Study of apparent hypersensitivity to Culicoides species in sheep in Israel

Between 1983 and 1997, a low incidence of seasonal allergic dermatitis was diagnosed in sheep in Israel aged eight months to eight years, in 10 flocks of a variety of breeds, but not the local Awassi breed. The 40 affected animals reacted with scratching, biting and vigorous stamping of the feet from April to November each year. The principal skin lesions were hyperkeratosis, spongiosis and infiltrations of eosinophils and mononuclear cells, typical of an allergic dermatitis which was probably a hypersensitivity reaction to biting insects. The epidemiological findings in relation to the area of Israel affected, the breeds of sheep and the distribution of the lesions suggested that the sheep's hypersensitivity was due to the bites of several species of Culicoides.     

Immunopathogenesis and immunotherapy of Culicoides hypersensitivity in horses: an update

Culicoides hypersensitivity (CH) is a strongly pruritic dermatitis of horses, characterised histologically by mixed perivascular to diffuse cellular infiltrates of mononuclear cells and eosinophils, and caused by helper T cell (Th)2, immunoglobulin (Ig)E-mediated reactions to antigens present in the saliva of biting Culicoides midges. As treatment of CH is still not satisfactory, a better knowledge of the pathogenesis of CH is needed for the development of new therapeutic modalities. Accordingly, availability of pure Culicoides allergens instead of crude whole body extracts is essential for the improvement of in vitro diagnosis of CH and development of a more effective allergen immunotherapy (AIT). Beside the Th2 cytokines interleukin (IL)-4 and IL-13, recent studies have shown the involvement of thymic stromal lymphopoietin, IL-5 and IL-31 in the pathogenesis of CH. The importance of IL-5 and IL-31 has been confirmed through targeting these cytokines by active immunisation of CH patients, resulting in a significant decrease of CH lesion scores. Thirty Culicoides salivary allergens have been described over the last 10 years and produced as pure recombinant (r-) proteins. Use of a protein microarray has allowed the identification of the most relevant Culicoides r-allergens for CH in central and northern Europe. An immunisation protocol has been developed for preventive AIT against CH. Small amounts of r-Culicoides allergens injected into the submandibular lymph nodes with Alum/MPLA as adjuvants seem to induce a suitable immune response for both preventive and therapeutic AIT. The next years will reveal whether these recent advancements will translate into an improved diagnosis, treatment and prevention of CH.     

Zinc and copper plasma levels in Icelandic horses with Culicoides hypersensitivity

Zinc concentration has been shown to have a potent immunomodulatory capacity, particularly influencing T helper cell organisation and cytokine secretion. Culicoides hypersensitivity (CHS) in horses resembles the early and late phase of type I hypersensitive reactions in man, characterised by a shift from T helper cell subtype 1 to T helper cell subtype 2 cytokine profile. In this pilot study, zinc and copper levels were measured in the plasma of 48 CHS-affected and 56 healthy Icelandic horses age 4-25 years (mean approximately 11 years) kept on 7 farms. Affected horses were divided into 3 groups according to the severity of disease. Time of blood collection and feeding management was constant. No differences in zinc or copper plasma levels and plasma copper/zinc ratio were determined among CHS horses and controls by univariate analysis of variance. Therefore, the most significant influences on zinc and copper plasma levels were affected by the location of housing. However, Spearman correlation showed a negative coefficient between the plasma zinc concentration and the severity of CHS (r = -0.31). Due to a probability value of P = 0.002 the null hypothesis r = 0 is rejected, although only 9% of the total variation of plasma zinc is presently explained by its relationship to CHS. In contrast, the Spearman correlation coefficient between plasma copper levels and severity of CHS was not significant (r = -0.14; P = 0.16). The minor deviations in plasma zinc concentrations in association with the severity of CHS may be real or due to neurohumoral or cytokine-mediated mechanisms, but appear too minimal to be relevant.     

Culicoides species attracted to horses with and without insect hypersensitivity

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.     

Field observations in Israel on hypersensitivity in cattle, sheep and donkeys caused by Culicoides

SUMMARY Hypersensitive lesions in the skin of sheep, cattle and donkeys from various regions in Israel are described. No cause other than insect bites was found. The animals were free of ticks, lice, fleas and blackflies, and of all the prevailing insect groups; Culicoides spp were considered as the most probable cause of the hypersensitivity. Lesions on the legs and abdomen were attributed mainly to C obsoletus Melgen, C schultzel gp and C puncticollis Becker, and less to C Imicola Kieffer. Lesions on the back, flanks, neck and head were attributed mainly to C Imicola, less to C newsteadl Austen and rarely to C pullcaris Linnaeus.     

A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity

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.     

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity)

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.     

Flaxseed (Linum usitatissimum) supplementation associated with reduced skin test lesional area in horses with Culicoides hypersensitivity

The purpose of this study was to quantify the effect of flaxseed (Linum usitatissimum) supplementation on the skin test response of atopic horses. Six horses that displayed a positive skin test for allergy to extract from Culicoides sp. participated in the 42-day, placebo-controlled, double-blind, cross-over trial. Results showed that supplementation with flaxseed for 42 days in our experimental horses reduced the mean skin test response to Culicoides sp. This observation was concurrent with a significant decrease in the long-chain saturated fatty acids; behenic acid (22:0) and lignoceric acid (24:0), in the hair of horses receiving flaxseed. There was also a significant decrease in aspartate aminotransferase, and increase in serum glucose in the treatment animals at specific sampling points. It was concluded that; in this small pilot study, flaxseed was able to reduce the lesional area of the skin test response of atopic horses, alter the fatty acid profile of the hair, reduce inflammation, and did not elicit any negative side-effects in the experimental horses.     

Lactoferrin, a glycoprotein with immunomodulatory and mast cell stabilising properties, in skin of horses suffering from Culicoides hypersensitivity

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.     

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch).

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.     

Culicoides hypersensitivity in the horse: 15 cases in southwestern british columbia

The investigation of a chronic, seasonal dermatitis of horses in southwestern British Columbia is described. Typically the history indicated an insidious onset, followed by a gradual progression in the severity of the signs each year. Lesions appeared during the warmer months of the year and tended to regress during the winter. The clinical signs consisted of areas of pruritus and excoriation, affecting predominantly the ventral midline, mane and tailhead. In all cases corticosteroid therapy relieved the pruritus and allowed the lesions to heal.

The salient pathological findings were hyperkeratosis, spongiosis and a dermal infiltration of eosinophils together with mononuclear cells. These changes are typical of an allergic dermatitis, which has been recognized in many parts of the world as a hypersensitivity reaction to the bites of Culicoides spp. In this instance, the epidemiological findings relating to the geographic area, the local insect population and the distribution of lesions implicated Culicoides obsoletus as the etiological agent.

     

Treatment of Culicoides hypersensitive horses with high-dose n-3 fatty acids: a double-blinded crossover study

In this double-blinded crossover study the clinical efficacy of high dose α-linolenic acid (linseed oil) for the treatment of Culicoides spp. hypersensitivity was evaluated. Seventeen privately owned horses from north Florida, USA completed the 18-week study. Criteria for inclusion included negative dermatophyte cultures, negative skin scrapes, failure to respond to ivermectin treatment, compatible clinical signs, positive skin test to Culicoides spp.antigen and a history of seasonal pruritus consistent with Culicoides spp. hypersensitivity. Horses were randomized and each horse's feed was supplemented with 200 mL of linseed oil (n-3 source) or 200 mL of corn oil (n-6 source) per day for a 6-week period. After a 6-week washout period, each horse was crossed over to the other supplement for an additional 6 weeks. Horses were evaluated every 3 weeks. The level of pruritus was determined by counting pruritic acts in a 30-min period. Total lesional surface area was determined by computerized evaluation of scanned photographs and documented physical examination findings. There was no significant change in level of pruritus observed between the groups ( P  = 0.89). Additionally, lesional surface area was not significantly different between the groups ( P  = 0.06). However, most owners stated that horses improved while supplemented with linseed oil.     

The effect of a topical insecticide containing permethrin on the number of Culicoides midges caught near horses with and without insect bite hypersensitivity in the Netherlands

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.     

Equine Culicoides hypersensitivity: evaluation of a skin test and of humoral response

Intradermal tests were carried out on 18 horses with clinical signs of Culicoides hypersensitivity (CHS) and 23 horses without clinical signs of CHS, and sera from these horses were analysed by SDS-PAGE and Western blotting (W-B). Intradermal injections of 0.1 ml of 25 microg/microl sterile Culicoides extract, 0.1 ml of 1:10,000 histamine (positive control) and 0.1 ml of physiological saline (negative control) were made in the dermis of the middle region of the neck. Analysis of reactions indicated that a 1 cm wheal and a skinfold thickness >10% at 24 h represented a valid cut-off between horses with and without CHS. In these conditions the test, even in winter when clinical signs were absent, had 100% sensitivity and specificity. The W-B was performed after running Culicoides extract on a 12% polyacrylamide gel. The test revealed the presence of several bands with molecular weight ranging from 6 to 200 kDa. In particular, a band of 65 kDa was predominantly found in hypersensitive horses by using an anti-IgE antibody while in normal horses the same band was mainly detected by using an anti-IgG antibody. Our results demonstrated that the skin test is a valid diagnostic test, with high sensitivity and specificity and that the band of about 65 kDa probably corresponds to the allergen involved in the pathogenesis of CHS.