Specific Dermatophagoides farinae extract for canine immunotherapy.

BACKGROUND: Canine atopic dermatitis (cAD) is a pruritic allergic skin disease most often caused by Dermatophagoides farinae. Differences in the sensitization profile to D. farinae have been reported between people and dogs. However, allergic dogs traditionally have been treated with extracts intended for human immunotherapy. HYPOTHESIS/OBJECTIVES: To develop a specific allergen immunotherapy for veterinary practice enriched in canine major allergens and to demonstrate its in vitro efficacy. ANIMALS: Twenty privately owned dogs, clinically diagnosed with cAD, and three healthy dogs. METHODS AND MATERIALS: A veterinary D. farinae allergen extract was manufactured and characterized compared to D. farinae extract used for human immunotherapy. The protein profile was analysed by SDS-PAGE and size exclusion chromatography and Der f 15 and Der f 18 allergens quantified by mass spectrometry. The allergenic profile was studied by immunoblot and the biological potency by enzyme-linked immunosorbent assay-inhibition assays. The extract's capacity to induce cytokine production [interleukin (IL)-10, interferon (IFN)-Ɣ] by peripheral blood mononuclear cells also was evaluated. RESULTS: The veterinary extract showed a higher content of high molecular weight proteins, preferentially recognized by atopic dog sera. The fold-increases in Der f 15 and Der f 18 with respect to the human extract were 2.07 ± 0.32 and 1.63 ± 0.15, respectively. The veterinary extract showed higher biological potency (0.062 versus 0.132 µg required for 50% inhibition of dogs sera) compared to the human extract and induced significantly higher levels of IL-10 (1,780 pg/mL) and IFN-Ɣ (50.4 pg/mL) with respect to the negative control. CONCLUSIONS AND CLINICAL IMPORTANCE: A veterinary D. farinae extract with a higher content of dog major allergens was developed and in vitro efficacy demonstrated by immunological parameters.

Effect of an anti-inflammatory pomegranate otic treatment on the clinical evolution and microbiota profile of dogs with otitis externa.

BACKGROUND: Canine otitis externa (OE) is a common disease characterised by inflammation of the epithelial tissue of the external ear canal. Secondary infections are frequent, and Malassezia pachydermatis and Staphylococcus pseudintermedius are routinely isolated and treated with antifungal and antibiotic compounds. HYPOTHESIS/OBJECTIVES: To analyse the otitis ear microbiome before and after a treatment with prednisolone plus pomegranate or antimicrobial drugs ANIMALS: 15 dogs with nonpurulent OE. METHODS AND MATERIALS: A 30 day, double-blinded, multicentre, randomized and controlled parallel-group (1:1) trial was conducted in 15 dogs with nonpurulent OE, following two different topical treatments (prednisolone plus pomegranate versus prednisolone plus antibiotic and antifungal drugs). On days (D)0, D15 and D30, serum and skin otic samples were collected, and clinical examination and microbiome analysis (bacteria and fungi) were performed. Results were compared with validated otitis clinical scores to assess the effectiveness of both treatments. RESULTS: Nine bacterial and four fungal families were detected during the three time-points tested. An increase in fungal diversity (Shannon index) and composition was the most significant change observed after both treatments. At treatment D15 and D30, the reduction in clinical signs was statistically significant in both treatment groups (P ≤ 0.05). Prednisolone plus pomegranate cleanser treatment was able to control the clinical signs of otitis as well as the bacterial and fungal overgrowth. CONCLUSIONS AND CLINICAL IMPORTANCE: Mild otitis cases associated with microbial overgrowth may be managed with topical antiseptic and anti-inflammatory agents without the need for antibiotic and/or antifungal compounds.

Clinical efficacy of sublingual allergen-specific immunotherapy in 22 cats with atopic dermatitis.

