Co-reactivity between related and unrelated environmental allergens in equine allergen-specific IgE serology testing in the UK.

BACKGROUND: Identification of environmental allergens in horses with allergic disease facilitates allergen avoidance and targeted immunotherapy. HYPOTHESIS/OBJECTIVES: To evaluate allergenic co-reactivity between 44 environmental allergens. ANIMALS: Horses with suspected allergic disease (n = 344) whose sera were submitted for environmental allergen testing. METHODS AND MATERIALS: Allergen-specific IgE serological assays were performed using 44 allergens divided into six taxonomically related groups: house dust/storage mites, moulds, insects, grass, tree and weed pollens. Using pairwise comparisons, odds ratios (ORs) were calculated for each environmental pair to determine if there was increased or decreased likelihood of a positive result for one allergen, given a positive result to another. The OR significance was set (using Holm-Bonferroni correction) at P < 0.00006 for all horses (n = 344) and P < 0.00005 for horses with at least one positive reaction (n = 239). Using one-way ANOVA with Tukey's post hoc tests (significance at P < 0.05), differences in mean log e ORs between three groups, taxonomically related allergens with a statistically significant association (related-associated), related allergens lacking a significant association (related-nonassociated) and unrelated allergens were tested. RESULTS: Statistically significant associations were found between both related and unrelated allergen pairs, the former being more frequent. For all horses (n = 344) and horses with at least one positive reaction (n = 239), co-reactivity ranged from 100% (grasses) to 0% (moulds). The weeds group was exceptional in having more co-reactions with another group (grasses). CONCLUSIONS AND CLINICAL IMPORTANCE: Co-reactivity was shown within and between certain related allergen groups. Further studies are required to determine whether this is the result of antigenic cross-reactivity.

Co-sensitization and cross-reactivity between related and unrelated food allergens in dogs - a serological study.

BACKGROUND: Knowledge of cross-reactivity between foods is useful so that potentially cross-reactive allergens can be avoided in diet trials. HYPOTHESIS/OBJECTIVES: To evaluate allergenic cross-reactivity in related foods. ANIMALS: Sera from 469 dogs with suspected adverse food reactions. METHODS: An IgE-based serological assay using 19 food allergens was performed in 469 dogs. Pairwise comparisons were used to calculate the odds ratios (ORs) for each food pair, with significance at P < 0.0002 by Holm-Bonferroni correction, both in all 469 dogs and in the 261 of 469 dogs with at least one positive reaction. One-way ANOVA with Tukey's post hoc tests (significance at P < 0.05) were used to test for differences between mean logE ORs in different food groups. Inhibition enzyme-linked immunosorbent assays (ELISAs) were performed to assess allergenic cross-reactivity between beef, lamb and cow's milk. RESULTS: Significant associations were observed between both related and unrelated food pairs. Associations were, however, more frequent and stronger among related than unrelated foods. In all 469 dogs, 38 of 43 related food pairs were significantly associated [mean (SD) logE OR 3.4 (0.9)] compared with 79 of 128 unrelated pairs [2.7 (1.0)], P < 0.0002. In positive dogs, 32 of 43 related pairs were significantly associated [2.7 [1.0)] compared with 49 of 128 unrelated pairs [1.8 (1.0)], P < 0.0002. Inhibition ELISAs confirmed the presence of cross-reactive IgE-binding epitopes in beef, lamb and cow's milk. CONCLUSIONS AND CLINICAL IMPORTANCE: The results suggest that related and potentially cross-reactive foods should be avoided in elimination diets.

Canine generalized demodicosis treated with varying doses of a 2.5% moxidectin+10% imidacloprid spot-on and oral ivermectin: parasiticidal effects and long-term treatment outcomes.

