Gribbles Veterinary Pathology and

Heska Corporation proudly present

Atopic Dermatitis Allergy

Testing and Management
Introducing the Heska
Allercept® IgE Test
Contents
Introduction 3
Atopic Dermatitis in the Dog: Allergy Testing and Management 4
- Definitions 4
- Pathophysiology of atopic dermatitis 4
Diagnosis of Atopic Dermatitis 7
- History and Clinical Signs 7
- Differential Diagnoses and Ruling Them Out 8
- Case Selection for Allergy Testing 10
- Case Selection for Avoidance Strategies 10
- Case Selection for Allergen-Specific Immunotherapy 10
Options for Allergy Testing 11
- Intradermal Testing 11
- Allergen-Specific IgE Serology 12
Controversies in Allergy Testing 13
- Why do some dogs with clinical AD have negative allergy tests? 13
- What’s wrong with allergen mixes in testing? 13
- Why is there a lack of correlation between IDT and ASIES? 14
- Is IDT or ASIES better at identifying allergens for 14
ASIT in seasonal allergic dogs?
- Does outcome of immunotherapy depend on the test used? 14
- What are the effects of glucocorticoids and cyclosporin on allergy testing? 14
Clinical Decision Making: IDT vs. ASIES 15
- When to perform allergen specific IgE serology with a view to 15
in-house ASIT or avoidance.
- When to refer for further workup or IDT. 15
Management of the atopic patient. 15
- Improving the epidermal barrier 16
- Controlling infections 17
- Controlling the allergy 19
Why are accurate results important in IgE testing ? 24
Submission of Samples for AllerceptR IgE Test 30

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Introduction
Gribbles Veterinary Pathology in conjunction with Heska Corporation is delighted to make
available to the veterinary practitioners of Australia the Allercept® IgE Test.

This document contains an overview of atopic dermatitis in the dog, allergy testing and
management and comparative data on the Allercept® IgE Test. Gribbles Veterinary
Pathology gratefully acknowledges the support of Specialist Veterinary Dermatologists in Aus-
tralia in the development of the test for Australian conditions and for their assistance in
providing information and presentations on atopic dermatitis.

Details on submitting samples for testing on the Allercept® IgE test are provided at the end of
this document. For further information please contact Gribbles Veterinary Pathology
on 1300 307 190.

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Atopic Dermatitis in the Dog: Allergy Testing and Management
David Robson, Rebecca Bassett and Greg Burton
Animal Skin Ear and Allergy Clinic
Melbourne Veterinary Specialist Centre
70 Blackburn Rd, Glen Waverley Vic 3150

Definitions

Atopic dermatitis (AD): a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic
clinical features including pruritus and secondary infections. It is associated most commonly with IgE antibodies to
environmental allergens.

Allergen-specific immunotherapy (ASIT): the practice of administering gradually increasing quantities of an aller-
gen extract to an allergic subject to ameliorate the symptoms associated with subsequent exposure to the causative
allergen

‘Allergy’ testing: a convenient but inaccurate phrase referring to intradermal testing or allergen-specific
IgE serology.

Intradermal testing (IDT): The process of introducing small quantities of allergen into the dermis of atopic patients
to try and assess specific hypersensitivities. Positive reactions may be classed as immediate phase or late phase.

Allergen-Specific IgE Serology (ASIES): Measurement of serum immunoglobulin E concentrations that can bind
to specific allergens.

Pathophysiology of atopic dermatitis

Overview

Atopy is a multifactorial disease in which genetically predisposed dogs may exhibit a combination of cutaneous
IgE-mediated immediate and late-phase reactions to environmental allergens and exhibit a myriad of immunological
changes leading to pruritus. Immunological abnormalities, antigenic stimuli, altered physiological and
pharmacological reactions and genetic predisposition all play a role in the pathogenesis. Atopy can be
considered in two phases; the acute phase and the chronic phase.

In the acute phase an epidermal barrier defect could facilitate contact of environmental allergens and microbes
with epidermal immune cells at skin sites of trauma and friction, and areas with increased surface humidity
(interdigital areas, external ear canals etc.). There are epidermal dendritic cells that partake in allergen processing
and initiation and augmentation of the inflammatory response. Leukocytes, keratinocytes are all involved in the
inflammatory reaction. There is an early influx of granulocytes and an allergen specific Th2 response, thus
promoting IgE production.

In the chronic phase there is a significant role from microbes, self-trauma and neuromediators all of which
contribute to the chronic inflammation. Chemokines are released in a continuous allergen-dependent and
independent cycle, with subsequent influx and activation of leukocytes and further chemokines and mediator
release. A Th1 profile may then predominate. The failure to down-regulate pro-inflammatory mechanisms leads to
self-perpetuating cutaneous inflammation. Exaggeration of the barrier dysfunction in lesional skin may result in
non-immunologically mediated pruritus due to environmental proteases and water loss (dry skin) as well as
predispose to further allergen absorption and irritable skin. Furthermore inflammation results in upregulation of
cellular and neurological receptors that result in increased sensitivity of neural pruritic pathways

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Role of Genetics and Heritability

Heritability is a measure of the degree to which inheritance plays a part in the aetiology of the disease and if this ap-
proaches 1, then the trait is considered to be entirely genetically determined. A study in 429 Labradors and Golden
retrievers revealed a heritability of 0.47. This means there is a significant contribution from the environment and
from genetic factors. In humans, the calculations vary from 0.425 to 0.495. A precise mode of inheritance has not
been found, but it is considered a polygenic disorder.

