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1.
We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.  相似文献   

2.
Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.  相似文献   

3.
In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy horses by i.d. injection of the purified serum IgE antibodies. The following day, Culicoides allergen was injected at the same sites. Skin reactions were not observed in response to allergen alone, and in two horses after stimulation at any previous IgE injection site. However, the other two horses showed an immediate skin reaction at the previous injection sites of IgE obtained from allergic horses. In addition, purified monoclonal antibodies to various equine immunoglobulin isotypes were injected i.d. into six healthy horses. Immediate skin reactions were observed in response to anti-IgE (6/6 horses) and anti-IgG(T) injections (5/6 horses). The specificities of both antibodies for IgE and IgG(T), respectively, were confirmed by enzyme linked immunosorbent assays. The results provide the first direct evidence that IgE mediates classical Type-I allergy in horses and plays a major role in the pathogenesis of summer eczema. The data also suggest that IgG(T) can bind to skin mast cells and might contribute to clinical allergy.  相似文献   

4.
Equine disease with an allergic etiology is common. Environmental antigens most often implicated as allergens in horses include molds, dusty hay, grass pollen, hay dust mites, and insect saliva. Although intradermal testing with allergen is a useful diagnostic tool for some species, skin testing frequently produces false positive results in horses. Allergen deprivation as a diagnostic tool is often impossible and at best it is ineffective at diagnosing the specific allergic reactivity. Synthesis of IgE after exposure to allergen is the instigator of the allergic process. While IgE exerts its effect after binding strongly to mast cell Fc receptors, the presence of free IgE in the serum can be used to quantify and determine the allergen specificity of the allergic disease. A lack of widely available reagents for detection of equine IgE has limited this approach in horses. We have used the nucleotide sequence of equine IgE to prepare a peptide-based immunogen to elicit equine epsilon chain-specific antisera. Selection of peptides was based on antigenic attributes of the deduced amino acid sequence of the equine epsilon chain. Six peptides were selected for conjugation to carrier molecules and rabbit immunization. Of these, one peptide elicited antisera that was successfully used in enzyme linked immunosorbant assay (ELISA) to screen horse serum from 64 allergic horses for allergen-specific IgE. Twenty-four of the 64 horses showed positive reactivity to one or more of the following allergens: grass, grain mill dust, mosquito, and horsefly. This study demonstrates the usefulness of peptide-based immunogens for development of antisera to rare or difficult to purify antigens such as IgE. Resultant antisera has great usefulness in diagnostic assays for equine allergy and as a research tool.  相似文献   

5.
The objective of this study was to compare and analyze three common diagnostic methods for summer eczema (SE) in horses, an allergic dermatitis caused by bites of Culicoides spp. Nine horses with a medical history of SE and nine control animals were intradermally challenged with whole body extracts (WBE) and the saliva of a native (C. nubeculosus) and exotic (C. sonorensis) Culicoides species. Blood and serum samples of the horses were examined for basophil reactivity by a histamine release test (HRT) and for Culicoides-specific serum immunoglobulin E (IgE) and G (IgG) by enzyme-linked immunosorbent assay (ELISA). The results of intradermal testing (IDT) at 30min (immediate reactivity) and 4h (late-phase reactivity) post challenge with most insect preparations revealed significant differences between horses with and without SE. Overall, the HRT showed the most accurate results with a sensitivity of 1.00 for all Culicoides preparations and specificities of 0.78 (WBE) and 1.00 (saliva). By contrast, delayed reactions of the IDT (24h), and levels of Culicoides-specific IgE and IgG in the native serum showed little or no distinction between allergic and non-allergic horses. However, the use of purified serum IgE and IgG indicated the possibility for elevated titers of insect-specific serum immunoglobulins in horses with SE. The IDT and HRT did not reveal obvious differences in onset and intensity of positive reactions for the native verses exotic Culicoides species, whereas the ELISA showed slightly higher numbers of positive reactions for serum IgG with the indigenous species. Saliva, as compared to WBE, was found to have improved sensitivity and/or specificity for the HRT and for the late-phase immune reactions as measured by the IDT. Overall, the results indicate that allergy tests utilizing effector cells (mast cells, basophils) are more accurate in diagnosing SE in horses than serological analysis by ELISA.  相似文献   

