Veterinary immunology and immunopathology2010; 139(2-4); 200-209; doi: 10.1016/j.vetimm.2010.10.015

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity.

Schaffartzik, A
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Marti, E
·
Torsteinsdottir, S
·
Mellor, PS
·
Crameri, R
·
Rhyner, C

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.

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Publication Date: 2010-10-15 PubMed ID: 21071100DOI: 10.1016/j.vetimm.2010.10.015Google Scholar: Lookup

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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.

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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