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1.
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   

2.
REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.  相似文献   

3.
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.  相似文献   

4.
Insect bite hypersensitivity (IBH) is an allergic dermatitis of the horse caused by bites of insects of the genus Culicoides and is currently the best characterized allergic disease of horses. This article reviews knowledge of the immunopathogenesis of IBH, with a particular focus on the causative allergens. Whereas so far hardly any research has been done on the role of antigen presenting cells in the pathogenesis of IBH, recent studies suggest that IBH is characterized by an imbalance between a T helper 2 (Th2) and regulatory T cell (T(reg)) immune response, as shown both locally in the skin and with stimulated peripheral blood mononuclear cells. Various studies have shown IBH to be associated with IgE-mediated reactions against salivary antigens from Culicoides spp. However, until recently, the causative allergens had not been characterized at the molecular level. A major advance has now been made, as 11 Culicoides salivary gland proteins have been identified as relevant allergens for IBH. Currently, there is no satisfactory treatment of IBH. Characterization of the main allergens for IBH and understanding what mechanisms induce a healthy or allergic immune response towards these allergens may help to develop new treatment strategies, such as immunotherapy.  相似文献   

5.
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   

6.
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   

7.
Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.  相似文献   

8.
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   

9.
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10.
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   

11.
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   

12.
Rabbits exposed to feeding tsetse flies developed cutaneous hypersensitivity responses to fly bites. These responses had characteristics of immediate and delayed type hypersensitivity. Saliva components from the tsetse fly Glossina morsitans centralis were electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major salivary proteins of 160, 92, 66, 64, 55, 42, 33, 28, and 15 kilodaltons were identified. Separated salivary components were transferred to nitrocellulose filters and probed with lectins and with whole sera and purified IgG from rabbits which had been exposed, via fly feeding, to tsetse antigens for variable periods. Many of the salivary proteins were identified as glycoproteins. Several major salivary proteins were recognized by antibodies from sensitized rabbits.  相似文献   

13.
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   

14.
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15.
Insect bite hypersensitivity (IBH) is a seasonal allergic skin disease in horses caused by bites of certain Culicoides spp. The aim of our study was to investigate the maternal effect on IBH and to estimate the heritability and repeatability of IBH in the Dutch Friesian horse population. Data consisted of 3,453 Dutch Friesian broodmares with 3,763 visual observations on IBH clinical symptoms scored by 12 inspectors during organized foal inspections in 2004 and 2008. Nine percent of the mares (n = 310) were scored in both years. Mares descended from 144 sires and 2,554 dams and 26.2% of the dams (n = 669) had more than 1 offspring in the data set (range: 2 to 6). Insect bite hypersensitivity was analyzed as a binary trait with a threshold animal model with and without a maternal effect, using a Bayesian approach. Observed IBH prevalence in Dutch Friesian broodmare population was 18.2%. Heritability on the liability scale was 0.16 (SD = 0.06); heritability on the observed scale was 0.07; and repeatability was 0.89 (SD = 0.03). Maternal effect was 0.17 (SD = 0.06) and significantly differed from zero, although the animal model without a maternal effect fitted the data better. These results show that genetic and permanent environmental factors affect IBH in Dutch Friesian horses. The dam affected the IBH development of her offspring through an additive genetic influence but also by being part of their rearing environment.  相似文献   

16.
Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.  相似文献   

17.
Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from horses affected by CHS prior to and 4h following intradermal challenge with a commercial C. nubeculosus extract is modified in comparison to skin biopsies from non-affected horses. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. In comparison to non-affected horses, higher variation in LF mRNA levels both prior to and post-intradermal challenge with C. nubeculosus extract was seen in horses affected by CHS. However, the statistical analysis demonstrated that LF mRNA expression was not significantly different between CHS affected and non-affected horses prior to intradermal challenge with C. nubeculosus extract. Intradermal injection of C. nubeculosus extract did not result in local upregulation of LF mRNA at 4h post-injection. LF mRNA expression was therefore not significantly different pre- or post-intradermal challenge with C. nubeculosus extract in either group. Our data indicate that clinically normal skin of horses affected by CHS is not characterized by modified maintenance levels of LF mRNA. In contrast to human skin allergen challenged sites, LF mRNA levels in horses affected by CHS are not significantly different to that of control sites at 4h post-injection of C. nubeculosus extract.  相似文献   

18.
Insect bite hypersensitivity (IBH) is a seasonal recurrent allergic reaction of horses to the bites of certain Culicoides spp. and is found throughout the world. The aim of our study was to estimate the heritability and repeatability of IBH in the Dutch Shetland pony population. A total of 7,924 IBH scores on 6,073 mares were collected during foal inspections in 2003, 2005, and 2006. Mares were scored for clinical symptoms of IBH from June until February by 16 inspectors. Of all mares, 74.4% (n = 4,520) had a single observation, 20.7% (n = 1,255) had 2 observations, and 4.9% (n = 298) had 3 observations in different years. The overall mean IBH prevalence was 8.8%. Heritability was 0.08 (SE = 0.02) on the observed binary scale and 0.24 (SE = 0.06) on the underlying continuous scale. Repeatability was 0.30 (SE = 0.02) and indicates that including repeated observations of the clinical symptoms of IBH will improve the accuracy of breeding values for IBH. We conclude that IBH, based on clinical symptoms, is a heritable trait in the Dutch Shetland pony population. Therefore, the IBH prevalence in this population can be decreased by selection.  相似文献   

19.
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20.
Icelandic horses in Austria are commonly affected by an allergic inflammatory skin disease recurring during the summer seasons, which shares characteristic features with Culicoides hypersensitivity. However, the causative agents have not yet been identified. Therefore, intradermal skin testing (IDST) with a standardised extract of Culicoides variipennis and 21 other allergens relevant within Austria was performed in 81 Icelandic horses. All horses included into the study were treated regularly with ivermectin and had no history of administration of anti-inflammatory drugs. Forty-three of these horses were affected by summer seasonal recurrent dermatitis (SSRD). No history or signs of any other disease were evident in any horse. Pruritic dermatitis due to ectoparasites, bacteria and dermatophytes were ruled out by means of fungal culture, skin scraping and biopsy. Culicoides variipennis antigens evoked a positive cutaneous reaction in 1 of 38 normal and 3 of 43 SSRD horses at the proposed dilution of 1:50,000 or 1:25,000, and in 24 of 38 normal and 13 of 43 SSRD horses at a dilution of 1:10,000. Furthermore, no significant differences in onset or intensity of skin reactions to the 21 other allergens, including pollens, moulds, mites and insects, except deerfly and horsefly, were obvious between the 2 groups. Efficiency (percentage of correct results) for the used antigens in the skin test was 0.47-0.60. Maximal sensitivity was 0.51. Altogether, 38 of 43 SSRD horses and 28 of 38 normal horses were positive 4 h after allergen administration. The divergence between IDST results and manifestation of clinical signs found in this study underlines the difficulties associated with establishing a skin test protocol in horses within a geographic area. Whether the outcome of this study would have been influenced significantly by using Culicoides spp. present in Austria has to be clarified in future research.  相似文献   

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