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Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity.
Abstract: 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.
Copyright © 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-10-15 PubMed ID: 21071100DOI: 10.1016/j.vetimm.2010.10.015Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Allergies
- Animal Science
- Animal Studies
- Biotechnology
- Cloning
- Culicoides
- Diagnosis
- Disease Diagnosis
- Disease Management
- Disease Treatment
- Equine Diseases
- Equine Health
- Equine Science
- Experimental Methods
- Immunoglobulin E
- Immunology
- Insect Bite Hypersensitivity
- Molecular biology
- Pathogenesis
- Veterinary Medicine
- Veterinary Research
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research article deals with the identification, production, and characterization of proteins found in the salivary glands of the Culicoides nubeculosus species that are associated with allergic reactions in horses. The ultimate aim is to form a foundation for the development of specific diagnoses of Insect Bite Hypersensitivity (IBH), and potentially, personalized immunotherapy for horses based on their individual sensitization patterns.
Study Objective and Methodology
- The primary objective of this research was to identify and characterize the allergenic proteins present in the salivary glands of the species Culicoides nubeculosus. These proteins have been suggested as primary allergens causing Insect Bite Hypersensitivity (IBH), an allergic skin condition in horses, which gets triggered due to insect bites.
- To achieve this, the researchers used phage surface display technology, a technique that allows the study of protein interactions. They created a cDNA library with the mRNA derived from the salivary glands of C. nubeculosus and displayed it on the M13 filamentous phage surface. They then enriched the clones that showed binding to serum IgE (Immunoglobulin E – an antibody associated with allergic reactions) in horses affected by IBH.
Results & Findings
- The researchers were able to isolate ten cDNA inserts encoding putative salivary gland allergens, which were then termed as Cul n 2 to Cul n 11.
- The cDNA sequences were amplified and subcloned into high-level expression vectors and expressed as fusion proteins in Escherichia coli. Preliminary ELISA tests confirmed their specific binding to serum IgE of affected horses.
- The researchers further isolated the complete open reading frames from BLAST analyses using RACE-PCR and subcloned them into expression vectors. The full-length proteins expressing in E. coli agreed with the molecular masses predicted from their sequences.
- Western blot analyses of all recombinant allergens revealed their ability to specifically bind to serum IgE of sensitized horses. Depending on the allergen, the frequency of sensitization ranged between 13% to 57%.
- Intradermal skin tests further verified the in vivo relevance of eight of the recombinant allergens. For allergens Cul n 6 and Cul n 11, sensitized horses were not available for testing.
- Control horses that did not exhibit clinical signs of IBH did not show any relevant immediate hypersensitivity reactions to the allergens.
Significance and Impact of the Study
- The results from the study contribute significantly by providing a collection of recombinant salivary gland allergens from C. nubeculosus that might be involved in the pathogenesis of IBH.
- These findings serve as a vital start towards the development of a method for the serologic diagnosis of IBH. It opens up the possibility of developing personalized immunotherapy for horses based on their individual sensitization patterns, thereby improving treatments for this condition.
Cite This Article
APA
Schaffartzik A, Marti E, Torsteinsdottir S, Mellor PS, Crameri R, Rhyner C.
(2010).
Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity.
Vet Immunol Immunopathol, 139(2-4), 200-209.
https://doi.org/10.1016/j.vetimm.2010.10.015 Publication
Researcher Affiliations
- Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Obere Strasse 22, CH-7270 Davos, Switzerland.
MeSH Terms
- Allergens / immunology
- Allergens / metabolism
- Animals
- Base Sequence
- Ceratopogonidae
- Cloning, Molecular
- DNA / genetics
- Gene Library
- Horse Diseases / immunology
- Horses
- Hypersensitivity / immunology
- Hypersensitivity / veterinary
- Immunoglobulin E
- Insect Bites and Stings / immunology
- Insect Bites and Stings / veterinary
- Intradermal Tests / veterinary
- Mice
- Protein Binding
- Salivary Glands / metabolism
- Skin Tests / veterinary
Citations
This article has been cited 21 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).
- Simonin EM, Wagner B. IgE-binding monocytes upregulate the coagulation cascade in allergic horses. Genes Immun 2023 Jun;24(3):130-138.
- Simonin EM, Babasyan S, Tarsillo J, Wagner B. IgE+ plasmablasts predict the onset of clinical allergy. Front Immunol 2023;14:1104609.
- Wyler M, Sage SE, Marti E, White S, Gerber V. Protein microarray allergen profiling in bronchoalveolar lavage fluid and serum of horses with asthma. J Vet Intern Med 2023 Jan;37(1):328-337.
