Culicoides obsoletus allergens for diagnosis of insect bite hypersensitivity in horses

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.</p

Factors associated with Culicoides Obsoletus complex spp.-specific IgE reactivity in Icelandic horses and Shetland ponies

Insect bite hypersensitivity (IBH) is a common allergic skin disease in horses, caused by biting insects of the Culicoides spp. In The Netherlands, Culicoides spp. of the Obsoletus complex are the most important midges involved in IBH. The aim of the present study was to identify and quantify associations between several endogenous (host) and exogenous (environmental) factors and immunoglobulin E (IgE) reactivity against Obsoletus complex-derived whole body extract or seven recombinant allergens, measured by ELISA. Data from 143 Icelandic horses and 177 Shetland ponies were analysed using multivariable models. In addition, the relationship between IgE reactivity and severity of clinical signs in IBH-affected horses was examined. Positive correlations were found between Obsoletus complex-specific IgE and severity of clinical signs. Disease status (IBH affected or control), breed and the interaction between IBH status and breed were significantly associated with IgE reactivity against several Obsoletus complex allergens. Significantly greater IgE reactivity was seen in IBH-affected horses compared to controls. The differences in IgE values between cases and controls were most pronounced in Icelandic horses. Shetland pony controls had significantly greater IgE reactivity compared to Icelandic horse controls, while differences in IgE values comparing Shetland pony cases and Icelandic horse cases were not significant. Severity of clinical signs and IgE reactivity in IBH-affected horses against several Obsoletus complex allergens appeared to be related. Consideration of the factors associated with Obsoletus complex-specific IgE in horses might further improve interpretation and accuracy of IgE ELISA test results within these breeds, although further research is required

Evaluation of a diagnostic ELISA for insect bite hypersensitivity in horses using recombinant Obsoletus complex allergens

Culicoides spp. of the Obsoletus complex belong to the most important species of midge, involved in causing insect bite hypersensitivity (IBH) in horses in The Netherlands. The aim of the current study was to evaluate seven different Obsoletus complex-derived recombinant allergens (Cul o 1–Cul o 7) and to compare these with Obsoletus complex whole body extract (WBE) in an IgE ELISA, using sera of 194 clinically-confirmed cases of IBH and 175 unaffected horses. The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISAs 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. Combi-1 resulted in the best sensitivity and specificity, both 89%. Both Combi-1 and Combi-2 performed less well with samples collected in winter, but over 70% of the IBH-affected horses could still be identified. In conclusion, a combination of three Obsoletus complex recombinant allergens (Cul o 3, 5 and 7) could potentially replace Obsoletus complex WBE in an IgE ELISA for diagnosis of IBH in horses

Staart- en maneneczeem steeds verder ontrafeld!

Staart- en maneneczeem is een veel voorkomende allergische aandoening bij paarden en pony’s en veroorzaakt veel leed. De afgelopen jaren hebben onderzoekers van Wageningen University en Universiteit Utrecht met steun van technologiestichting STW, Vereniging Koepel Fokkerij en Artu Biologicals onderzoek gedaan naar meerdere aspecten van deze aandoening. De belangrijkste resultaten van het onderzoek worden toegelicht in dit artikel

Seasonal differences in cytokine expression in the skin of Shetland ponies suffering from insect bite hypersensitivity

Insect bite hypersensitivity (IBH) in horses is a seasonal, IgE-mediated, pruritic skin disorder primarily caused by Culicoides spp. We hypothesize that a mixed Th2/Th1-type immune status, off season, alters into Th2-dominated immune reactivity in the skin of IBH-affected ponies in the IBM season. To study these immune response patterns Culicoides-specific IgE levels, skin histopathology and cytokine and transcription factor mRNA expression (IL4, IL10, IL13, IFN gamma, FoxP3 and CD3(zeta)) in lesional and non-lesional skin of ponies affected by IBH in the IBH season were compared with those of the same animals off season and those in skin of healthy ponies in both seasons. The present study revealed a significantly higher histopathology score in lesional skin of affected ponies than in non-lesional skin and skin of healthy ponies in the IBH season. Culicoides obsoletus-specific IgE serum levels of ponies with IBH were significantly higher than those in healthy ponies in both seasons. Interestingly, C. obsoletus-specific IgE serum levels within each group were the same in the IBH season and off season. The expression of IL4, IL13 and IFN gamma mRNA in skin biopsies in the IBH season showed a significant increase compared to off season in both skin derived from healthy control ponies (n = 14) as well as in lesional and in non-lesional skin from IBH-affected animals (n = 17). This apparently general up-regulation of cytokine expression during the IBH season directly correlated with an increased CD3(zeta) mRNA expression in the skin, indicating an overall increased T cell influx during the summer months. The only significant difference observed between lesional skin from IBH-affected animals as compared to skin from healthy control animals in the IBH season was a lower expression of IL13/CD3(zeta) in the affected animals. FoxP3 and IL10 levels were unaffected, except for a lower expression of FoxP3 in healthy control skin in the IBM season as compared to off season, In addition, the increased level of C obsoletus-specific IgE did not correlate with higher histological scores in LE skin. In summary, our data indicate a general immune activation in the skin of both healthy and IBH-affected ponies during the IBH season that potentially obscures the Culicoides-specific immune reaction pattern, even in lesional skin of IBH-affected animals. (C) 2012 Elsevier B.V. All rights reserved