BACKGROUND: Sublingual immunotherapy (SLIT) has been deployed in humans and dogs; to the best of the authors' knowledge, there are no published studies about the use of SLIT in cats. OBJECTIVES: Evaluate the clinical efficacy of SLIT in atopic cats sensitized to dust and storage mites, assessing immunological changes associated with SLIT treatment. ANIMALS: Twenty-two client-owned cats with clinical signs compatible with feline atopic dermatitis (fAD) and serum allergen-specific immunoglobulin (Ig)E against house dust and storage mites. METHODS AND MATERIALS: Prospective, multicentre, open-label clinical trial. Individualized mite-specific SLIT was administered orally for 12 months. All cats underwent clinical examination to record SCORing feline allergic dermatitis (SCORFAD), pruritus Visual Analog Scale (pVAS) and serum allergen-specific IgE and IgG, every three months for 12 months. RESULTS: Sixteen of 22 cats (73%) completed the study and three of six cats withdrawn from the study were included in an intention-to-treat analysis. SCORFAD and pVAS values decreased significantly from baseline (T0) to the third month of treatment (P = 0.0004 and P = 0.0013, respectively), with median total values ranging from 19 (6-44) (T0) to 2.5 (0-17) (T12) (P = 0.0001), and from 8 (6-10) (T0) to 2.3 (0-8) (T12) (P = 0.0001), respectively. Allergen-specific IgE values decreased significantly from the ninth month (T9) of treatment (P = 0.0032), with median scores decreasing from 56 (12-729) (T0) to 34 (0-158) (T12) (P = 0.0208). No significant differences in allergen-specific IgG values were observed throughout the study. No adverse effects related to the use of SLIT were reported. CONCLUSIONS AND CLINICAL IMPORTANCE: Sublingual immunotherapy should be considered a rapid, effective, safe and well-tolerated treatment in cats with feline atopic dermatitis fAD.

An investigation of cross-reactivity between the poultry red mite and house dust and storage mites in dogs with contact with chickens infested with red mites.

BACKGROUND: The European poultry red mite (PRM) Dermanyssus gallinae, a common ectoparasite of laying chickens and pigeons; it also can feed on other birds, humans and domestic animals, causing clinical signs ranging from mild discomfort to severe dermatitis. Little is known about possible hypersensitivity to PRM or cross-sensitization with house dust or storage mites. HYPOTHESIS/OBJECTIVES: Knowledge on possible PRM immunoglobulin E (IgE)-mediated allergy and possible cross-sensitization with house dust and storage mites may facilitate the clinical approach. The aim herein was to clarify possible evidence of type I hypersensitivity to PRM in dogs and possible occurrence of cross-sensitization with house dust and storage mites. ANIMALS: Sixteen dogs with chronic contact with PRM-infested chickens from traditional bird houses and 10 control dogs with no contact with birds. METHODS AND MATERIALS: Dogs were subjected to intradermal testing (IDT) and serum specific IgE (sIgE) determination for house dust and storage mites and D. gallinae. RESULTS: The highest wheal score was obtained with 0.1 mg/mL D. gallinae extract. Positive IDT reactions to PRM were found in four of 10 control dogs and in 10 of 16 from the chicken-exposed group. SIgE to PRM was detected in one control and in seven dogs exposed to chickens. No significant correlation was found between IDT or sIgE scores to PRM and house dust and storage mites. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Contact with PRM-infested chickens may lead to sensitization without allergy, independently from sensitization to house dust and storage mites.