6.
In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.  相似文献   

7.
Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE receptors to the surfaces of mast cells and basophils induces the release of inflammatory mediators. One of these mediators is histamine. The histamine was then detected by a colorimetric enzyme-linked immunosorbent assay. The histamine assay was used to test 33 horses with skin hypersensitivity and 20 clinically healthy control animals for histamine release from their peripheral blood basophils after stimulation with Culicoides allergen extract or monoclonal anti-IgE antibody. An increased histamine release was observed in the horses with skin hypersensitivity compared to the control group after allergen-specific stimulation with Culicoides extract (p=0.023). In contrast, stimulation with anti-IgE induced similar amounts of released histamine in both groups (p=0.46). For further evaluation of the HRA, we prepared a receiver operating-characteristic (ROC) curve and performed a likelihood-ratio analysis for assay interpretation. Our results suggested that the assay is a valuable diagnostic tool to identify sensitization to Culicoides allergens in horses. Because some of the clinically healthy horses also showed sensitization to Culicoides extract, the assay cannot be used to distinguish allergic from non-allergic animals. The observation that sensitization is sometimes detectable in non-affected animals suggested that clinically healthy horses use immune mechanisms to control the reaction to Culicoides allergens that are different or absent in allergic horses.  相似文献   

8.
Insect bite hypersensitivity (IBH), an allergic reaction to the saliva of Culicoides, occurs in all horse breeds and is a severe problem. In this study, we aimed at exploring whether exterior conditions, such as duration of allergenic exposure during the first summer and age of the horses at the time of import, influence the development of IBH. Additionally, data were analyzed regarding the period IBH started after birth and after import. Datasets for 582 horses were collected. The horses were locally born Icelandic horses and horses of other breeds with IBH, as well as imported Icelandic horses, both affected and nonaffected. For locally born horses, time of birth and duration of allergen exposure during the first summer had no influence on the prevalence of IBH. The disease started mostly in their third year of life. The majority of the imported horses affected caught IBH during their first year in Central Europe. Older imported horses seemed to develop IBH quicker than younger animals. Animals imported in their first winter are exposed to the allergen when they are at least 7 months old. They experienced approximately the same low risk of developing IBH as locally bred horses. The risk of IBH increased with the horses’ age at import. From our data, we conclude that the period to develop successful immune tolerance goes beyond the perinatal phase and is longer than formerly supposed.  相似文献   

9.
The high-affinity receptor for IgE (FcepsilonRI) plays a central role in IgE-mediated allergic reactions. Cross-linking of FcepsilonRI by IgE-antigen complexes results in the activation of mast cells and basophils and is thought to contribute to the immunopathology of Heaves, a chronic obstructive pulmonary disease of horses. Recombinant protein corresponding to the extra-cellular portion of the FcepsilonRI alpha subunit, cloned and sequenced previously, was expressed using both mammalian cells and insect cells. The yield of expressed protein was considerably greater using insect cells and the baculovirus expression system. The recombinant proteins differed in size between the two systems, presumably due to differences in the extent of glycosylation. However, recombinant protein from both cell systems bound equine IgE present in bronchoalveolar lavage fluid from horses with Heaves. These results suggest that the recombinant extra-cellular part of FcepsilonRI should be a useful tool with which to study equine IgE responses.  相似文献   

10.
The aims of this study were to determine (1) which species of Culicoides is most commonly attracted to horses, (2) whether horses suffering insect hypersensitivity attract more Culicoides spp. than unaffected horses, and (3) the times when Culicoides spp. are most active. Horses affected by insect hypersensitivity and unaffected horses were placed inside mosquito netting tents for 30 min at different times of the day. All Culicoides spp. trapped inside the tents were collected and identified. C. obsoletus was the most common species found, followed by C. pulicaris. Healthy horses attracted slightly more midges than horses that were affected with insect hypersensitivity. All of the Culicoides species were most active at sunset, less so at sunrise and very few or no midges were trapped in the afternoon or at night.  相似文献   

11.
Due to characteristic clinical signs, immunoglobulins of isotype E (IgE) are believed to be involved in several allergic diseases of the horse. To date, closer investigations have been hampered by the fact that neither purified equine IgE nor anti-equine IgE monoclonal antibodies were available for IgE isotype determination. As an approach to solve this problem, we constructed a stable cell line (EqE6) that expresses recombinant equi-murine IgE specific for 4-(hydroxy-3-nitro-phenyl) acetyl (NP). Biochemical analysis of the purified protein revealed a highly glycosilated IgE monomer of approximately 230,000 Da. The biological ability of the NP-IgE to mediate histamine release after crosslinking with antigen was demonstrated in vitro using equine blood leucocytes. In vivo, the intradermal application of NP-IgE followed by antigen crosslinking induced a type I hypersensitivity skin reaction in horses. Both results indicate that the recombinant NP-IgE contains an intact and functional Fc(epsilon) RI binding site and mediates effector functions in equine basophils and cutaneous mast cells. This equi-murine IgE can be used for the production of IgE-specific polyclonal and monoclonal antibodies. In addition, the NP specificity allows the antigen-specific activation of equine Fc(epsilon)-receptor-expressing cells, such as mast cells and basophils. This property could be used to investigate IgE-mediated mechanisms for a better understanding of equine type I allergic diseases.  相似文献   

12.
Sulfidoleukotrienes (sLT) generated in vitro after incubation of equine peripheral blood leukocytes (PBL) with different inducing agents were determined in 18 healthy and 16 insect bite dermal hypersensitivity (IDH)-affected horses. PBL from these 32 horses were stimulated with Concanavalin A, Parascaris equorum, Culicoides nubeculosus and Simulium extracts, and with a six-Grass mix. The cells of all but four horses generated sLT after incubation with Concanavalin A; these four horses did also not produce sLT with the other inducing agents. Of the 28 remaining horses (12 affected with IDH and 16 healthy), all but three generated sLT with the P. equorum extract. The six-Grass mix did not induce sLT production in any of the tested horses. sLT generation with Concanavalin A and Parascaris was statistically not different between IDH-affected and healthy horses. PBL of the diseased horses, however, produced significantly more sLT with the Culicoides (p < 0.01) and Simulium (p < 0.05) extracts than those of the healthy animals. Additionally, sLT generation with the Culicoides extract was measured at different times of the year in one IDH-affected animal and remained high even in winter, when the horse was asymptomatic. sLT and histamine release were determined in 10 horses in parallel. Positive correlations of 0.81 and 0.82 for Concanavalin A and Parascaris (p < 0.01 and p < 0.05, respectively), and of 0.95 and 0.94 for Culicoides and Simulium (p < 0.01) were found between sLT and histamine release. These results indicate that, alike in humans, sLT are released in vitro from equine basophils along with histamine in response to various stimuli and that immediate type hypersensitivity reactions to Culicoides and Simulium are often involved in the pathogenesis of IDH. Thus, sLT generation from equine basophils offers an in vitro diagnostic tool for IDH even in sensitised but asymptomatic horses.  相似文献   

13.
The pathogenesis of equine urticaria is not well understood. In man, urticaria has been associated with immunological and nonimmunological mechanisms leading to the release of various mediators by mast cells. Skin biopsies of 32 horses with a history of urticaria were stained with toluidine blue, a double-labelling method for chymase and tryptase, and immunohistochemistry for immunoglobulin (Ig)E. These horses were compared with horses with pemphigus foliaceus, insect bite hypersensitivity and control horses with healthy skin. Neither formalin fixation time nor biopsy site influenced the staining methods. No chymase-positive cells were found. In all groups of horses, cells staining with toluidine blue and for tryptase and IgE were found in the epidermis and hair follicle papilla and significantly more positively staining cells were observed in the subepidermal dermis compared with the deep dermis. Horses with urticaria had significantly more IgE-bearing cells in the subepidermal dermis than control horses. However, horses with urticaria had significantly fewer toluidine-blue-stained mast cells in both subepidermal and deep dermis compared with the insect bite hypersensitivity and pemphigus foliaceus groups. This study suggests that IgE-mediated reactions play a role in the pathogenesis of urticaria.  相似文献   

14.
Insect bite hypersensitivity (IBH) and atopy can both be causes of pruritus in horses and are associated with allergen-specific IgE to biting insects and environmental allergens respectively. Information with respect to differences in IgE levels in diseased and healthy animals is crucial in enabling an understanding of the clinical relevance of results of allergen-specific IgE tests. The aim of this study was (i) to evaluate and compare levels of allergen-specific IgE, using an ELISA method, in Icelandic horses, with and without IBH, from Iceland and Sweden respectively; (ii) to investigate patterns of allergen-specific IgE to insects, pollens, moulds and mites in those groups of horses; and (iii) to investigate the clinical significance of employing two different cut-off levels for the ELISA. The study compromised a total number of 99 horses from Iceland and Sweden, with and without IBH, divided in 5 groups. Sera from the horses were analysed blindly with the use of Allercept , a non-competitive, solid-phase ELISA-test, designed to detect the presence of allergen-specific IgE in sera using the recombinant alpha chain of the high-affinity IgE receptor (FcepsilonR1alpha). The distribution of the ELISA values was shown for each insect, mould, mite and pollen allergen, in the different groups using 10th, 50th and 90th percentiles. The use of two cut-off levels, 150 EA and 300 EA, did not eliminate the false positives. Horses with IBH had a higher number of positive reactions, counting all the 29 allergens, than healthy controls and this was borderline significant (P=0.053). In this study it was shown that serological testing with an ELISA that uses the high-affinity IgE receptor (FcepsilonR1alpha) is presently not suitable as a tool for establishing a diagnosis of IBH or equine atopy. The importance of establishing a correct cut-off level for the ELISA for the different allergens is emphasised.  相似文献   

15.
Using an ELISA test with specific equine antisera, resting and post-exercise serumIgE concentrations were determined in 20 horses exhibiting varying degrees of exercise- induced pulmonary hemorrhage (EIPH), and in 10 horses identified endoscopically as non-bleeders.

Significantly higher serum IgE concentrations (P<0.01) were present in horses which bled profusely than in those which bled mildly or not at all. Reductions in serum IgE concentrations were evident following exercise in most horses and, even though these reductions were not statistically significant, this suggested the possibility that circulating IgE had become bound to basophils or to bronchial mast cells as a result of the exercise.  相似文献   


16.
Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.  相似文献   

17.
Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a > or =50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.  相似文献   

18.
Insect bite hypersensitivity (IBH) in horses is most likely caused by Culicoides species, although other insects may also play a role. Until now no effective cure has been found for this condition, although numerous therapeutic and preventive measures have been used to control insect hypersensitivity. One such method is to apply a topical insecticide to horses. In this study, the effect of a topical insecticide containing permethrin (3.6%) was examined in seven pairs of horses. The horses were placed inside a tent trap to collect Culicoides spp. and other insects attracted to the horses on two subsequent evenings. On the first evening, both horses were untreated. After the end of this session, one horse of each pair was treated with the pour-on insecticide; treated horses were kept separate from untreated horses. The next evening the pairs of horses were again placed inside the tent trap and insects were collected. Similar percentages of Culicoides were trapped as in earlier studies (C. obsoletus 95.34% and C. pulicaris 4.54%), with healthy horses attracting more Culicoides than horses affected by IBH. The number of Culicoides, the percentage of blood-fed Culicoides obsoletus, and the total number of insects attracted to horses 24 hours after treatment with permethrin were reduced but the reduction was not statistically significant. No negative side effects of permethrin administration were observed.  相似文献   

19.
Insect bite dermal hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. and sometimes Simulium spp. The aim of the investigation presented here was to identify allergens causing IBH. A cDNA library expressing recombinant Culicoides nubeculosus proteins was screened using affinity-purified serum from an IBH-affected horse. Screening of the library resulted in identification of one immunoreactive clone. The sequence of the cDNA insert was determined and revealed a 600 bp insert with an open reading frame coding for a 78 amino acid long protein, called rCul n 1. Analysis of the deduced amino acid sequence revealed an identity of 67-78% to the C-terminal part of the 318 amino acid long ribosomal P0 protein from other Diptera. Furthermore, the 38 C-terminal amino acids displayed an identity of 57% with the C-terminal part of the acidic ribosomal protein P2 from Aspergillus fumigatus. The cDNA insert was subcloned and expressed as a [His]6-tagged protein in Escherichia coli and purified using Ni2(+)-chelate affinity chromatography. The 10kDa recombinant Cul n 1 protein bound the affinity-purified antibody fraction used for screening the expression library. Determination of IgE and IgG levels against rCul n 1 by ELISA in sera from 19 IBH-affected and 18 Swiss control horses and in sera from eight control horses living in Iceland showed no significant differences between the three groups of horses (median IgE levels = 60, 49 and 44 relative ELISA units, respectively). rCul n 1 did not induce sulfidoleukotriene (sLT) release from peripheral blood leukocytes of IBH-affected horses (N = 5), although sLT release was induced with the Culicoides whole body extract.  相似文献   

20.
Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera were analyzed by ELISA for IgE antibodies to the same allergens. The kappa test of concordance was used for comparison of the results of both tests. Out of 61 allergen extracts, only two (Timothy and Quack) had kappa values greater than 0.60, suggesting a substantial agreement between skin testing and IgE ELISA. The statistical comparison of the remaining 59 allergens showed little or no concordance between the tests beyond chance. To identify parameters that may influence the sensitivity of the ELISA, the assay was modified to detect allergen-specific IgGb and IgG(T) in serum, and the protein content in all allergen extracts was determined by SDS-PAGE. The commercial allergen extracts revealed a high variation in detectable protein. High concentrations of allergen-specific IgG in horse serum were found to compete with IgE for binding to the plates. In conclusion, an ELISA using whole serum and crude allergen preparations provides limited diagnostic information in horses. The reliable diagnosis of allergens in equine skin hypersensitivity is essential to improve allergen-specific treatments, such as hyposensitization, or the development of allergy vaccines.  相似文献   

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