Chapter 1.3
Serum anti-Staphylococcus pseudintermedius IgE and IgG antibodies in dogs with atopic dermatitis and nonatopic dogs
Background – Dogs and humans with atopic dermatitis (AD) are predisposed to colonization and recurrent infection with Staphylococcus spp. Studies in humans suggest that staphylococcus-specific immunoglobulin E (IgE) plays a key role in disease pathogenesis. Few such studies have been undertaken in dogs.
Hypothesis/Objectives – The aim of this study was to compare levels of staphylococcus-specific IgE and immunoglobulin G (IgG) in dogs with AD, nonatopic dogs with staphylococcal pyoderma, and nonatopic and noninfected control dogs.
Animals – Sera were collected from 108 dogs with AD, 39 nonatopic dogs with staphylococcal pyoderma secondary to different underlying conditions, 67 age-matched nonatopic control dogs, and nine control dogs reared in minimal disease conditions.
Methods – Serum Staphylococcus pseudintermedius-specific IgE and IgG antibodies were measured by enzyme-linked immunosorbent assay.
Results – Dogs with AD had significantly higher levels of anti-staphylococcal IgE than nonatopic dogs with staphylococcal pyoderma and the two groups of control dogs. Levels of anti-staphylococcal IgG were significantly higher in atopic dogs and nonatopic dogs with pyoderma compared with nonatopic control dogs and control dogs reared in minimal disease conditions, but there was no significant difference in levels of anti-staphylococcal IgG between dogs with AD and nonatopic dogs with pyoderma.
Conclusions and clinical importance – A significantly increased IgE response to S. pseudintermedius antigens in atopic dogs suggests an immunopathogenic role for anti-staphylococcal IgE. The finding of elevated IgE and IgG in atopic dogs is also important as a prelude to studies on antigenic specificity and possible correlations with disease phenotype.
Introduction
Staphylococcal infection is a major complicating factor in both canine and human atopic dermatitis (AD).1,2 Furthermore, it is increasingly recognized that the immunoglobulin E (IgE) response to staphylococcal antigens in humans plays an important role in the pathogenesis of the disease.3 There are, in fact, two conditions in humans associated with anti-staphylococcal IgE and recurrent staphylococcal skin infections, namely AD and hyper-IgE syndrome.1 However, no condition analogous to the latter has yet been reported in dogs.
Numerous studies have investigated the IgE response to Staphylococcus aureus antigens in human AD.1,4-6 Amongst the atopic diseases, the production of IgE directed against microbial antigens appears to be limited to AD, and there is no such response in patients suffering from atopic respiratory diseases.6,7 In most studies, allergen-specific IgE levels correlate with disease severity, which is suggestive of a significant immunopathogenic role.5,6 More recently, attention has focused on soluble staphylococcal toxins, particularly superantigens, which are documented in >50% of cases.6,8 Not only are superantigens capable of inducing inflammation through polyclonal B and T cell activation, but they also provide a rich source of allergen to the skin immune system.8,9 It is suggested that the combination of superantigen-producing staphylococci and the subsequent IgE response may be very important factors in determining the severity of AD.6,8,9
The major canine skin pathogen is Staphylococcus pseudintermedius,10 and its complete genomic sequence has been recently published.11 It is known that dogs suffering from canine AD have a higher rate of carriage of S. pseudintermedius than do normal dogs, and that lesional skin of affected dogs is frequently colonized by this organism.12 Staphylococcus pseudintermedius produces a range of exotoxins, including superantigens. In one study, 25 of 96 isolates from cases of canine pyoderma in the UK were shown to produce multiple superantigens, usually staphylococcal enterotoxin A (SEA) and C (SEC).13 In another study, a novel enterotoxin-related gene, se-int, found in all 44 isolates examined, shared >50% homology with both SEC and staphylococcal enterotoxin B (SEB).14
There are few published studies on the immune response to staphylococcal antigens in canine AD and other canine skin conditions. One study examined the IgE and IgG response to staphylococcal antigens in 31 dogs with pyoderma secondary to AD, 34 cases of recurrent idiopathic pyoderma, 14 cases of idiopathic deep recurrent pyoderma, 15 cases of single-episode pyoderma, and 39 healthy control dogs that were not age matched.15 Both dogs with AD and those with recurrent superficial pyoderma had significantly higher levels of anti-staphylococcal IgE than did the healthy dogs. Levels of IgG antibody appeared to be much higher than those of IgE, and were found to increase with increasing chronicity of the infection. A later study examined the IgG and IgA response to staphylococcal antigens in dogs suffering from AD with and without concomitant pyoderma, as well as cases of idiopathic deep pyoderma, pustular demodicosis, flea-allergy dermatitis with pyoderma and anal furunculosis, and in healthy control dogs (albeit not age matched).16 All affected animals had significantly higher levels of IgG antibody than the healthy control animals, but no significant differences were found between the affected groups. However, the significance of this study, which did not assess IgE levels, for the pathogenesis of AD is unclear.