Seasonal differences in cytokine expression in the skin of Shetland ponies suffering from insect bite hypersensitivity

Insect bite hypersensitivity (IBH) in horses is a seasonal, IgE-mediated, pruritic skin disorder primarily caused by Culicoides spp. We hypothesize that a mixed Th2/Th1-type immune status, off season, alters into Th2-dominated immune reactivity in the skin of IBH-affected ponies in the IBM season. To study these immune response patterns Culicoides-specific IgE levels, skin histopathology and cytokine and transcription factor mRNA expression (IL4, IL10, IL13, IFN gamma, FoxP3 and CD3(zeta)) in lesional and non-lesional skin of ponies affected by IBH in the IBH season were compared with those of the same animals off season and those in skin of healthy ponies in both seasons. The present study revealed a significantly higher histopathology score in lesional skin of affected ponies than in non-lesional skin and skin of healthy ponies in the IBH season. Culicoides obsoletus-specific IgE serum levels of ponies with IBH were significantly higher than those in healthy ponies in both seasons. Interestingly, C. obsoletus-specific IgE serum levels within each group were the same in the IBH season and off season. The expression of IL4, IL13 and IFN gamma mRNA in skin biopsies in the IBH season showed a significant increase compared to off season in both skin derived from healthy control ponies (n = 14) as well as in lesional and in non-lesional skin from IBH-affected animals (n = 17). This apparently general up-regulation of cytokine expression during the IBH season directly correlated with an increased CD3(zeta) mRNA expression in the skin, indicating an overall increased T cell influx during the summer months. The only significant difference observed between lesional skin from IBH-affected animals as compared to skin from healthy control animals in the IBH season was a lower expression of IL13/CD3(zeta) in the affected animals. FoxP3 and IL10 levels were unaffected, except for a lower expression of FoxP3 in healthy control skin in the IBM season as compared to off season, In addition, the increased level of C obsoletus-specific IgE did not correlate with higher histological scores in LE skin. In summary, our data indicate a general immune activation in the skin of both healthy and IBH-affected ponies during the IBH season that potentially obscures the Culicoides-specific immune reaction pattern, even in lesional skin of IBH-affected animals. (C) 2012 Elsevier B.V. All rights reserved

Effects of Polysaccharides from Different Species of Dendrobium (Shihu) on Macrophage Function

Dendrobium spp. are precious medicinal plants, used in China for thousands of years as health foods and nutrients. Polysaccharides are the main effective ingredients in Dendrobium plants. In this study, the chemical characteristics and the effects of crude polysaccharides (CPs) from five species of Dendrobium on macrophage function were investigated and compared in vitro for the first time. Chemical characteristic studies showed that CPs from different species of Dendrobium were diverse, displaying widely varied Mw distributions and molar ratios of monosaccharides. Their effects on macrophage functions, such as promoting phagocytosis, release of NO and cytokines IL-1α, IL-6, IL-10 and TNF-α, were also different. Moreover, CPs from D. officinale, especially collected from Yunnan Province, exerted the strongest immunomodulatory activities and could be explored as a novel potential functional food. The diverse chemical characteristics of CPs from different species of Dendrobium might contribute to their varied effects on macrophage functions, which should be further investigated

Culicoides obsoletus extract relevant for diagnostics of insect bite hypersensitivity in horses

Insect bite hypersensitivity (IBH) is an allergic dermatitis in horses caused by the bites of Culicoides species. The aim of the present study was to evaluate the applicability of whole body extracts of C. obsoletus (the main species found feeding on horses in the Netherlands), C. nubeculosus (rarely found in The Netherlands) and C. sonorensis (typical for North America) for diagnosis of IBH in horses in The Netherlands. Blood and serum samples of 10 clinically confirmed IBH affected and 10 healthy control horses were used to evaluate the IgE titers (ELISA) against the Culicoides whole body extracts of the three Culicoides species. Basophil degranulation was assessed by histamine release test (HRT) after stimulation with these extracts at 5, 0.5 and 0.05 µg/ml. IBH affected horses had significantly higher IgE titers against C. obsoletus than against C. nubeculosus and C. sonorensis. Furthermore, C. obsoletus induced significantly higher histamine release in whole blood of IBH affected horses compared to the other extracts at 0.5 µg/ml. Western blot data revealed IgE binding to many proteins in C. obsoletus extract. This interaction was absent or weak in C. nubeculosus and C. sonorensis extracts for IBH affected horses. Results on individual level indicate that the HRT is more sensitive than ELISA in diagnosing IBH. However, ELISA is more practical as a routine test, therefore the ELISA was further evaluated using C. obsoletus extract on 103 IBH affected and 100 healthy horses, which resulted in a test sensitivity and specificity of 93.2% and 90.0%, respectively. The IgE ELISA readings enabled the analysis of the predicted probability of being IBH affected. From an optical density 450 nm value of 0.33 onwards, the probability of IBH affected was more than 0.9. The results presented in this paper show that the use of native Culicoides spp. that feed on horse, is important for improved diagnosis and that the described ELISA based on C. obsoletus can be used routinely to diagnose IBH in countries where this species is the main Culicoides feeding on horse