- Cvitas I, Oberhaensli S, Leeb T, Marti E. Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: Just another brick in the wall?. PLoS One 2022;17(8):e0266263.
- Birras J, White SJ, Jonsdottir S, Novotny EN, Ziegler A, Wilson AD, Frey R, Torsteinsdottir S, Alcocer M, Marti E. First clinical expression of equine insect bite hypersensitivity is associated with co-sensitization to multiple Culicoides allergens. PLoS One 2021;16(11):e0257819.
- 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.
- Novotny EN, White SJ, Wilson AD, Stefánsdóttir SB, Tijhaar E, Jonsdóttir S, Frey R, Reiche D, Rose H, Rhyner C, Schüpbach-Regula G, Torsteinsdóttir S, Alcocer M, Marti E. Component-resolved microarray analysis of IgE sensitization profiles to Culicoides recombinant allergens in horses with insect bite hypersensitivity. Allergy 2021 Apr;76(4):1147-1157.
- Larson EM, Babasyan S, Wagner B. Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity. PLoS One 2020;15(5):e0233537.
- François L, Hoskens H, Velie BD, Stinckens A, Tinel S, Lamberigts C, Peeters L, Savelkoul HFJ, Tijhaar E, Lindgren G, Janssens S, Ducro BJ, Buys N, Schurink AA. Genomic Regions Associated with IgE Levels against Culicoides spp. Antigens in Three Horse Breeds. Genes (Basel) 2019 Aug 8;10(8).
- Verdon M, Lanz S, Rhyner C, Gerber V, Marti E. Allergen-specific immunoglobulin E in sera of horses affected with insect bite hypersensitivity, severe equine asthma or both conditions. J Vet Intern Med 2019 Jan;33(1):266-274.
- Torsteinsdottir S, Scheidegger S, Baselgia S, Jonsdottir S, Svansson V, Björnsdottir S, Marti E. A prospective study on insect bite hypersensitivity in horses exported from Iceland into Switzerland. Acta Vet Scand 2018 Nov 3;60(1):69.
- Einhorn L, Hofstetter G, Brandt S, Hainisch EK, Fukuda I, Kusano K, Scheynius A, Mittermann I, Resch-Marat Y, Vrtala S, Valenta R, Marti E, Rhyner C, Crameri R, Satoh R, Teshima R, Tanaka A, Sato H, Matsuda H, Pali-Schöll I, Jensen-Jarolim E. Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer. Allergy 2018 Jul;73(7):1436-1446.
- Bordon KC, Wiezel GA, Amorim FG, Arantes EC. Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology. J Venom Anim Toxins Incl Trop Dis 2015;21:43.
- Rádrová J, Vlková M, Volfová V, Sumová P, Cêtre-Sossah C, Carpenter S, Darpel K, Rakotoarivony I, Allène X, Votýpka J, Volf P. Hyaluronidase Activity in Saliva of European Culicoides (Diptera: Ceratopogonidae). J Med Entomol 2016 Jan;53(1):212-6.
- Mueller RS, Janda J, Jensen-Jarolim E, Rhyner C, Marti E. Allergens in veterinary medicine. Allergy 2016 Jan;71(1):27-35.
- Kügler J, Zantow J, Meyer T, Hust M. Oligopeptide m13 phage display in pathogen research. Viruses 2013 Oct 16;5(10):2531-45.
- Simonin EM, Torsteinsdóttir S, Svansson V, Björnsdóttir S, Freer H, Tarsillo J, Wagner B. Early allergen introduction overrides allergy predisposition in offspring of horses with Culicoides hypersensitivity. Front Immunol 2025;16:1654693.
- Jebbawi F, Olomski F, Inversini V, Keller G, Rhiner T, Waldern N, Lam J, Pantelyushin S, Canonica F, Birkmann K, Johansen P, Kündig TM, Fettelschoss-Gabriel A. Anti-IL-5 Vaccination Dampens Allergen-Specific IgE Levels and Modulates IL-4 and IL-5 Th2 Cytokines in Skin Allergy of Mice and Horses. Allergy 2025 Dec;80(12):3377-3390.
- Jonsdottir S, Stefansdottir SB, Mirkovitch J, Ziegler A, Torsteinsdottir S, Marti E. Culicoides allergens expressed in insect cells induce sulphidoleukotriene release from peripheral blood leukocytes of horses affected with insect bite hypersensitivity. Front Immunol 2025;16:1597233.
- Yoshida S, Matsuda A, Iwata E, Ono T, Hisaeda K, Ohzawa E, Hiasa Y, Kitagawa H. Allergen-specific immunoglobulin-Es for dermatitis in the Japanese native Noma horses. J Vet Med Sci 2024 Sep 1;86(9):938-945.
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