Advocate(®) (2.5% moxidectin+10% imidacloprid) (Bayer HealthCare, Leverkusen, Germany) is a multiparasiticidal spot-on authorized for treating canine demodicosis in many countries. This blinded, randomized three-phase clinical trial compared its efficacy employing different dosing regimens with that of ivermectin. In the blinded first phase, 58 dogs suffering from generalized demodicosis were randomly assigned to one of four groups and treated with monthly, biweekly or weekly applications of Advocate(®), or with oral ivermectin (IVR) at 500 µg/kg daily. Dogs were evaluated clinically and multiple skin scrapings undertaken every 4 weeks until parasitological cure was achieved (defined as two consecutive series of deep skin scrapings at monthly intervals negative for all life forms). Forty dogs completed the 16-week initial blinded phase, with 5 cases achieving parasitological cure. Five dogs were deemed treatment failures and subsequently treated with ivermectin. The treatment protocol was then changed for the remaining 35 dogs and this cross-over phase (Phase 2) was maintained for a further 8 weeks with an additional 9 dogs achieving parasitological cure. Thereafter, all remaining animals were treated with IVR until cured (Phase 3). Overall, 26 dogs achieved parasitological cure during the clinical investigation. Of these, 23 remained disease-free for at least 12 months while two were lost to follow up and one died of unrelated causes. A total of 32 (55.2%) dogs were withdrawn at various stages of the investigation including the 5 dogs that were judged treatment failures. Other reasons for withdrawal included: non-compliance, lost to follow-up, ivermectin toxicity or reasons unrelated to the investigation. No adverse effects were attributable to the use of Advocate(®). Parasiticidal efficacy was assessed by changes in mite counts (live adult, juvenile and egg) and skin lesion extent & severity scores. Statistical significance was assessed using ANCOVA with initial mite counts or skin scores used as the covariate to account for variations in disease severity. Planned pairwise comparisons were used to identify differences between treatment groups. The efficacy of Advocate(®) increased with its rate of application across all measures of efficacy. Although ivermectin was shown to be more effective than Advocate(®) applied once weekly, both treatment protocols produced clinically satisfactory results. It was concluded that weekly application of Advocate(®) can be recommended as effective for the treatment of canine generalized demodicosis without the potential for toxicity associated with ivermectin.

Serum anti-Staphylococcus pseudintermedius IgE and IgG antibodies in dogs with atopic dermatitis and nonatopic dogs.

BACKGROUND: Dogs and humans with atopic dermatitis (AD) are predisposed to colonization and recurrent infection with Staphylococcus spp. Studies in humans suggest that staphylococcus-specific immunoglobulin E (IgE) plays a key role in disease pathogenesis. Few such studies have been undertaken in dogs. HYPOTHESIS/OBJECTIVES: The aim of this study was to compare levels of staphylococcus-specific IgE and immunoglobulin G (IgG) in dogs with AD, nonatopic dogs with staphylococcal pyoderma, and nonatopic and noninfected control dogs. ANIMALS: Sera were collected from 108 dogs with AD, 39 nonatopic dogs with staphylococcal pyoderma secondary to different underlying conditions, 67 age-matched nonatopic control dogs, and nine control dogs reared in minimal disease conditions. METHODS: Serum Staphylococcus pseudintermedius-specific IgE and IgG antibodies were measured by enzyme-linked immunosorbent assay. RESULTS: Dogs with AD had significantly higher levels of anti-staphylococcal IgE than nonatopic dogs with staphylococcal pyoderma and the two groups of control dogs. Levels of anti-staphylococcal IgG were significantly higher in atopic dogs and nonatopic dogs with pyoderma compared with nonatopic control dogs and control dogs reared in minimal disease conditions, but there was no significant difference in levels of anti-staphylococcal IgG between dogs with AD and nonatopic dogs with pyoderma. CONCLUSIONS AND CLINICAL IMPORTANCE: A significantly increased IgE response to S. pseudintermedius antigens in atopic dogs suggests an immunopathogenic role for anti-staphylococcal IgE. The finding of elevated IgE and IgG in atopic dogs is also important as a prelude to studies on antigenic specificity and possible correlations with disease phenotype.

