 7 recombinant allergens allows to determine for which exact components of C. obsoletus the IBH horses are allergic (“component resolved diagnosis”). This will enable a tailor made composition of (recombinant) allergens for use in immunotherapy.