Topical polyhydroxy acid treatment for autosomal recessive congenital ichthyosis in the golden retriever: a prospective pilot study.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) in golden retrievers is due to a PNPLA1 gene mutation, which plays a role in epidermal lipid organization and metabolism. Topical therapies are used to reduce scaling; however, there are few published efficacy studies. OBJECTIVES: To examine the efficacy of topical treatment based on gluconolactone, a polyhydroxy acid with known beneficial effects on stratum corneum structure. ANIMALS: Sixteen golden retriever dogs with clinical signs of ARCI and PCR-confirmed PNPLA1 gene mutation. METHODS: This was a prospective, multicentre, noncontrolled study. Dogs were treated with a shampoo and lotion containing gluconolactone and other hydroxyl acids. Treatments were administered initially twice weekly for two weeks, then once weekly for two weeks and finally once monthly. Examinations were performed prior to and at 14 and 30 days of treatment to assess scaling, presence of other skin lesions and pruritus. In two dogs, pre- and 30 day post-treatment, skin biopsies were obtained. RESULTS: The extent and size of the scales were reduced by 60% and 75% after 14 and 30 days of treatment, respectively (P < 0.001). In 20% of the dogs, scaling was no longer observed after the first 30 days of treatment. No other skin lesions or pruritus were observed in any dog. Post-treatment biopsies showed normalization of the stratum corneum morphology and reduced hyperpigmentation. CONCLUSION AND CLINICAL IMPORTANCE: The frequent use of a shampoo and lotion containing gluconolactone may be an effective measure to improve skin scaling in golden retrievers with ARCI.

Development and characterization of an equine skin-equivalent model.

BACKGROUND: There is increasing interest in the biological and pathological study of equine skin owing to the high prevalence of cutaneous diseases in horses. However, knowledge of equine skin cell biology and cultures is limited by the low number of in vitro studies in the literature. HYPOTHESIS/OBJECTIVES: The objective of the study was to develop and characterize an in vitro equine skin equivalent. METHODS: Cultures of pure equine keratinocytes and dermal fibroblasts were obtained by enzymatic digestion of skin biopsies. Fibroblasts were embedded into type I collagen matrices to obtain dermal scaffolds, the surface of which was seeded with keratinocytes. The three-dimensional cultures were exposed to the air-liquid interface to enable epidermal stratification. RESULTS: After 14 days in air-exposed conditions, histological analysis showed that keratinocytes underwent differentiation into a multilayered epidermis. Immunohistochemical studies revealed the expression of epidermal cytokeratin in keratinocytes, whereas vimentin was expressed in dermal fibroblasts, as expected in equine skin. Immunostaining of Ki67 showed proliferative keratinocytes in the stratum basale. A continuous basement membrane at the dermo-epidermal junction was also detected immunohistochemically through the expression of its major components (type IV collagen and laminin 5). Ultrastructural analysis by electron microscopy showed desmosomes located among keratinocytes in all layers and hemidesmosomes among the basal keratinocytes and lamina densa. CONCLUSIONS AND CLINICAL IMPORTANCE: This study reports, for the first time, the development of an in vitro equine skin-equivalent model that resembles equine skin morphologically, immunohistochemically and ultrastructurally.

Inhibitory effect of topical adelmidrol on antigen-induced skin wheal and mast cell behavior in a canine model of allergic dermatitis.

BACKGROUND: Adelmidrol is a semisynthetic derivative of azelaic acid and analogue of the anti-inflammatory compound palmitoylethanolamide (PEA). Based upon its physicochemical properties, adelmidrol is suitable for topical application. The main objective of the present study was to evaluate the efficacy of a topical adelmidrol emulsion on early and late inflammatory responses in hypersensitive dogs. Repeated intradermal injections of Ascaris suum extract were performed in both lateral thoracic areas of six conscious hypersensitive Beagle dogs, topically treated during 8 consecutive days. Adelmidrol (2%) was applied to one side and vehicle to the other. 24 hours after the last antigen challenge, two biopsies (adelmidrol- and vehicle-treated side) were obtained for each dog at the antigen injection site. RESULTS: A significant reduction in the antigen-induced wheal areas was observed on the 4th and 7th day of adelmidrol treatment. Moreover, cutaneous mast cell numbers were significantly decreased in biopsies obtained after 8 consecutive days of topical adelmidrol treatment. CONCLUSIONS: The results obtained in the present study show that topical treatment with adelmidrol might represent a new therapeutic tool in controlling the early and late allergic inflammatory skin responses in companion animals.

