Immunoglobulin-E-bearing cells in skin biopsies of horses with insect bite hypersensitivity.

The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis of acute IBH lesions than in the dermis of healthy skin (median = 2.8 vs. 0.0 cells/mm2; P < or = 0.01). Significantly, more mast cells were detected with metachromatic (median = 160 vs. 62 cells/mm2; P < or = 0.001) and tryptase-specific stainings (median = 120 vs. 69 cells/mm2; P < or = 0.001) in the dermis of acute IBH biopsies compared to healthy skin. No chymase+ mast cells were found in any skin biopsy. IBH lesions fixed in PFA for >24 h were compared to dermatomycosis (DM) lesions; IBH biopsies contained a similar number of IgE-protein+ cells to DM biopsies (median = 249 vs. 192 cells/mm2; P = 0.08) but had significantly more IgE-mRNA+, metachromatic and tryptase+ mast cells than DM biopsies. This study suggests an involvement of IgE-mediated immune reactions in the pathogenesis of IBH as well as, sometimes, in dermatomycosis. Using double labelling, cells which expressed IgE protein and contained mast cell enzymes were detected.     

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria

Background – Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. Hypothesis/Objective – The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. Animals – Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. Methods – The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B cells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T‐helper 2 cytokines (interleukins IL‐4, IL‐5 and IL‐13), a T‐helper 1 cytokine (interferon‐γ), IL‐4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT‐PCR. Results – In subepidermal lesional skin of RU‐affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79‐positive (P ≤ 0.01), MAC387‐positive (P ≤ 0.01) and tryptase‐positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU‐affected horses contained more eosinophils (P ≤ 0.05) and tryptase‐positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL‐4 (P ≤ 0.01), IL‐13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL‐4 receptor α (P ≤ 0.05) was increased in lesional skin of RU‐affected horses compared with control horses. Expression of IL‐4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. Conclusions and clinical importance – Analysis of cytokine expression and inflammatory infiltrate suggests that T‐helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.     

Lymphoid nodules in skin biopsies from dogs, cats, and horses with nonneoplastic dermatoses

In a retrospective histopathologic study of nonneoplastic dermatoses, lymphoid nodules were found in 0.3% of 3,408 canine, 5.1% of 469 feline, and 4.5% of 325 equine skin biopsies. In all 3 species, the majority of cases wherein lymphoid nodules were found were diseases of presumed immune-mediated nature. In cats and horses, the majority of cases were also diseases characterized by tissue eosinophilia.     

Alterations of epidermal proliferation and cytokeratin expression in skin biopsies from heavy draught horses with chronic pastern dermatitis

We report the historical, clinical and histopathological characteristics of skin lesions in biopsies from 37 heavy draught horses with chronic pastern dermatitis. The skin lesions were divided into four macroscopic groups: scaling (group I, n=5), hyperkeratotic and hyperplastic plaque-like lesions (group II, n=14), nodular skin masses (group III, n=16) and verrucous skin lesions (group IV, n=2). The principal histological findings were hyperkeratosis and epidermal hyperplasia. There was a gradual increase in epidermal hyperplasia from groups I to IV, suggesting that the lesions represent different stages of disease. In all cases, there was perivascular dermatitis dominated by T lymphocytes with an increase in MHC class II-positive dendritic-like cells. Immunohistochemical labelling for cytokeratins CK5/6(4), CK10 and CK14 indicated a change in their expression pattern. This correlated with the degree of epidermal hyperplasia, indicating abnormal differentiation of keratinocytes. There was a statistically significant correlation between the severity of skin lesions and several other factors including increasing age, increasing cannon circumference, prominence of anatomical structures such as fetlock tufts of hairs, ergots and chestnuts, and bulges in the fetlock region.     

Immunoglobulin A monoclonal gammopathy in two horses with multiple myeloma

The clinical findings in two horses with secretory multiple myeloma and secondary immunoglobulin A (IgA) monoclonal gammopathy were non-specific and included weight loss, pale mucous membranes, limb oedema and bacterial respiratory tract infection. Consistent laboratory abnormalities included hyperproteinaemia, hyperglobulinaemia, hypoalbuminaemia and hypercalcaemia. The diagnosis was based on the presence of IgA monoclonal gammopathy in serum and urine and bone marrow plasmacytosis (> 10 per cent). One horse was euthanased; it had neoplastic plasma cell infiltrates in its kidneys, spleen, liver, bone marrow, myocardium and adrenal glands. The other horse was treated for a bacterial pneumonia and was still alive six months after it was first examined.     

Results of intradermal tests in horses without atopy and horses with atopic dermatitis or recurrent urticaria.

OBJECTIVE: To compare results of intradermal tests (IDT) for environmental allergens at 30 minutes and 4, 6, and 24 hours after injection in horses without atopy and horses with atopic dermatitis (AD) or recurrent urticaria (RU). ANIMALS: 22 horses without atopy, 10 horses with RU, and 7 horses with AD. PROCEDURE: In all horses, medical history was obtained, and results of physical examination, hematologic examination, serum biochemical analyses, examination of bronchoalveolar lavage fluid, and IDT with 73 allergens were examined. RESULTS: Horses with AD or RU had a significantly greater mean number of positive reactions for IDT, compared with horses without atopy. Horses with AD had a significantly greater number of positive reactions than horses without atopy in every allergen group at all time periods, except for molds at 4 and 24 hours. Horses with RU had a significantly greater number of positive reactions than horses without atopy in every allergen group, except for molds at 30 minutes and 4 and 6 hours, trees at 4 and 6 hours, and grasses at 4 hours. CONCLUSIONS AND CLINICAL RELEVANCE: A significantly greater number of positive reactions for IDT in horses with AD or RU, compared with horses without atopy, provides evidence of type-I IgE-mediated hypersensitivity for these diseases. Evaluation of results of IDT performed in horses with AD or RU is useful in determining specific allergens for the formulation of immunotherapy along with providing identification of allergens that could be useful when creating avoidance strategies.     

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.     

Response to immunotherapy in six related horses with urticaria secondary to atopy

Urticaria secondary to atopy may be a familial problem in some horses. Immunotherapy using a vaccine containing antigens selected on the basis of history and results of intradermal testing can be an effective method of managing atopy in horses; a response to therapy may be seen within 2 months.     

Diagnosis of equine arteritis virus infection in two horses by using monoclonal antibody immunoperoxidase histochemistry on skin biopsies

Two 5-year-old grade male horses presented with epiphora, rhinorrhea, conjunctival and nasal mucosal hyperemia, and dorsal and thoracic macropapular rash. Skin biopsies were collected from the affected areas, and serial sections were evaluated following hematoxylin and eosin and immunoperoxidase histochemistry staining by using a murine monoclonal antibody of the immunoglobulin G2A isotype recognizing the 30-kDa membrane protein of equine arteritis virus (EAV). In both horses, lesions consisted of mild to moderate diffuse superficial dermal edema and vasculitis with mild perivascular lymphocytic infiltrates, occasional endothelial hypertrophy, and single-cell necrosis of tunica media myocytes. Immunohistochemically, a few endothelial cells, myocytes, and pericytes containing intracytoplasmic EAV antigen were identified. Immunoperoxidase histochemistry of skin biopsies can be used as an ancillary test for the clinical diagnosis of equine viral arteritis in horses, especially when a cutaneous macropapular rash is evident.     

Intradermal testing of horses with chronic obstructive pulmonary disease and recurrent urticaria.

Six horses with chronic obstructive pulmonary disease (COPD) and 8 horses with recurrent urticaria were skin tested with 67 extracts from 58 allergens, including pollens, epidermals, cultivated farm plants, dusts, molds, and insects. Reactions were evaluated 3 times over a 24-hour period immediately after the injections. Results were compared with those obtained from 11 clinically normal horses. All horses had positive skin test reactions. Significant differences was evident between horses with COPD and clinically normal horses for only 3.0% of the possible extract reactions, and between horses with urticaria and clinically normal horses for only 4.5% of the possible extract reactions. Horses with COPD or urticaria had greater total percentage of allergen extract reactions than did clinically normal horses. Positive reactions were observed at all 3 evaluation periods, and late-onset reactions were not always preceded by positive reaction at earlier periods. All horses with COPD or urticaria had at least 1 skin test reaction that exceeded the mean +/- 2 SD, as calculated for each of the 67 extracts for the group of clinically normal horses.     

Intradermal testing in healthy horses and horses with chronic obstructive pulmonary disease, recurrent urticaria, or allergic dermatitis.

OBJECTIVE: To compare responses to a variety of intradermally injected allergens among healthy horses and horses with chronic obstructive pulmonary disease (COPD), recurrent urticaria (RU), and atopic dermatitis-insect hypersensitivity (allergic dermatitis [AD]). DESIGN: Case-control study. ANIMALS: 86 horses. PROCEDURE: Results of intradermal testing for horses with COPD, RU, or AD were compared with results for healthy horses. RESULTS: Compared with healthy horses, horses with COPD, RU, and AD were significantly more likely to have positive (> or = 3+) reactions to intradermal allergens (molds, weeds, trees, grasses-crops, and insects) 30 minutes (immediate reaction), 4 hours (late-phase reactions), and 24 hours (delayed-phase reactions) after exposure. In addition, diseased horses reacted to a significantly higher number of allergens in each allergen group than did healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE: Reactions to individual allergens should not be used to determine that horses have hypersensitivity. Overall patterns of reactivity to intradermal allergens may be helpful in management when used in conjunction with a compatible history and evidence of potential exposure to allergens in horses with conditions associated with hypersensitivity to environmental allergens.     

In vitro allergen-induced degranulation of pulmonary mast cells from horses with recurrent airway obstruction (heaves).

OBJECTIVE: To determine the capacity of pulmonary mast cells (PMC) to degranulate in response to various potential allergens and other secretagogues in horses with recurrent airway obstruction (heaves) and clinically normal horses before and after exposure to moldy hay. ANIMALS: 5 horses with heaves and 5 clinically normal horses. PROCEDURES: Heaves was characterized as an increased clinical respiratory score and maximum change in transpulmonary pressure of > 20 cm H2O after exposure. Bronchoalveolar lavage was performed during each period. Washed and resuspended cells were exposed for 20 minutes at 37 C with whole reconstituted freeze-dried preparations of Aspergillus fumigatus, Alternaria tenuis, and Ambrosia elatior, fungal extracts of Aspergillus fumigatus, Alternaria tenuis, and Micropolyspora faeni; A23187; and compound 48/80. Histamine release (HR) was used as a marker of degranulation. RESULTS: Compared with clinically normal horses, HR was significantly greater from PMC from horses with heaves during remission and exacerbation in response to whole preparations and extracts of Aspergillus fumigatus and whole preparations of Alternaria tenuis. Extracts of Alternaria tenuis caused significantly greater HR from PMC from horses with heaves during exacerbation. Histamine was also released from PMC in response to A23187 and to changes in osmolality of the medium, but only as a result of cell lysis by compound 48/80. CONCLUSIONS: Increased degranulation of PMC after antigenic challenge may contribute to the pathogenesis of heaves in horses. CLINICAL RELEVANCE: Strategies for prevention and treatment that attenuate degranulation of PMC may assist in the clinical management of horses with heaves.     

A multicenter retrospective study of 151 renal biopsies in horses

Background: Renal biopsies are uncommonly performed in horses and little is known about their diagnostic utility and associated complication rate. Objective: To describe the techniques, the complication rate, risk factors, and histopathology results; as well as evaluate the safety and diagnostic utility of renal biopsy in the horse. Animals: One hundred and forty‐six horses from which 151 renal biopsies were obtained. Animals ranged in age from 48 hours to 30 years. Methods: Multicenter retrospective study, with participation of 14 institutions (1983–2009). Results: Renal biopsy in horses was associated with a similar rate of complications (11.3%) to that occurring in humans and companion animals. Complications were generally associated with hemorrhage or signs of colic, and required treatment in 3% of cases. Fatality rate was low (1/151; 0.7%). Biopsy specimens yielded sufficient tissue for a histopathologic diagnosis in most cases (94%) but diagnoses had only fair (72%) agreement with postmortem findings. Risk factors for complications included biopsy specimens of the left kidney (P= .030), a diagnosis of neoplasia (P= .004), and low urine specific gravity (P= .030). No association with complications was found for age, sex, breed, institution, presenting complaint, other initial clinicopathologic data, biopsy instrument, needle size, or use of ultrasonographic guidance. Conclusions and Clinical Importance: Renal biopsy in horses has low morbidity and results in a morphological histopathologic diagnosis in 94% of cases. However, this procedure might result in serious complications and should only be used when information obtained would be likely to impact decisions regarding patient management and prognosis.     

Intradermal skin testing in Icelandic horses in Austria

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.     

Glomus tumours in the skin and subcutis of three horses

Three horses presented with variably painful, nonulcerated masses of the head or neck that were diagnosed as glomus tumours. Grossly, they were fleshy, pink to tan masses ranging from 0.4 to 9 cm in diameter, involving either the deep dermis and subcutis or the subcutis and underlying skeletal muscle. Microscopically, neoplastic epithelioid cells were arranged in sheets, cords and packets within lobules. The neoplastic cells frequently abutted and formed nodular bulges into large endothelium-lined vascular spaces, especially around the tumour periphery. Large nerve branches were associated with each tumour. As determined by immunohistochemistry, the neoplastic cells consistently expressed α-smooth muscle actin and vimentin, and some cells in two of the cases expressed desmin. A laminin- or collagen IV-positive basement membrane was demonstrated around individual tumour cells or small groups of cells in all three cases. Morphological features and immunohistochemistry supported the diagnosis of glomus tumour, most consistent with the solid type in humans. Applying a classification system used in humans, two of these tumours met criteria of malignancy (glomangiosarcomas). One horse was euthanized due to complications associated with recurrence and treatment-related necrosis and secondary infection.