Peripheral blood mononuclear cell responses to major and minor Dermatophagoides allergens in canine atopic dermatitis.

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans. Most cases involve hypersensitivity to the house dust mites (HDM) Dermatophagoides farinae and Dermatophagoides pteronyssinus. Human atopic dermatitis is associated with the HDM derived allergens Der f 1 and 2, and Der p 1 and 2. Serological data, however, suggest that a 98/104kD protein is the most important allergen in dogs with atopic dermatitis. The aim of this study was to characterise the specificity of circulating T-cells in canine atopic dermatitis for HDM derived allergens. Peripheral blood mononuclear cells (PBMCs) from dogs with atopic dermatitis that were skin test positive for D. farinae and D. pteronyssinus were cultured with crude extracts of D. farinae, D. pteronyssinus and D. microceras, a 98/104kD allergen purified from D. farinae, Der f 1 and Der f 2. There was significantly greater responsiveness of PBMCs to the D. farinae and D. pteronyssinus extracts compared to the D. microceras extract, and similarly to the purified 98/104kD allergen compared to Der f 1 and Der f 2. The close association between serological findings and PBMC proliferation implies that the 98/104kD HDM protein is a major target of immune recognition and that T-cells also participate in the pathogenesis of canine atopic dermatitis by supporting IgE production.     

Characterization of house dust mite and scabies mite allergens by use of canine serum antibodies

OBJECTIVE: To identify the major allergenic proteins from the 3 main species of dust mites to which dogs react (Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei) and evaluate the potential cross-reactivity of dust mite allergens with antigens from the ectoparasitic mite Sarcoptes scabiei var canis. SAMPLE POPULATION: Sera from 83 dogs with atopic dermatitis. PROCEDURE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting using serum from atopic dogs was used to identify IgE-binding proteins in extracts of the 4 mite species. RESULTS: Sera of atopic dogs contained IgE against 23, 17, 25, and 17 allergens from D. farinae, D. pteronyssinus, E. maynei, and S. scabiei, respectively. Unlike the situation for humans, the major allergens for dogs are mostly proteins that are larger than 90 kd molecular weight. Dermatophagoides farinae and E. maynei appear to be more allergenic for dogs than is D. pteronyssinus. Some dogs with serum IgE against dust mites also had IgE against antigens of S. scabiei var canis. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple dust mite allergens induce an IgE response in dogs. These allergens are mostly greater than 90 kd molecular weight.     

Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.     

Serum immunoglobulin E against storage mite allergens in dogs with atopic dermatitis

OBJECTIVE: To determine the prevalence of serum IgE against the storage mites Acarus siro, Blomia tropicalis, and Tyrophagus putrescentiae in a population of dogs with atopic dermatitis. SAMPLE POPULATION: Sera from 84 dogs with atopic dermatitis residing in various regions of the United States and Europe. PROCEDURE: Immunoblotting of sera from atopic dogs was used to identify proteins in mite extracts that bound IgE. RESULTS: 94% of the dogs had serum IgE against proteins in extracts of 1 or more of the storage mite species. Ninety-five, 92, and 89% of the storage mite-sensitive dogs had serum IgE against proteins in extracts of A siro, B tropicalis, and T putrescentiae, respectively. Eighty-two percent had serum IgE against at least 1 protein in all 3 species. Most of the major allergens had molecular weights > 80 kd. A greater percentage of the dog sera had IgE against storage mite proteins, compared with proteins of the house dust mites Dermatophagoides farinae and D pteronyssinus. CONCLUSION AND CLINICAL RELEVANCE: Many dogs with atopic dermatitis have serum IgE against many allergens of storage mites. Most of these allergens, like allergens of dust mites, had molecular weights > 80 kd. Storage mite sensitivity in dogs may be as important, if not more important, than dust mite sensitivity.     

IgE sensitivity and cross-reactivity to crude and purified mite allergens (Der f 1, Der f 2, Der p 1, Der p 2) in atopic dogs sensitive to Dermatophagoides mite allergens

The present study investigates IgE-reactivity to crude and purified mite allergens by intradermal skin test (IDST), Immunodot method, and ELISA in atopic dogs sensitive to mite allergens, as well as the allergenic cross-reactivity between Dermatophgoides (D) farinae (DF) and D. pteronyssinus (DP) in dogs by IgE-ELISA inhibition. IDST and Immunodot method for crude mite allergens were performed for atopic dogs and 16 atopic dogs showed sensitivity to mite allergens. Of the 16 dogs, all dogs had anti-DF IgE and 11 had anti-DP IgE. We measured specific IgE to purified major allergens (Der f 1, Der f 2, Der p 1, Der p 2). Of the 16 atopic dogs, six had anti-Der f 1 IgE and seven had anti-Der f 2 IgE. Similarly, of the 16 dogs, six had anti-Der p 1 IgE and seven had anti-Der p 2 IgE. However, eight dogs had no specific IgE to these mite allergens. These dogs may be sensitive to other major mite allergens except Der 1 and Der 2. In the dogs that had both anti-DF and DP IgE, IgE binding to DF was greatly inhibited by DP, and reciprocal inhibition was observed. Based on these data, it appears that there is a strong cross-reactivity between DF and DP in dogs. Similarly, a cross-reactivity between DF and DP in purified allergens was also observed. IDST and Immunodot method are useful methods for the diagnosis of atopic diseases in dogs, and ELISA is a useful method for further investigation of IgE-reactivity for the allergens.     

Reactivity to intradermal injection of extracts of Dermatophagoides farinae, Dermatophagoides pteronyssinus, house dust mite mix, and house dust in dogs suspected to have atopic dermatitis: 115 cases (1996-1998)

OBJECTIVE: To compare reactivities to intradermal injection of extracts of Dermatophagoides farinae, Dermatophagoides pteronyssinus, house dust mite mix, and house dust in dogs suspected to have atopic dermatitis. DESIGN: Retrospective study. ANIMALS: 115 dogs. PROCEDURES: Records of all dogs suspected to have atopic dermatitis that underwent intradermal testing between October 1996 and July 1998 were reviewed. Reactivities to intradermal injection of crude mixed house dust mite (1:25,000 wt/vol) and crude house dust (25 PNU/ml) extracts were compared with reactivities to intradermal injection of individual extracts of D farinae and D pteronyssinus (1:50,000 wt/vol). RESULTS: Ninety dogs were confirmed to have atopic dermatitis including 61 of the 69 dogs with positive reactions to either or both of the individual house dust mite extracts. Intradermal testing with the mixed house dust mite extract had sensitivity of 75%, specificity of 96%, and accuracy of 83%. Intradermal testing with the house dust extract had sensitivity of 30%, specificity of 93%, and accuracy of 56%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that use of crude mixed house dust mite and crude house dust extracts for intradermal testing in dogs is not as accurate a method of determining house dust mite hypersensitivity as is the use of individual D farinae and D pteronyssinus extracts mainly because of the high percentage of false-negative results. Extracts of individual house dust mites are recommended for intradermal testing of dogs suspected to have atopic dermatitis.     

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.     

Dermatophagoides farinae-specific IgG responses in atopic dogs undergoing allergen-specific immunotherapy with aqueous vaccines

The molecular and immunologic mechanisms associated with successful allergen-specific immunotherapy (ASIT) have not been completely elucidated. The aim of this study was to characterize the changes in Dermatophagoides farinae -specific IgG in atopic dogs undergoing ASIT using aqueous vaccines. Fifteen atopic dogs with a positive skin test reaction to D. farinae were treated with aqueous vaccines for a minimum of 2 months following a standard protocol. Serum samples were collected before and during therapy and used to probe Western blots containing separated proteins of D. farinae . IgG responses were detected using a polyclonal goat anticanine IgG antibody and a chromogenic substrate 3,3'-diaminobenzidine. The blots were analysed using a semiquantitative digital image analysis system that evaluated the number and molecular weight of bands, as well as their intensity, which was related to IgG concentration. Prior to ASIT, all dogs showed allergen-specific IgG responses to various antigens of D. farinae . During ASIT, there was a significant increase in the total quantity of D. farinae -specific IgG antibodies to various antigens from the mite ( P  = 0.015). Significant increases were observed for a 98-kDa band ( P  = 0.015), likely to be Der f 15; bands with molecular weights between 50 and 70 kDa ( P  = 0.012); and bands between 30 and 45 kDa ( P  = 0.035). These findings provide support for the hypothesis that ASIT induces IgG blocking antibodies to allergens known to be relevant in canine atopic dermatitis.     

Comparison between an intradermal skin test and allergen-specific IgE-ELISA for canine atopic dermatitis

The aim of this study was to compare the results of an intradermal skin test (IDST) with those of an allergen-specific IgE-ELISA in 210 dogs with atopic dermatitis. All the dogs had a clinical diagnosis of atopic dermatitis and underwent an IDST. The sera of all dogs were analysed for allergen-specific IgE by ELISA using the monoclonal antibody D9 against dog IgE. IDST was used as the standard assay. In both methods, the following antigens provided a positive test result: Dermatophagoides farinae, Acarus siro, Tyrophagus putrescentiae, ragweed, mugwort and Lepidoglyphus destructor. ELISA had an overall sensitivity of 82.4% and an overall specificity of 93.8%. The overall accuracy of the ELISA was 91.3%. The evaluated monoclonal D9 ELISA was found to be a reliable tool for the diagnosis of those allergens that cause clinical atopy, and can be recommended for use in dogs when immunotherapy is a therapeutic option.     

IgG responses to antigens from Dermatophagoides farinae in healthy and atopic dogs

In this study, we used a semi-quantitative electrophoresis and immunoblotting technique to characterise the IgG response to antigens from Dermatophagoides farinae in 20 healthy and 20 atopic dogs. Both groups mounted an IgG response to multiple antigens from the mite. There was no significant difference in the number of bands recognised, or the molecular weights of the bands, between the two groups. The two most obvious bands in both groups were proteins with molecular weights of 98 kDa (likely to be the high molecular weight allergen Der f 15) and 44 kDa, although dogs in both groups recognised a similar pattern of other antigens. The magnitude of the IgG response was greater in the atopic group although this was not statistically significant. The results indicate that the immune system of both healthy and atopic dogs generates an IgG response to multiple antigens from D. farinae. As some of these antigens (such as the 98 and 44 kDa proteins) are also targeted by IgE in atopic dogs, immunoglobulin class switching in response to Th2 cytokines may not be as dominant a process as has been proposed.     

Comparison of intradermal and serum testing for allergen-specific IgE using a Fcepsilon RIalpha-based assay in atopic dogs in the UK

Atopic dermatitis in dogs is a common allergic skin disease that affects substantial numbers of dogs in the UK. The purpose of this study was to compare the results of an intradermal test (IDT) and an in vitro test in a large cohort of dogs. Dogs were intradermal tested with Greer allergens (Greer Labs Inc, Lenoir, NC, USA) using standard techniques. At the same time blood samples were drawn and submitted for evaluation by ELISA using the ALLERCEPT Definitive Allergen Panels for allergen-specific IgE, a commercial assay that uses a biotinylated recombinant extracellular domain of the high affinity Fc-epsilon receptor alpha chain protein (Fcepsilon RIalpha). The allergens used in the two tests included grass, tree and weed pollens, moulds, flea saliva/whole flea extract and house dust mite species. The optical density readings from the ELISA for each allergen were compared with the results of the IDT for 265 dogs. The prevalence of positive reactions in the ELISA was equal to or greater than the results of the IDT in the case of almost all of the allergens, but two notable exceptions were the house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus. These two allergens were the most common positive reactions by IDT (prevalence D. farinae 78.9%, D. pteronyssinus 66.4%). The results of the two tests were significantly different (McNemar's test, P<0.05) for 16 of the 22 allergens. The sensitivities of the ELISA compared to the IDT (where there were more than 3 dogs with positive reactions in both tests) varied between 19.3 and 77.1% (D. pteronyssinus 19.3% and D. farinae 67.9%) and the specificities varied between 64.2 and 96.6% (D. pteronyssinus 96.6% and D. farinae 89.3%).     

Assessment of cross-reactivity among five species of house dust and storage mites

In vitro cross-reactivity among two house dust (Dermatophagoides farinae, D. pteronyssinus) and three storage (Acarus siro, Tyrophagus putrescentiae, Lepidoglyphus destructor) mites was examined in 20 mite-sensitive dogs with natural occurring atopic dermatitis (group A), 13 high-IgE beagles experimentally sensitized to D. farinae (group B), and five healthy beagles (group C). Intradermal testing (IDT) and serology for allergen-specific IgE demonstrated that co-sensitization for all possible pairs of the five mites was generally 45% or higher among group A dogs. In the same dogs, enzyme-linked immunosorbent assay cross-inhibition results indicated that each one of D. farinae, A. siro and T. putrescentiae was a strong inhibitor of all the remaining mites, whereas D. pteronyssinus was a strong inhibitor of L. destructor. A high number of positive IDT and serology test results for D. pteronyssinus, A. siro, T. putrescentiae and L. destructor were recorded among group B dogs. No conclusive evidence of exposure to these mites was found upon analysis of dust samples from their environment and their food for the presence of mites and guanine. Also, the number of positive test results was generally higher among group B than among group C dogs. Enzyme-linked immunosorbent assay cross-inhibition revealed that D. farinae was a strong inhibitor of D. pteronyssinus, A. siro and T. putrescentiae. Collectively, these results demonstrated extensive in vitro cross-reactivity among house dust and/or storage mites that can explain false-positive results upon testing of dust mite-sensitive dogs with atopic dermatitis.     

Sensitivity patterns to house dust mites and forage mites in atopic dogs: 150 cases

This study investigated intradermal test reactions to extracts of six species of mites in 150 dogs with atopic dermatitis. At least one positive reaction was seen in 120 animals (80%). Dermatophagoides farinae attracted the highest number of positive reactions (108 dogs, 90% of dogs and 72% of atopic dogs showing positive reactions). Positive reactions to other mites were not uncommon, with many dogs testing positive for Dermatophagoides pteronyssinus (32% of dogs tested), Acarus siro (35%), Tyrophagus putrescentiae (30%), Glycyphagus domesticus (27%) and Lepidoglyphus destructor (23%). Sensitivity to D. farinae alone occurred commonly (57% of cases), but multiple sensitivities were seen frequently with the other mites. Cases of sensitivity to only one mite were also seen: D. pteronyssinus (five cases), T. putrescentiae (one case) and G. domesticus (one case). Further studies are needed to appreciate more clearly the precise role played by the different species of mite in canine atopic dermatitis.     

Determination of antigenic proteins of housedust mites in 90 dogs suffering from atopic dermatitis

Housedust mites, Dermatophagoides pteronyssinus (D. pteronyssinus) and Dermatophagoides farinae (D. farinae), are the important causative agents of allergic diseases in human and animals. By using 165 dogs suffering from atopic dermatitis (AD), serum levels of immunogloblin E (IgE) antibody against 25 kinds of allergen including housedust mites were determined. Housedust mites were the most frequent allergen against which 90 of the 165 allergic dogs (54.5%) by IMMUNODOT assay. With the further analysis of immunoblotting assay in the 90 dogs sensitized with housedust mites, antigenic proteins of housedust mites recognized by IgE antibodies were with the apparent molecular masses of 15, 76, 90, 98, and 170-kD. Among them, the 15-kD protein that might be identical to Group 2 antigens (Der f2, Der p2) was prominently observed (52/90). This study indicates that about a half of dogs with AD were sensitized to housedust mites, suggesting that Group 2 antigens of housedust mites may be a major allergen in canine AD.     

Stratum corneum removal facilitates experimental sensitization to mite allergens in atopic dogs

In humans with atopic dermatitis and in mouse models of IgE-mediated allergic diseases, evidence is mounting that the stratum corneum (SC) provides an important barrier against environmental allergens. At this time, it is not known whether the SC has a similar role in dogs, especially in those with atopic dermatitis. The objectives of this pilot study were to determine whether SC removal led to earlier and stronger sensitization of atopic dogs to Dermatophagoides farinae (Df) house dust mites. Five Maltese-beagle atopic (MBA) dogs were sensitized epicutaneously after the SC was removed with ten tape strips (TS group), while sensitization was done without tape strips in five other MBA dogs (nontape stripping; NTS group). During this 16 week study, sensitization was assessed with allergen-specific IgE serology, intradermal testing with Df allergens and determination of stimulation indices of blood mononuclear cells cultured with Df and stained for CD4 and the activation markers CD25 or CD30. Compared with dogs from the NTS group, those of the TS group exhibited earlier rises in Df-specific IgE serum levels, usually had higher allergen-specific IgE titres, showed higher intradermal test reactivity and had earlier increases and higher percentages of CD25- or CD30-positive activated allergen-specific peripheral CD4-positive T lymphocytes. These observations implicate a role of the SC as a barrier limiting sensitization to exogenous allergens in this experimental atopic dog model.     

House dust and forage mite allergens and their role in human and canine atopic dermatitis

This article reviews the literature regarding the role of house dust and forage mite allergens in canine atopic dermatitis. The presence of immunoglobulin E (IgE) to these mites, especially to Dermatophagoides farinae, is common in both normal and atopic dogs. Exposure of dogs to the different mites is described both in the direct environment and in the coat of animals for house dust mites and in the food for forage mites. Allergens causing allergic disease in dogs seem to be different from those in humans. Dogs seem to react to high molecular weight allergens, compared to the low molecular weight group 1 and group 2 proteases that are commonly implicated in humans with atopic diseases. Despite numerous published studies dealing with this subject, a number of questions still need to be addressed to better understand the exact role of these mites in the pathogenesis of canine atopic dermatitis and to improve the quality of the allergens used in practice.     

Evaluation of the usefulness of sensitization to aeroallergens as a model for canine atopic dermatitis in genetically predisposed Beagles

OBJECTIVE: To evaluate a model for atopic dermatitis (AD) and to measure the effect of sensitization in Beagles genetically predisposed to produce high serum concentrations of allergen specific IgE. ANIMALS: 22 laboratory Beagles. PROCEDURE: Seventeen dogs were sensitized from birth to 3 allergens (recombinant birch pollen, Dermatophagoides pteronyssinus, and D farinae). Five nonsensitized dogs from the same litters served as controls. Clinical scoring, regular intradermal testing, measurement of serum concentrations of allergen-specific IgE, and collection of biopsy specimens of skin at 23, 32, and 43 weeks of age were performed. Serial tissue sections were stained for identification of IgE+ cells, mast cells and their subtypes, T-cells, Langerhans cells, and major histocompatibility complex class-II+ cells. At the age of 15 months, dogs were continuously exposed to 2 microg of mite allergen/g of dust. RESULTS: Sensitized dogs had positive intradermal test reactions and significantly higher serum concentrations of allergen specific IgE, compared with nonsensitized dogs. In sensitized and nonsensitized dogs, a significantly higher number of mast cells was found at predilection sites, compared with the control biopsy site. The number of mast cells at predilection sites increased with age. Sensitization significantly increased the number of epidermal Langerhans cells by 23 weeks of age. The number of epidermal Langerhans cells significantly increased in nonsensitized dogs by 32 weeks of age. Clinical scoring only revealed mild transient erythema in some dogs. CONCLUSIONS: increases in concentrations of serum allergen-specific IgE and exposure to allergens is not sufficient to induce clinical signs of AD in genetically predisposed dogs.     

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.     

Total IgE and allergen-specific IgE and IgG antibody levels in sera of atopic dermatitis affected and non-affected Labrador- and Golden retrievers

Canine atopic dermatitis (CAD) is an allergic skin disease associated with IgE and IgG antibodies (Ab) to environmental allergens. The aim of this study was to determine which other factors influence serum Ab levels in CAD-affected and non-affected dogs as this has only been poorly investigated in dogs so far. Total and allergen-specific IgE levels and Dermatophagoides farinae (DF)-specific IgG1 and IgG4 were measured by ELISA in sera of 145 CAD-affected and 271 non-affected Labrador- and Golden retrievers. A multivariable logistic regression analysis including the factors age, breed, gender, castration, clinical CAD status and allergen-specific immunotherapy (ASIT) was performed. Golden retrievers had more frequently total (OR=1.87, 95% CI=1.26-2.87, p<0.01) and specific IgE levels above the threshold value than Labrador retrievers, suggesting that genetic factors influence IgE levels in dogs. Castration was generally associated with low Ab levels (OR=0.43-0.65, p<0.05). Surprisingly, dogs with CAD did not have increased odds for high IgE against any of the allergens tested. ASIT with DF was associated with high DF-specific IgG1 (OR=4.32, 95% CI 1.46-12.8, p<0.01) but was not associated with DF-specific IgG4 or decreased IgE levels. Further studies are needed to understand the role of allergen-specific IgE in CAD and of IgG1 in ASIT.     

Peripheral blood mononuclear cell responses to Dermatophagoides farinae in canine atopic dermatitis

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans that is characterised by the presence of allergen-specific IgE. Data from skin tests and serological analysis suggest that the house dust mite Dermatophagoides farinae is the most important allergen in dogs with atopic dermatitis. The aim of this study was to determine if D. farinae specific peripheral blood mononuclear cell (PBMC) responses could be detected in dogs with atopic dermatitis. PBMCs were isolated by the density centrifugation from dogs with atopic dermatitis that were skin test positive for D. farinae, dogs with atopic dermatitis that were skin test negative for D. farinae, and healthy dogs. Cells were cultured with increasing concentrations of the D. farinae extract, no antigen, vaccine antigens or concanavalin A (ConA). There was significantly greater responsiveness of PBMCs from the D. farinae positive dogs than from either the D. farinae negative or healthy dogs (ANOVA, P<0.05). In contrast, no significant differences were observed in the control responses between the three groups. This is the first study to demonstrate that D. farinae specific circulating memory cells are involved in the pathogenesis of canine house dust mite hypersensitivity.