Food allergen-specific serum IgG and IgE before and after elimination diets in allergic dogs.

Serum food allergen-specific antibody testing is widely offered to identify suitable ingredients for diets to diagnose adverse food reaction (AFR) in dogs with allergic skin disease. Antibody concentrations in blood samples obtained during an unsuccessful diet to help in the choice of diet changes may be influenced by the previous diet. The objective of this paper was to measure food antigen-specific IgE and IgG for the most commonly used 16 food antigens before and after an elimination diet. Levels of food-specific serum IgE and IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Dogs had detectable IgE antibodies to beef, pork, lamb and cows' milk; and detectable IgG antibodies to beef, pork, lamb, cows' milk, chicken and turkey. Of 19 dogs with complete data sets, 14 dogs showed clear improvement during diet and in 7 dogs AFR could be diagnosed by deterioration on rechallenge and subsequent improvement on refeeding the diet. Serum was obtained before and 6-8 weeks after beginning such a diet. There was no significant difference in pre- and post-diet levels for any of the individual allergens nor for the total IgE and IgG concentrations of all antigens (P=0.55 and P=0.53 respectively). In these 19 dogs in which an elimination diet was used for the diagnosis of food allergy and in which 14 were probably food allergic and 7 were proven food allergic there were no significant differences in food-specific antibodies before and after an elimination diet of 6-8 weeks.

Treatment of canine-generalized demodicosis: a blind, randomized clinical trial comparing the efficacy of Advocate(Bayer Animal Health) with ivermectin.

Advocate (moxidectin 2.5% + imidacloprid 10%) is a multiparasiticidal agent authorized for treating canine demodicosis in many countries. This blind, randomized clinical trial assessed the efficacy of Advocate at varying treatment intervals and compared it with that of oral ivermectin. Fifty dogs with generalized demodicosis were randomly assigned to one of four treatment groups: oral ivermectin (500 microg/kg once daily), Advocate applied at the authorized dose monthly (ADV1), every 2 weeks (ADV2) or weekly (ADV4). Each dog was evaluated every 4 weeks for 4 months or until negative scrapings at all sites resulted on two successive evaluations (parasitological cure). Miticidal efficacy was determined through deep skin scrapings taken from the same three sites on each occasion. Total numbers of live and dead adult mites, juveniles and eggs were determined. Thirty-five dogs completed the 4-month trial. Parasiticidal efficacy was assessed using several parameters including reduction in live adult mite counts. ancova analysis for this parameter confirmed that there were differences in efficacy among the treatment groups (P < 0.002). Tukey-Kramer all pairwise multiple comparison tests revealed that ADV4 was more effective than ADV1 (P = 0.016). Ivermectin was more effective than ADV1 (P = 0.003). Both ivermectin and ADV4 showed clinically substantial reductions in adult mite counts (89% for ADV4 and 98% for ivermectin). In conclusion, the efficacy of Advocate increased with the rate of application and weekly application may represent a new approach to the treatment of caninegeneralized demodicosis.

Levels of house dust mite-specific serum immunoglobulin E (IgE) in different cat populations using a monoclonal based anti-IgE enzyme-linked immunosorbent assay.

Levels of serum immunoglobulin E (IgE) specific for the house dust mites (HDMs) Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP) in 58 cats with clinical signs suggestive of atopic dermatitis (allergic dermatitis cats), 52 cats with no history of allergic or immunological disease (nonallergic cats) and 26 specific pathogen-free (SPF) cats were measured using a monoclonal anti-IgE enzyme-linked immunosorbent assay. Reactivity to both native and reduced HDM allergens was compared. SPF cats had significantly lower levels of HDM-specific serum IgE than cats with allergic dermatitis and nonallergic cats. The difference in levels of HDM-specific IgE in the serum of cats with allergic dermatitis and nonallergic cats was significant for native DF allergen, but not for native DP allergen or reduced HDM allergens. The results suggest that DF in its native form may be a significant allergen in cats with allergic dermatitis. The clinical relevance of these reactions, however, remains to be proven.

