Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses.
Abstract: 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.
Copyright © 2013 Elsevier B.V. All rights reserved.
Publication Date: 2013-03-19 PubMed ID: 23561552DOI: 10.1016/j.vetimm.2013.03.005Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article is focused on the development and testing of allergens from the insect Culicoides obsoletus in diagnosing hypersensitivity reactions in horses caused by insect bites. This was accomplished by identifying, cloning, and expressing these allergens and then evaluating their effectiveness in identifying affected and healthy horses.
Objective and Methodology
- The study aimed to identify and create recombinant allergens from C. obsoletus, a species that often feeds on horses in the Netherlands. This was achieved by comparing sequences in the C. obsoletus specific RNA database with previously known allergens from other Culicoides species, C. nubeculosus and C. sonorensis.
- The research team used a technique called Basic Local Alignment Search Tool (BLAST) to find similarities in the genes coding for allergens in C. obsoletus. Seven different genes were identified which corresponded to the allergens named Cul o 1 through Cul o 7.
- These identified allergens were then expressed as hexahistidine tagged recombinant proteins in E. coli.
- A specific allergen, Cul o 1, and a counterpart from C. sonorensis, Cul s 1, were expressed using a different method, the baculovirus expression system in insect cells, to evaluate differences between species and expression systems.
Results and Findings
- The study demonstrated that horses affected by Insect Bite Hypersensitivity (IBH) in the Netherlands had higher IgE levels to Cul o 1 than to the counterpart allergen Cul s 1. This was determined by an IgE ELISA test, a common procedure for detecting antibodies in the blood.
- Interestingly, the research showed the allergen Cul o 1, when generated in E. coli, was as good in diagnosing horses with IBH as the same allergen produced in the baculovirus/insect cell system.
- Further, the allergens were evaluated for their ability to differentiate between IBH affected and healthy horses using ELISA, and also tested for inducing Type I hypersensitivity reaction in horses by an intradermal test.
- The accuracy of testing ranged from 38% to 67% for the different allergens. However, when results were combined across all allergens (Cul o 1 to Cul o 7), the test achieved a sensitivity of 92% and specificity of 85%.
Conclusion and Implications
- The research concluded that the allergens expressed in E. coli from C. obsoletus could be valuable as diagnostic tools in determining allergen-specific sensitivity in horses with IBH.
- This research may contribute to a more efficient diagnosis of IBH and potentially aid in the development of new immunotherapy treatments for horses affected by this condition, particularly in areas where C. obsoletus is most abundant.
Cite This Article
APA
van der Meide NM, Roders N, Sloet van Oldruitenborgh-Oosterbaan MM, Schaap PJ, van Oers MM, Leibold W, Savelkoul HF, Tijhaar E.
(2013).
Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses.
Vet Immunol Immunopathol, 153(3-4), 227-239.
https://doi.org/10.1016/j.vetimm.2013.03.005 Publication
Researcher Affiliations
- Cell Biology and Immunology Group, Wageningen University, Wageningen, The Netherlands.
MeSH Terms
- Allergens / drug effects
- Allergens / genetics
- Animals
- Baculoviridae / genetics
- Ceratopogonidae / immunology
- Cloning, Molecular
- Escherichia coli / genetics
- Horse Diseases / diagnosis
- Horses
- Hypersensitivity / diagnosis
- Hypersensitivity / veterinary
- Immunoglobulin E / blood
- Insect Bites and Stings / diagnosis
- Insect Bites and Stings / immunology
- Insect Bites and Stings / veterinary
- Recombinant Proteins
Grant Funding
- BBS/E/I/00001701 / Biotechnology and Biological Sciences Research Council
Citations
This article has been cited 17 times.- Pessoa VC, Branco-Ferreira M, Jónsdóttir S, Marti E, Tilley P. Comparison of Skin Prick Tests (SPT), Intradermal Tests (IDT) and In Vitro Tests in the Characterization of Insect Bite Hypersensitivity (IBH) in a Population of Lusitano Horses: Contribution for Future Implementation of SPT in IBH Diagnosis. Animals (Basel) 2023 Aug 28;13(17).
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- Larson EM, Babasyan S, Wagner B. IgE-Binding Monocytes Have an Enhanced Ability to Produce IL-8 (CXCL8) in Animals with Naturally Occurring Allergy. J Immunol 2021 May 15;206(10):2312-2321.
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