Allergen-specific immunotherapy in dogs with atopic dermatitis: is owner compliance the main success-limiting factor?

BACKGROUND: While the efficacy of allergen-specific immunotherapy (ASIT) to treat canine atopic dermatitis has been well established, it remains unclear why not all dogs show the same response to treatment. The goal of the study was to determine the relationship between duration of ASIT and two measurements of success: disease severity and concomitant medication sparing effect. METHODS: Data were retrospectively compiled for 145 dogs with atopic dermatitis treated with ASIT. As a measure of treatment compliance, cases were stratified into dogs treated for less than 12 months or for at least 12 months. Treatment efficacy, defined as a reduction in disease severity score (scale 0-10), was compared between both groups, and correlations between treatment success and several related factors were examined. RESULTS: ASIT treatment duration was strongly correlated with treatment efficacy. Animals treated for less than 12 months showed lower efficacy rates (22 per cent) than those treated for at least 12 months (65 per cent). Further, in animals treated for at least 12 months, concomitant medications were reduced more (87 per cent) than in animals treated for less than 12 months (39 per cent). CONCLUSION: A lack of owner compliance emerged as the main factor explaining the reduced effectiveness of ASIT. To improve treatment adherence, veterinarians and owners need to be better informed about ASIT mechanisms of action before starting treatment.

Importance of house dust and storage mites in canine atopic dermatitis in the geographic region of Galicia, Spain.

Sensitisation to mites is frequent in atopic dogs. The main mite genus involved in canine atopic dermatitis is Dermatophagoides. The importance of storage mite allergens in dogs has been controversial. The aim of this study was to evaluate the sensitisation rates against storage mites (Lepidoglyphus destructor and Tyrophagus putrescentiae) and house dust mites (Dermatophagoides farinae and D. pteronyssinus) in atopic dogs from Galicia, a highly humid and temperate region of Spain, using a FcepsilonRIalpha-based immunoglobulin E (IgE) in vitro test. The study was performed on 95 dogs suffering from atopic dermatitis and presenting detectable specific serum IgE levels: 91.6% of the dogs tested positive for storage mites, whereas sensitisation to house dust mites was detected in 87.4%. These results indicate the importance of storage mites in this specific geographic area.

Immunologic responses against hydrolyzed soy protein in dogs with experimentally induced soy hypersensitivity.

OBJECTIVE: To assess whether dogs with experimentally induced type I hypersensitivity against soy protein would respond to soy hydrolysate and develop cutaneous or gastrointestinal tract reactions after intradermal and oral challenge exposure. ANIMALS: 12 naïve Beagle pups (9 sensitized and 3 control dogs). PROCEDURE: 9 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization period, serum concentrations of soy-specific IgE were determined and an intradermal test was performed to confirm the dogs were sensitized against soy protein. An intradermal challenge test and an oral challenge test with native and hydrolyzed soy protein were conducted on 6 sensitized and 2 control dogs. RESULTS: High serum concentrations of soy-specific IgE and positive results for the intradermal test were observed for the 9 sensitized dogs after completion of the sesitization process. Sensitized dogs challenge exposed with hydrolyzed soy protein had a reduced inflammatory response after intradermal injection and no clinical response after an oral challenge exposure, compared with responses after intradermal and oral challenge exposure with native soy protein. CONCLUSIONS AND CLINICAL RELEVANCE: Soy-sensitized dogs did not respond to oral administration of hydrolyzed soy protein. Thus, hydrolyzed soy protein may be useful in diets formulated for the management of dogs with adverse reactions to food.

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity.

OBJECTIVE: To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. ANIMALS: 8 naïve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). PROCEDURES: 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. RESULTS: In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.

Immunoproteomic characterization of a Dermatophagoides farinae extract used in the treatment of canine atopic dermatitis.