Assessment of the ability of Malassezia pachydermatis to stimulate proliferation of canine keratinocytes in vitro.

OBJECTIVE: To investigate the direct interaction between canine keratinocytes and live Malassezia pachydermatis and thereby determine the role of these organisms in the pathogenesis of epidermal hyperplasia associated with Malassezia dermatitis in dogs. SAMPLE POPULATION: Primary canine keratinocyte cultures established from skin samples obtained from clinically normal dogs. PROCEDURE: The proliferative response of keratinocytes co-cultured with Malassezia organisms for 1, 2, or 3 days was assessed by use of direct manual counting (to determine the number of keratinocytes in both the monolayer and the medium) and immunohistochemical staining techniques involving antibodies against proliferating cell nuclear antigen (PCNA) and another cellular proliferation marker, Ki-67. The potential cytotoxic effect of Malassezia organisms was investigated by use of an apoptosis detection kit to label keratinocytes co-cultured with M. pachydermatis that underwent apoptosis. RESULTS: No stimulatory effect of Malassezia organisms on canine keratinocyte proliferation was detected via cell counting and immunohistochemical techniques. However, there was a significant increase in dead keratinocytes in the medium with increasing numbers of Malassezia organisms in the co-culture. More apoptotic cells were observed in keratinocyte monolayers co-cultured with high numbers of M. pachydermatis than there were in monolayers cultured without Malassezia organisms, and the number increased after prolonged incubation. CONCLUSIONS AND CLINICAL RELEVANCE: M. pachydermatis did not stimulate canine keratinocyte proliferation in vitro. The results suggested that the epidermal hyperplasia observed in dogs with Malassezia dermatitis is unlikely to be caused by a direct effect of the organism on the keratinocyte cell cycle, but is likely to involve other mechanisms.

Failure of extracts from Malassezia pachydermatis to stimulate canine keratinocyte proliferation in vitro.

Epidermal hyperplasia is one of the major histopathological features seen in dogs with Malassezia dermatitis. The aim of this study was to investigate the effects of extracts and culture supernatants from Malassezia pachydermatis on the proliferation of canine keratinocytes. Keratinocyte cultures were established from normal dog skin, and cell monolayers were co-cultured with Malassezia extracts (prepared either with or without protease inhibitors) and supernatants derived from organisms grown in liquid culture. The proliferation of keratinocytes was measured using a colourimetric assay. Neither the culture supernatants nor the Malassezia extracts had significant effects on the proliferation rate of canine keratinocytes, regardless of whether protease inhibitors were present or not. The results indicate that the epidermal hyperplasia seen in Malassezia dermatitis is unlikely to be caused directly by secretion of products from the organism.

Identification of major allergens of Malassezia pachydermatis in dogs with atopic dermatitis and Malassezia overgrowth.

We have previously shown that both atopic and normal dogs generate an IgG response to antigens of Malassezia pachydermatis. The aim of this study was to compare IgE responses to separated proteins of M. pachydermatis in 28 atopic dogs with Malassezia dermatitis and 22 clinically normal dogs using Western immunoblotting. Six different detection systems were evaluated in order to assess sensitivity and eliminate nonspecific binding and cross-reactivity. The protocol yielding the best results utilized a monoclonal mouse antidog IgE, an alkaline phosphatase conjugated goat antimouse IgG which had been passed through a canine IgG column 3 times, a chemiluminescent substrate and a digital imaging system. Proteins of 45, 52, 56 and 63 kDa were recognized by more than 50% of the atopic dog sera and thus represented major allergens. Only a minority of normal dogs showed faint IgE binding to these proteins. The results indicate that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease.