Evaluation of IgG subclass responses against Dermatophagoides farinae allergens in healthy and atopic dogs

A semiquantitative chemiluminescent Western blot analysis system was developed and validated to evaluate antigen-specific IgG subclass responses to electrophoretically separated proteins of Dermatophagoides farinae in healthy and atopic dogs. Both groups mounted similar D. farinae-specific IgG1 and IgG4 responses to multiple antigens, but IgG2 and IgG3 responses were difficult to detect. The most commonly recognized bands in both groups were 18 and 98 kDa antigens for IgG1 and 18, 45, 66, 98, 130 and 180 kDa for IgG4. The number of bands recognized per dog did not differ significantly, but significantly more atopic dogs had an IgG1 response to a 180 kDa protein. The overall D. farinae-specific IgG1 and IgG4 responses were slightly higher, but not significantly different, in the healthy group. The results suggest that some antigens produced by D. farinae can induce different subclass responses. However, as most of these responses are seen in both healthy and atopic dogs, they are likely to merely represent recognition of foreign proteins presented to the immune system, rather than involvement in the pathogenesis of atopic dermatitis. The role of the 180 kDa antigen warrants further study.     

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.     

Immunoglobulin G responses in 21 dogs with skin diseases to antigens from different isolates of Staphylococcus intermedius

The aim of this study was to characterise the immunoglobulin G (IgG) response in 21 dogs with or without pyoderma to antigens from six isolates of Staphylococcus intermedius. The staphylococcal proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred electrophoretically on to a membrane and subjected to immunoblotting with the dogs' serum. Gels containing separated proteins from the six isolates revealed 29 to 33 distinct bands with molecular weights ranging from 20 to 230 kDa. All the dogs' sera contained IgG that recognised 12 to 24 bands (mean 17), regardless of whether the dogs had pyoderma. The recognised proteins had molecular weights ranging from 20 to 198 kDa but the majority had molecular weights below 75 kDa. The most intense band in all six isolates had a molecular weight of 28 to 29 kDa. The antibody responses to the six isolates were essentially similar except that there were significantly more bands in the response to isolate 2 than to isolate 6, and occasional differences in the intensity of individual bands. All 21 dogs mounted an IgG response to multiple antigens in S intermedius, which differed only marginally between the six isolates. This lack of variation provides evidence that the host's response to different isolates of S intermedius is not a major factor in canine pyoderma.     

Characterisation of major and minor Dermatophagoides allergens in canine atopic dermatitis

Atopic dermatitis is a well-recognised chronic inflammatory skin disease of humans and dogs. Most atopic dogs are sensitised to Dermatophagoides mites. The aim of this study was to characterise allergens in different Dermatophagoides species using polyclonal and monoclonal antibodies to canine IgE. Western blots were prepared from crude extracts of D farinae, D pteronyssinus and D microceras, and purified group 1 and 2 allergens under reducing and non-reducing conditions. They were probed with sera from atopic (n = 33) and healthy (n = 27) dogs. There was no significant difference in the sensitivity or specificity between the polyclonal and monoclonal sera in detecting Dermatophagoides -specific IgE. Major allergens common to both D farinae and D pteronyssinus were detected at 97-98 kDa, 103-104 kDa and 134-139 kDa on both reducing and non-reducing blots. Major allergens at 84-85 kDa, 65-69 kDa and 44-45 kDa were only recognised on reducing blots, suggesting that these are fragments of the larger allergens. Only a few sera recognised group 1 or 2 allergens on blots of crude extracts or purified allergens. These results confirm that, in atopic dogs, high molecular weight allergens are the most important Dermatophagoides allergens, rather than the low molecular weight group 1 and 2 proteins.     

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.     

The immunoglobulin G response to Malassezia pachydermatis extracts in atopic and non-atopic dogs

IgG immunoreactivity to Malassezia pachydermatis was compared in atopic and non-atopic dogs. Malassezia pachydermatis proteins with a molecular weight of 98 kDa were recognized at a significantly higher frequency in the sera of atopic dogs. Most of the atopic dogs with Malassezia dermatitis had a greater IgG response than did normal dogs.     

SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis

Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.     

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.     

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

Abstract 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.     

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.     

Serum IgE and IgG responses to food antigens in normal and atopic dogs,and dogs with gastrointestinal disease

In human food allergy, with or without concurrent atopy, there may be significant increases in serum allergen-specific IgE. Serological methods have been tried but are not currently recommended for diagnosis of suspected food allergy in dogs. The aim of this study was to investigate humoral immune responses to food antigens in dogs. Serum IgG and IgE antibodies specific for food antigens were measured by enzyme linked immunosorbent assay (ELISA) using polyclonal anti-dog IgG and IgE reagents. Antigens tested were beef, chicken, pork, lamb, chicken, turkey, white fish, whole egg, wheat, soybean, barley, rice, maize corn, potato, yeast and cow's milk. Three groups were examined: normal dogs, dogs with atopic dermatitis (AD); and dogs with one of four types of gastrointestinal (GI) disease: small intestinal bacterial overgrowth (SIBO), inflammatory bowel disease (IBD), food-responsive disease, and infectious diarrhoea. Statistically significant differences in food-specific antibodies were not detected between the GI subgroups. There were statistically significant differences in the IgE concentration between the normal dogs, and dogs with atopic or GI disease, for all of the antigens tested. There were statistically significant differences in the average IgG concentrations between the normal dogs, and dogs with atopic or GI disease, for all of the antigens tested, except egg and yeast. The relationship of antigen responses for pooled data was analysed using principle component analysis and cluster plots. Some clustering of variables was apparent for both IgE and IgG. For example, all dogs (normal and diseased) made a similar IgG antibody response to chicken and turkey. Compared with other groups, atopic dogs had more food allergen-specific IgE and this would be consistent with a Th(2) humoral response to food antigens. Dogs with GI disease had more food allergen-specific IgG compared with the other groups. This may reflect increased antigen exposure due to increased mucosal permeability which is a recognised feature of canine intestinal disease.     

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.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

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.     

Western blot analysis of the IgG response of sheep vaccinated with S48 Toxoplasma gondii (Toxovax)

The IgG antibody responses of sheep vaccinated by the subcutaneous injection of live tachyzoites of ‘incomplete’ strain S48 toxoplasma (Toxovax) were analysed by Western blotting. Antibodies corresponding to a range of tachyzoite antigens (13 to 48 kD) were detected, but the response was dominated by antibody recognising a 30 to 32 kD band. Unvaccinated ewes challenged orally with oocysts of the ‘complete’ M3 toxoplasma strain had a more complex IgG response that recognised antigens in six dominant bands of similar intensity as those in sheep vaccinated with S48 tachyzoites and then challenged with M3 oocysts. No differences were detected between the antigenic structures of the S48 tachyzoites and RH strain tachyzoites when the antigens were probed with immune ovine sera. Many of the anitgens of the S48 tachyzoites that were recognised had molecular weights similar to those of antigens that have been identified in other strains of toxoplasma.     

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.     

Late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with house dust mite-induced atopic dermatitis

OBJECTIVE: To determine the prevalence of late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with atopic dermatitis and an immediate reaction to D farinae. ANIMALS: 6 healthy dogs and 20 dogs with atopic dermatitis and immediate reactions to D farinae. PROCEDURE: ntradermal tests were performed with D farinae at 1:1,000 wt/vol and 1:50,000 wt/vol concentrations, and skin reactivity was evaluated after 0.25, 6, and 24 hours. Serum D farinae-specific IgE antibodies were assayed. Extent of lesions (atopy index) and pruritus (visual analogue scale) were evaluated in dogs with atopic dermatitis. RESULTS: Late-phase reactions were observed in healthy dogs at 6 hours (n = 2 dogs) and 24 hours (1) with the 1:1,000 wt/vol concentration, and at 6 hours (1) and 24 hours (1) with the 1:50,000 wt/vol concentration of allergen. Late-phase reactions in healthy dogs were only observed in dogs with an immediate reaction to D farinae. Late-phase reactions were observed in 11 of 20 dogs with atopic dermatitis at 6 and 24 hours with the 1:1,000 wt/vol concentration and in 10 of 20 at 6 and 24 hours with the 1:50,000 wt/vol concentration of allergen. There was no difference in mean atopy index, mean visual analogue scale of pruritus, or mean serum D farinae-specific IgE concentration of dogs with a late-phase reaction, compared to dogs without a late-phase reaction. CONCLUSIONS AND CLINICAL RELEVANCE: Late-phase reactions may be observed after an immediate reaction to intradermal skin testing in healthy and allergic dogs but are more commonly observed in dogs with atopic dermatitis.     

Immunoglobulin G responses to Malassezia pachydermatis in healthy dogs and dogs with Malassezia dermatitis

Serum immunoglobulin G (IgG) responses of healthy dogs and dogs with Malasseziapachydermatis dermatitis were compared by Western immunoblotting. M pachydermatis CBS 1879 was disrupted mechanically and its proteins were separated and blotted on to nitrocellulose membranes before being incubated with sera from eight healthy beagles, eight Irish setters with gluten-sensitive enteropathy, 15 healthy basset hounds, and 30 dogs with Mpachydermatis-associated dermatitis, 20 of which were basset hounds. The mean (se) numbers of bands of immunoreactivity observed in the seborrhoeic basset hounds (10.7 [0.4]) and affected mixed-breed dogs (9.4 [0.9]) were significantly greater than in the beagles (3-0 [1.0]), Irish setters (5.5 [1.1]) and healthy basset hounds (5.6 [0.7]). The number of bands identified was correlated (r(s) = 0.76, P < 0.001) with the anti-M pachydermatis IgG values measured by ELISA in a previous study. Most of the dogs were immunoreactive towards the 132, 66 and 50 to 54 kDa proteins and the affected dogs were also usually reactive towards the 219, 110, 71 and 42 kDa proteins.     

Electrophoretic and antigenic characterisation of Dermatophilus congolensis extracellular products

Dermatophilus congolensis is the causative agent of bovine dermatophilosis and lumpy wool in sheep. Two field isolates of D. congolensis, one each from a cow in Ghana and a sheep in Scotland, were cultured for 24–72 h in a synthetic medium based on RPMI-1640. Culture filtrates were examined by SDS-PAGE and considered to contain extracellular products released by growing hyphae and filaments. Electrophoretic profiles of culture filtrates of the two isolates contained common bands and bands that were unique to each isolate. The composition of extracellular products altered with increasing culture periods indicating that specific products were released at different stages of growth. Culture filtrate prepared in the presence of serine protease and metalloprotease inhibitors contained more and better defined bands than that prepared without protease inhibitors indicating the presence of proteases in culture filtrates. Western blot analysis of extracellular products using a panel of sera showed that the two isolates from different host species and distant geographical locations contained cross-reactive antigens. Natural and experimental infections stimulated antibody responses to antigens in culture filtrates, sera from animals that were disease free but in-contact with dermatophilosis-infected animals also contained antibodies to extracellular antigens. The antigens recognised by most sera had molecular weights of 200 kDa in the bovine isolate, 170 kDa in the ovine isolate and 67, 27 and 52–55 kDa in both isolates. The number of antigenic bands of both isolates was positively correlated with the intensity of challenge and the severity of infection: antibodies in sera from disease-free cattle in Ghana recognised more antigens than sera from disease-free sheep in Scotland and more antigens were recognised by sera from chronically-infected Ghanaian cattle than by sera from experimentally-infected calves and sheep. The latter developed antibodies to antigens of 27 and 24 kDa during the course of infection. The electrophoretic profiles of extracellular products of D. congolensis are less complex than those of other structures of the bacterium yet they exhibit differences between the two isolates. Extracellular products contain antigens recognised by sera from naturally exposed and experimentally-infected animals that may be involved in immunity to D. congolensis or immunopathogenesis of dermatophilosis.