Canine AD and human AD share many common features.17 The most recent research in human AD has emphasized the very important role of staphylococcal infection and the subsequent immune response; thus, there is a need to revisit this in canine AD, employing age-matched control animals and more refined methods of antigen extraction. The aim of this study was to measure levels of staphylococcus-specific IgE and IgG in a large population of dogs suffering from AD and compare them with levels in dogs suffering from staphylococcal infection secondary to other causes and in two control groups with no history of skin disease or infection. We hypothesized that if an immunopathogenic role for anti-staphylococcal IgE in canine AD exists, anti-staphylococcal IgE levels would be greater in atopic dogs than in age-matched nonatopic dogs, irrespective of their pyoderma status.
Materials and methods
Serum samples
Sera were assayed from the following groups.
The number, sex and ages of dogs in each group are shown in Table 1. The study was approved by The University of Liverpool School of Veterinary Science Ethics Committee. In the case of Groups 1–3, all sera were derived from excess serum that was drawn for diagnostic purposes. In the case of Group 4, sera were obtained and supplied under the appropriate licence (Charles River Laboratories via the Minister of Health and Children, Ireland).
Table 1. Number, sex and age of dogs in the study
Antisera
An alkaline phosphatase-conjugated monoclonal antibody (mAb, clone 5.91; North Carolina State University, Raleigh, NC, USA) was used to detect staphylococcus-specific IgE. This antibody has been shown to be specific for canine IgE with no cross-reactivity to IgG.20 Staphylococcus-specific IgG was detected using alkaline phosphatase-conjugated goat anti-dog IgG (γ-chain specific; Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA).
Preparation of Staphylococcus pseudintermedius antigen extract
Fifteen S. pseudintermedius isolates from cases of canine pyoderma were used in initial studies. Soluble and cell wall-associated antigens from both stationary and exponential growth phases were prepared as previously described.21 The extracted proteins were assessed for protein A content by PAGE and western blotting using a chicken anti-protein A (IgY; Gallus Immunotech, Fergus, ON, Canada). The specificity of the chicken anti-protein A was shown using S. aureus Newman SpA (protein A-producing) and S. aureus Newman Δ SpA (nonprotein A-producing) positive and negative controls, respectively. Three strains that were protein A negative using this criterion were further assessed for IgG binding. The preparation derived from a strain (8012) that showed minimal binding to serum from a MD dog was selected for use in the study.
Assessment of Staphylococcus pseudintermedius strain 8012 for se-int gene
Strain 8012 was assessed for the presence of the superantigen se-int gene by PCR. Genomic DNA was extracted from 500 μL of stationary-phase culture grown in tryptic soy broth at 37°C using the Edge Biosystems PurElute Bacterial Genomic kit (Edge Biosystems, Gaithersburg, MD, USA) following the manufacturer’s instructions, with the addition of 250 μg/mL (final concentration) of lysostaphin (Ambi Products, Lawrence, NY, USA) prior to incubation. The PCR was performed using Promega GoTaq DNA Polymerase (M8301) with the Promega dNTP mix (U1511; Promega UK Ltd, Southampton, UK), employing the following primers (Invitrogen, Life Technologies Ltd, Paisley, UK): se-int forward 5′-TATAGGTACCCTTGGACTTTTGGATG-3′; and se-int reverse 5′-TGGCGAGCTCCAAATCCATTAGCC-3′. Appropriate positive (genomic DNA from S. pseudintermedius strain ED99) and negative controls (no template) were included.
Enzyme-linked immunosorbent assay (ELISA)
Serum staphylococcus-specific antibodies were assayed by ELISA using protocols modified from previous studies.20,22 Briefly, microtitre plates (Greiner Labortechnik, Frickenhausen, Germany) were coated with S. pseudintermedius extract diluted 1:1000 (IgE ELISA) and 1:3000 (IgG ELISA) in 0.05 M carbonate/bicarbonate buffer, pH 9.6, and blocked with phosphate-buffered saline containing 0.5% polyvinylpyrrolidone (average molecular weight 10,000) and 0.5% sucrose. All dilutions of serum and secondary antibodies were in Tris-buffered saline containing 0.05% Tween-20 (TBST) and 0.5% human serum albumin. Samples were assayed in duplicate at dilutions of 1:10 (IgE) and 1:400 (IgG). Immunoglobulin E was detected with 1 μg/mL phosphatase-conjugated mAb (clone 5.91); IgG was detected with 0.125 μg/mL phosphatase-conjugated goat anti-dog IgG (γ-chain specific). Between incubation steps, plates were washed three times with TBST using an automated plate washer (Bio-Tek, Winooski, VT, USA). Colour was developed using AP Yellow One Component Microwell Substrate (pNPP; BioFx Laboratories, Owings Mills, MD, USA) for 30 min at room temperature before the reaction was stopped by the addition of 1 M NaOH. Absorbances were determined at 405 nm using a microplate reader (Tecan, Männedorf, Switzerland). Results were expressed as optical density (OD) units at 405 nm, determined as an average of duplicate results after correction by subtracting the mean OD value of duplicate blank wells.
Statistical analysis
All data were analysed using SPSS 20.0 (IBM UK Ltd, Portsmouth, UK). Differences were considered statistically significant when the P-value was <0.05. After testing for normality, differences in age and levels of antibodies between groups were tested by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons, assuming unequal variance between groups. Linear regression was used to assess whether there was any statistically significant association between duration of staphylococcal infection and levels of anti-staphylococcal antibodies.
Results
PCR results for strain 8012
The PCR results were positive for the superantigen gene se-int.
Reproducibility
From the assay controls, the mean intra-assay variations were 6.3 and 9.7% for IgE and IgG ELISAs, respectively. Mean interassay variations were 11.6 and 12.2% for IgE and IgG ELISAs, respectively.
Serum anti-Staphylococcus pseudintermedius IgE
There was a highly significant difference in anti-staphylococcal IgE levels between the four groups (P < 0.009, ANOVA; Figure 1). Group 1 atopic dogs had significantly higher levels of anti-staphylococcal IgE than Group 2 nonatopic dogs with pyoderma (P < 0.05, Dunnett’s post hoc test) and both the age-matched Group 3 nonatopic control dogs and Group 4 MD control dogs (both P < 0.05, Dunnett’s post hoc test). Significantly higher levels of anti-staphylococcal IgE were also seen in the Group 2 nonatopic dogs with pyoderma and Group 3 nonatopic dogs compared with the Group 4 MD dogs (both P < 0.05, Dunnett’s post hoc test). There was no significant difference in IgE levels between the Group 2 nonatopic dogs with pyoderma and the Group 3 nonatopic dogs without pyoderma.
Figure 1. Anti-staphylococcal IgE in Group 1 dogs with atopic dermatitis, Group 2 nonatopic dogs with pyoderma, Group 3 nonatopic control dogs, and Group 4 minimal disease control dogs. Bars indicate the mean of each group. a Significantly different from Group 2, P < 0.05; b significantly different from Group 3, P < 0.05; and c,d,e significantly different from Group 4, P < 0.05.
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