BACKGROUND: Canine atopic dermatitis is a pruritic allergic skin disease. House dust mites have been identified as the main non-seasonal responsible agent. Unlike in human allergic patients, groups 1 and 2 antigens have been described as minor allergens in dogs, while groups 15 and 18 are considered the major allergens. Despite these differences, allergic dogs have traditionally been treated using extracts intended for human immunotherapy. OBJECTIVES: To investigate the immunological characteristics and the allergen reactivity of dogs with atopic dermatitis using a Dermatophagoides farinae commercial extract. METHODS: Eighteen dogs diagnosed with atopic dermatitis and 3 healthy control dogs from the Iberian Peninsula were included in the study. All the animals were older than 12 months, from both sexes and different breeds and showed positive specific IgE against D. farinae (>2500 ELISA Absorbance Units). The D. farinae allergenic extract used in this study was manufactured and characterized. The allergenic profile of the dogs was investigated by immunoblot and specific IgE, IgG, IgG1 and IgG2 measured by direct ELISA. Allergen identity was confirmed by immunoblot inhibition and mass spectrometry analyses. RESULTS: The results confirmed the relevance of groups 15 and 18 antigens, but also groups 1, 2 and other medium molecular weight allergens in the sensitization of dogs with atopic dermatitis. Immunoblot inhibition and mass spectrometry assays confirmed these results. Relevant allergens were quantified by scanning densitometry (Der f 1: 17µg/mg, Der f 2: 20.3µg/mg, Der f 15: 18.1µg/mg and Der f 18: 9.4µg/mg). Concerning immunoglobulins profile, differences in IgE and IgG1 levels were observed between non-atopic and atopic dogs. CONCLUSIONS: The commercial D. farinae extract characterized in this study contains the major allergens involved in the sensitization of dogs with atopic dermatitis, representing a suitable candidate for its use in the diagnosis and immunotherapy of mite allergic dogs.

Effects of sphingolipid extracts on the morphological structure and lipid profile in an in vitro model of canine skin.

Ceramides (CER) are essential sphingolipids of the stratum corneum (SC) that play an important role in maintaining cutaneous barrier function. Skin barrier defects occur in both human beings and dogs affected with atopic dermatitis, and have been associated with decreased CER concentrations and morphological alterations in the SC. The aim of the present study was to investigate the changes induced by three different sphingolipid extracts (SPE-1, SPE-2 and SPE-3) on the morphological structure and lipid composition of canine skin, using an in vitro model, whereby keratinocytes were seeded onto fibroblast-embedded collagen type I matrix at the air-liquid interface. Cell cultures were supplemented with SPE-1, SPE-2, SPE-3 or vehicle (control) for 14 days. The relative concentrations of lipids were determined by ultra-performance liquid chromatography coupled to mass spectrometry. The ultrastructural morphology of samples was examined by transmission electron microscopy. SPE-1 induced significant elevation in total CERs, CER[NS], CER[NDS], CER[NP], CER[AS], CER[AP], CER[EOS] and CER[EOP] subclasses, whereas SPE-2 induced a significant elevation in total CER, CER[AP] and CER[EOS] compared with control conditions. Ultrastructural analysis revealed an increase in lamellar-lipid structures in the SC of SPE-1-treated samples. The findings demonstrated that SPE-1 stimulates production of CERs, as shown by changes in lipid composition and ultrastructural morphology. Thus, SPE-1 contributes to the formation of a well-organised SC and represents a potential therapeutic target for improving skin barrier function in atopic dermatitis.

Proficiency monitoring of monoclonal antibody cocktail-based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs.

The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergen-specific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA.

Evaluation of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture.

