A.H.M. van den Broek

Canine atopic disease: the prevalence of positive intradermal skin tests at two sites in the north and south of Great Britain

Results of intradermal skin test responses to the same panel of 53 allergens were compared in 118 dogs with atopic disease presented at two geographical centres, Edinburgh (87 cases) and London (31 cases). The allergens most commonly positive at both centres were human dander and Dermatophagoides farinae, but positive tests to all of the allergens used occurred in at least one case. The mean number of allergens to which positive tests resulted in atopic dogs was 5.126 (Edinburgh) and 5.129 (London). The majority of animals were sensitive to allergens from more than one group. A significantly higher number of positive reactors to house dust allergen was observed at London than at Edinburgh (P < 0.05), while a significantly higher number of positive reactions to grass pollens was detected at Edinburgh than in London (P < 0.05). Sensitivity to Dermatophagoides pteronyssinus, in the absence of sensitivity to D. farinae, was uncommon and therefore both of these mite allergens should be incorporated in intradermal skin testing panels in Great Britain.

Retrospective survey of allergen immunotherapy in canine atopy

The clinical records of 277 cases of canine atopy treated with specific allergen immunotherapy were reviewed. A good response was defined as control with immunotherapy either alone or with topical agents, a partial response as control with immunotherapy and other systemic agents, and a poor response as no perceived benefit and the immunotherapy discontinued. The mean follow-up period was 29.2 months (range 10 to 85 months). Ninety-one cases (33 per cent) were lost to follow-up or failed to comply with the therapeutic protocol. Of the remaining 186 cases, 40 (21.5 per cent) had a good response to immunotherapy, 74 (39.8 per cent) had a partial response, and 72 (38.7 per cent) had a poor response. Immunotherapy was therefore of long-term benefit in 114 dogs (61.3 per cent). No significant differences in response rates were associated with the breed or sex of the dog, or the age of onset of the disease, or with the type or number of allergens included in a vaccine. Dogs which had clinical signs for more than 61 months before immunotherapy had a significantly poorer response rate (23.5 per cent, P<0.05). In-house cases had a significantly better response rate (95.2 per cent, P<0.05) than externally managed cases.

Identification of tropomyosin, paramyosin and apolipophorin/vitellogenin as three major allergens of the sheep scab mite, Psoroptes ovis

Analysis by one‐dimensional (1‐D) SDS‐PAGE/Western blotting of whole mite extract (larval and adult stages) with sheep sera taken 0–84 days after infection with the sheep scab mite, Psoroptes ovis revealed a progressive IgE antibody response, with a dominant high molecular weight allergen (MW 180 kDa) detected early during infection. Further analysis by two‐dimensional (2‐D) SDS‐PAGE/Western blotting with post‐infection sera, revealed a more complex picture with numerous (> 20) IgE reactive spots. Trypsin digest and Maldi‐ToF analyses of these spots identified two house dust mite allergen homologues, namely tropomyosin (Der p 10) and paramyosin (Der p 11), and analysis of a third spot indicated an apolipoprotein‐like IgE reactive protein (Der p 14). Further 1‐D and 2‐D SDS/Western blotting, with specific antibodies to the house dust mite allergens Der p 10, 11, and to the IgE reactive peptide of the high molecular weight house dust mite allergen, Der p 14 (vitellogenin/apolipophorin), provided firm evidence for the presence of these three allergens in extracts of the Psoroptes mite. These studies show for the first time that homologues of the house dust mite 10, 11 and 14 group allergens are represented as immunodominant allergens of the sheep scab mite, and may represent important vaccine candidates.

Cutaneous hypersensitivity reactions to Psoroptes ovis and Der p 1 in sheep previously infested with P. ovis--the sheep scab mite

We have previously shown that infestation with Psoroptes ovis induces an IgE response and intense tissue eosinophilia, typical of a Type I hypersensitivity response [Parasite Immunol. 22 (2000) 407]. Intradermal tests (IDSTs) suggest that there are also delayed and Arthus-type responses to this parasite. In order to study the nature of ovine cutaneous reactions to P. ovis, naïve controls and experimentally infested sheep (n = 5) were challenged intradermally with mite antigen. Challenge elicited immediate (P < 0.001) and delayed (P < 0.005) wheal reactions in sensitised sheep. At 6 (P < 0.02) and 30 h (P < 0.001) the predominant infiltrating cells were eosinophils. To explore the role of circulating antibodies, naïve sheep (n = 5) were subjected to Prausnitz-Kustner (PK) tests. These elicited immediate (P < 0.02) but not delayed wheal reactions. At 6 h eosinophils (P < 0.001) dominated the infiltrate. These results suggest that P. ovis allergens provoke an IgE-dependent immediate and late phase response and a cell-mediated eosinophil-rich delayed-type hypersensitivity response (ER-DTH).

Attempts to immunize sheep against the scab mite, Psoroptes ovis.

Adult Psoroptes ovis were successively extracted in saline, 1% Tween, 1% Triton and 8 m urea +0·1% CHAPS. The Triton extract was separated into fractions which did or did not bind to ConA lectin. Using QuilA as adjuvant, both Triton fractions and the saline, Tween and urea extracts were tested separately as candidate protective antigens against a P. ovis challenge infestation in sheep. All induced circulating antibody responses, but the saline and Tween extracts also stimulated significant protective effects in those sheep which developed active disease. Compared to control sheep injected with adjuvant only, these immunized animals had more than two and seven‐fold reductions in mean lesion areas and mite numbers, respectively.

Identification of an antigen from the sheep scab mite, Psoroptes ovis, homologous with house dust mite group I allergens

Infestation of sheep with the ectoparasitic mite Psoroptes ovis, results in a severe allergic dermatitis. Currently, little is known about the allergens/antigens that stimulate the allergic response. We have isolated an 836‐bp cDNA from a P. ovis cDNA library which displays strong homology to cysteine proteases and, in particular, to the group I house dust mite allergens Der p 1, Der f 1 and Eur m 1. The cDNA was expressed in Escherchia coli, fused to a hexahistidine tag and the recombinant protein (Pso o 1) purified using a nickel‐affinity column. The recombinant Pso o 1 was tested for recognition by immunoglobulin (Ig)G and IgE in serum from P. ovis naïve and P. ovis infested sheep. Using Western blots, both classes of antibody to Pso o 1 were detected in postinfestation serum. In enzyme‐linked immunosorbent assays, a pronounced IgG‐antibody response to Pso o 1 was detected in five of five sheep 3 weeks postinfestation. The IgE‐antibody response to whole mite extract was poor in four of five animals. However, a marked IgE response occurred in the fifth animal, and IgE anti Pso o 1 was detected in the serum.

Infestation of sheep with Psoroptes ovis, the sheep scab mite, results in recruitment of Foxp3(+) T cells into the dermis.

Regulatory T cells (Tregs) play a central role in maintenance of immune homeostasis by controlling harmful immune responses to inappropriate antigens and are thought to play a key role in modulating hypersensitivity reactions. Infestation of sheep with Psoroptes ovis results in a pronounced cutaneous hypersensitivity‐type response, which appears to be crucial for mite survival. We hypothesize that (i) Tregs are involved in sheep scab lesions and (ii) Treg responses may crucially affect lesion development and subsequent mite survival. Foxp3 is a key transcription factor required for generation and maintenance of Tregs in rodents and humans, and is the most widely used marker for Tregs in these species. In this study, we sequence ovine foxp3 and show that it exhibits a high degree of homology with foxp3 from other species. Using a validated immunohistochemical staining technique, we demonstrate that infestation of sheep with P. ovis results in an influx of Foxp3+ T cells into the skin. Future work will investigate the regulatory function of ovine Foxp3+ T cells and determine whether the quality of the Treg response to P. ovis plays a role in individual susceptibility to the mite.

Epidermal and hepatic glucocorticoid receptors in cats and dogs.

Low capacity, high affinity [3H] dexamethasone binding receptors were identified in cytosolic preparations of liver (mean number 45 +/- 10.1 fmol mg-1 protein, apparent dissociation constant 0.4 +/- 0.1 nM) and skin (mean number 46.4 +/- 23.8 fmol mg-1 protein, apparent dissociation constant 1 +/- 0.2 nM) of clinically normal dogs. For clinically normal cats, approximately half these numbers of receptors with a lower affinity, were detected in liver (mean number 23.1 +/- 10.4 fmol mg-1 protein, apparent dissociation constant 3.2 +/- 0.9 nM) and skin (mean number 23.90 +/- 10.9 fmol mg-1 protein, apparent dissociation constant 2.2 +/- 1.5 nM). This difference between dogs and cats in [3H] dexamethasone binding receptors may contribute to the relative glucocorticoid resistance observed in cats.

Evaluation of a human generic formulation of ciclosporin in the treatment of canine atopic dermatitis with in vitro assessment of the functional capacity of phagocytic cells

To compare the efficacy, tolerability and safety of a generic formulation of ciclosporin for human beings with prednisone in the treatment of canine atopic dermatitis), human generic ciclosporin A (hgCsA) (5 mg/kg daily) and prednisone (1 mg/kg daily for seven days, followed by 1 mg/kg every second day) were administered to 13 and seven dogs with atopic dermatitis, respectively, for 42 days. Skin changes were assessed using a modified canine atopic dermatitis extent and severity index (mCADESI-01) and a pruritus intensity scale system. The in vitro functional capacity of phagocytic cells was assessed using the tetrazolium reductase activity and zymosan-stimulated tetrazolium reductase activity tests, as well as measurements of the percentage phagocytic activity and the ingestion capacity of phagocytic cells. Haematological and biochemical parameters were also monitored. There was a greater than or equal to 50 per cent reduction from the baseline in mCADESI-01 scores in 84.6 and 100 per cent of dogs, and a greater than or equal to 50 per cent reduction from the baseline in pruritus scores in 76.9 and 85.7 per cent of dogs, treated with hgCsA and prednisone, respectively. No important adverse physical, haematological or biochemical effects occurred with either drug and no statistically significant changes were detected in any of the four tests assessing the functional activity of phagocytes. The generic formulation of ciclosporin was effective in reducing the severity of physical signs of canine atopic dermatitis and was well tolerated.

Temporal pattern of isotype-specific antibody responses in primary and challenge infestations of sheep with Psoroptes ovis--the sheep scab mite

In sheep Psoroptes ovis provokes an allergic dermatitis with significant P. ovis antigen-specific IgE responses. The kinetics of the IgE response to primary and challenge infestations of P. ovis were reported earlier [Parasite Immunol. 22 (2000) 407]. The present study examines IgG, IgM and IgA responses to primary and challenge infestations of P. ovis and the profile of antigens/allergens reacting with IgG and IgE antibodies. Antigen-specific enzyme-linked immunosorbent assays (ELISAs) demonstrate that primary infestations elicited significant increases in levels of IgG and IgM but not IgA antibodies. IgG and IgM responses to primary and challenge infestations were not significantly different. Western blots of reduced P. ovis proteins indicate that IgG antibodies reacted with five major antigens following primary infestation and only three of these after challenge infestation. IgE antibodies bound to three major and five minor allergens after primary infestation and two additional minor allergens after challenge infestation. Immunodominant antigens >100 and <15 kDa and allergens >100 kDa were most consistent in stimulating substantial IgG and IgE antibody responses, respectively. These antigens/allergens may be exploited in immunodiagnosis and modulation of the host immune response.