OBJECTIVE: To assess expression and function of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture. SAMPLE POPULATION: C2 cells maintained in medium lacking IgE for up to 10 passages before being stored at -80 C. PROCEDURE: Cells were thawed, cultured in medium without IgE for 1 to 3 passages, sensitized for 7 days with IgE-rich serum from dogs naturally sensitized to Ascaris suum, and stimulated with antigen Asc S1 from A suum, goat polyclonal anti-canine IgE, or calcium ionophore and phorbol myristate acetate (PMA). Percentage of intracellular beta-hexosaminidase released and concentration of tumor necrosis factor-alpha (TNF-alpha) synthesized after stimulation were determined. Expression of cell-surface IgE receptors was assessed by use of a flow cytometry. RESULTS: Immunologic stimulation (antigen or anti-IgE) failed to induce release or synthesis of detectable amounts of beta-hexosaminidase or TNF-alpha. In contrast, nonimmunologic stimulation (calcium ionophore and PMA) led to release of beta-hexosaminidase (mean +/- SEM maximum release, 23.95+/-1.96%) and synthesis of TNF-alpha (maximum concentration, 34.34+/-2.34 pg/10(6) cells). As revealed by use of flow cytometry, C2 cells expressed surface IgE receptors that bound canine IgE in vitro. CONCLUSIONS: Continuous culture of the canine mastocytoma cell line C2 in medium without exogenous IgE or cytokines and other growth factors resulted in cell-surface expression of nonfunctional IgE receptors. However, C2 cells maintained in continuous culture may still be a useful tool for the evaluation of mast cell responses to nonimmunologic stimulation and IgE receptor differentiation and maturity.

Efficacy of a new topical cyclosporine A formulation in the treatment of atopic dermatitis in dogs.

Topical treatment with cyclosporine A (CsA) has recently become possible with the development of novel nanotechnology pharmaceutical formulations of CsA able to penetrate through the epidermis providing good absorption and dermal action. The aim of this multicentre, blinded, parallel, randomized, placebo controlled trial was to evaluate the efficacy of a new topical CsA formulation in dogs with atopic dermatitis (AD). Dogs (n=32) with severe and moderate clinical signs of non-seasonal AD, but few localized lesions, were randomly allocated to receive topical CsA (17 dogs) or placebo (15 dogs) and were treated twice a day for 6 weeks. Before and 21 and 45 days after starting the treatment, the severity of a previously selected skin lesion was evaluated according to a dermatological scoring system. Owners using a visual analogue scale also assessed pruritus weekly and effectiveness of the treatment was defined as a reduction of at least 50% in these variables after 45 days. After 21 and 45 days the lesion severity score in animals treated with CsA was significantly lower than at baseline (P<0.01, both times). In contrast, the animals on placebo showed no significant improvement at days 21 or 45. The percentage of dogs with an effective reduction in pruritus at the end of the trial was 87.5% and 28.6% in the CsA and placebo groups, respectively. These results suggest that topical administration of CsA is effective in reducing the severity of skin lesions and pruritus in dogs with moderate to severe AD as soon as 3 weeks after starting treatment.

Effects of palmitoylethanolamide on the cutaneous allergic inflammatory response in Ascaris hypersensitive Beagle dogs.

Palmitoylethanolamide (PEA) is an endogenous lipid mediator with anti-inflammatory and anti-hyperalgesic properties. The main objective of the present study was to evaluate the effects of PEA on the cutaneous allergic inflammatory reaction induced by different immunological and non-immunological stimuli in hypersensitive dogs. Six spontaneously Ascaris hypersensitive Beagle dogs were challenged with intradermal injections of Ascaris suum extract, substance P and anti-canine IgE, before and after a single oral administration of PEA at doses of 3, 10 and 30 mg/kg. A significant reduction in wheal area induced by both antigen and anti-canine IgE challenge was observed after PEA administration. No significant differences were observed between the two higher doses studied, suggesting that the 10 mg/kg dose had exerted the maximum inhibitory effect. When blood levels of PEA were compared with the effects at different times, an evident correlation was obtained. However, the anti-inflammatory effects of PEA were more long-lasting than their plasma concentrations. The intradermal injection of substance P did not reveal any skin reaction (wheal or erythema formation) at any of the concentrations tested. In conclusion, PEA might constitute a new therapeutic strategy for the treatment of allergic inflammatory skin diseases in companion animals.

Intra and inter-laboratory reproducibility of a monoclonal antibody cocktail based ELISA for detection of allergen specific IgE in dogs: proficiency monitoring of macELISA in six laboratories.

The purpose of this study was to evaluate the reproducibility of results yielded using a monoclonal antibody based ELISA for detection of allergen specific IgE when run in six separate affiliated laboratories. On two separate occasions, duplicate samples of 15 different sera pools were independently evaluated by each laboratory in a single blinded fashion. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.2% (range 2.6-18.2%), while the average intra-laboratory inter-assay variance was 12.1% (range 8.0-17.1%). The overall inter-assay inter-laboratory variance was consistent among laboratories and averaged 15.6% (range 15.1-16.6%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose-response profiles observed in each of the laboratories were indistinguishable. Considering positive/negative results, inter-assay inter-laboratory concordance of results exceeded 95%. Correlation of OD values between and among all laboratories was strong (r>0.9, p<0.001). Correlation of OD values between the two separate evaluations was also high for all allergens except olive, which was attributed to lot-to-lot differences of allergen coated wells. Collectively, the results demonstrated that the monoclonal antibody based ELISA for measuring allergen specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory, but between laboratories using the same monoclonal-based ELISA.

Evaluation of storage mite contamination of commercial dry dog food.

Storage mites may be considered important allergens in dogs with atopic dermatitis. High sensitization rates to Tyrophagus, Acarus, and Lepidoglyphus species have been reported in atopic dogs, and dry pet food has been suggested as a potential source of storage mite exposure. The aim of the present study was to evaluate commercial dry dog food for contamination with storage mites, and how storage time and conditions could influence the risk of contamination. Ten different premium commercial dry dog foods formulated for skin disorders were selected. Food bags were opened and stored for 6 weeks under two different environmental conditions. At different time points, samples from each bag were collected and analysed by microscopy, guanine test, storage mite-specific traps, and a modified flotation technique. On opening, two storage mites identified as Acarus siro were isolated from one of the 10 bags by flotation technique, indicating that storage mites can be present in packaged dry dog food bags. After 5 weeks of storage under environmental conditions optimal for mite growth (23.2 +/- 2.1 degrees C and 71 +/- 5.6% of relative humidity), mites were detected by microscopic observation in nine of the 10 diets. When mites were identified by the flotation technique, Tyrophagus spp. were found to be the most common contaminating species. These results show that dry dog food can be a suitable substrate for storage mite reproduction, and that environmental and storage conditions may influence food contamination and mite development.

Development and characterization of a canine skin equivalent.

The development of a complex cellular model, which incorporates the basic cell components of the dog skin, would be a useful tool to investigate the biology and pathology of canine skin and also to replace animal testing partially. The aim of the present study was to develop and characterize a canine skin equivalent. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies from healthy dogs. Fibroblasts were embedded into a bio-matrix from collagen type I matrix protein; this built the scaffold where the keratinocytes were seeded, at air exposed conditions. At 3, 7, 15 and 21 days of culture in special growth media, skin equivalents were analysed by histological, immunohistochemical and electron microscopical techniques. At 15 days, keratinocytes underwent differentiation to a multilayer epidermis with stratum basal, stratum spinosum, stratum granulosum and stratum corneum. Expression of epidermal cytokeratins in keratinocytes was detected by immunhistochemistry, and followed the same pattern than in the normal canine epidermis. Fibroblasts from the skin equivalent expressed vimentin as dermal fibroblasts do. A basement membrane (BM) was observed underneath the epidermis; ultrastructurally, it was similar to the normal canine BM and collagen IV and laminin 5 were detected immunohistochemically as major components of this structure. Skin equivalents developed from canine cutaneous cells presented a similar morphological structure than healthy canine skin. Moreover, the immunohistochemical analysis revealed the expression of the major markers of the epidermis (keratins), dermis (vimentin) and BM (collagen type IV, laminin 5).