The Hygiene Hypothesis

The hygiene hypothesis of atopic disease in humans suggests that environmental changes in the industrialised world
have lead to a decrease in microbial contact at an early age and thus resulted in the growing epidemic of atopic
eczema, allergic rhinoconjunctivitis, and asthma over and above what genetics alone can explain. More recently a
modified version of this original hypothesis has been developed. This suggests that it is not just a decreased
exposure to all infections that increases risk of atopic disease, but more specifically is related to establishment of
intestinal microbiota and subsequent development of oral tolerance in the infant. There is evidence to suggest
antibiotics early in life can risk allergic development.

Role of Leukocytes

There is a complex interplay between a variety of cell types such as mast cells, T cells, dendritic cells, eosinophils,
neutrophils, B cells, and macrophages through release of chemokines or inflammatory mediators.

Dendritic cells
Langerhans cells are the principle antigen presenting dendritic cell of the epidermis. There are higher numbers in
lesional and non-lesional AD skin. Langerhans cells express FcεRI (receptor for IgE) and process allergen and
present this to T cells. Subsequently, epidermal (including inflammatory dendritic epidermal cells or IDECs in
humans), as well as dermal dendritic cells may also have a significant role in skewing the T-cell response to the
absorbed antigen and may therefore be critical to the initiation of the atopic state.

T lymphocytes
T lymphocytes play a major role in lesional skin of dogs with AD. There are two subsets recognised in the patho-
genesis of atopic dermatitis; Th1 and Th2. In early lesions a Th2 profile tends to predominate and the cytokines
produced promote IgE production. It is considered that a Th1 profile predominates with chronicity. ASIT has been
shown to trigger a switch to a Th1 profile and induce regulatory T cells that can downregulate the severity of the
allergic reaction.

B lymphocytes
B cells are important in that they synthesise antigen specific antibodies esp. IgE and IgG. They are not a major
component of the atopic inflammatory infiltrate though so immunoglobulin synthesis probably occurs in
extracutaneous sites such as lymph nodes, spleen or bone marrow.

Mast Cells
Mast cells (MC) exert their effect by synthesising and releasing inflammatory mediators, e.g. histamine, proteases
and cytokines. In AD this is triggered by binding and crosslinking of allergen-specific IgE to MC bound IgE-specific
receptors (FcεRI). Upon degranulation, the granule contents cause the signs of allergic inflammation by
interaction with microvasculature and other inflammatory cells. Mast cells in the atopic individual have a higher
releasability by IgE-mediated degranulation than normal dogs

Mast cells are found in highest numbers where the body interfaces with the environment such as around blood
vessels of pinnae, interdigital areas and lowest density is on nasal planum. Mast cell numbers are also higher in
atopic lesional skin, but otherwise there is no difference between non-lesional atopic skin and normal skin.

5
Eosinophils
Eosinophils play a role in chemotactic stimuli in the inflammatory reaction. They have receptors on their cell surface
for IgG, IgE and other inflammatory mediators. Eosinophil cationic protein is a major pruritic mediator in human AD.

Keratinocytes
They can release cytokines and chemokines (chemotactic cytokines) that recruit leukocytes to the site of
inflammation. It is proposed in humans this may occur following aeroallergen or detergent exposure. Activated
keratinocytes can release mediators, including proteases that directly induce pruritus.

Role of Antibodies

IgE is not considered to be the mainstay of development or perpetuation of atopic dermatitis. In humans they
describe two groups of atopics, those who have high levels of allergen specific IgE (extrinsic atopics) and those
that do not (intrinsic atopics). It is also well accepted in dogs that not all atopic dogs will display allergen-specific
IgE either. Furthermore, allergen-specific IgE can occur in the normal dog, IgE concentrations do not necessarily
drop with successful allergen-specific immunotherapy and AD has been recognised in human atopics with
agammaglobulinaemia!

Allergen specific IgG has been identified in dogs with AD, but concentrations are generally higher in normal
individuals than atopics, so IgG may be playing a protective role. However, there are also IgG receptor bindingsites
on mast cells, just as there are for IgE, and binding of immunoglobulins to either can induce degranulation of the
mast cell.

Role of Barrier Dysfunction

Loss of the epidermal barrier facilitates penetration of bacteria, allergens and chemical irritants that further
enhance the inflammatory process. This may explain the high clusters of Langerhans cells at the site of allergen
penetration. Barrier defects have been well described in humans and there is evidence to suggest similar
intercellular lipid defects in the stratum corneum of atopic dogs and probable barrier defects.

The skin consists of epidermis and dermis and provides the protective barrier to the environment. The outermost
layer of the epidermis is the stratum corneum (cornified layer) and disruption to this layer reduces the barrier to
pathogens and allows moisture loss. Disruption to the outer layer may result from environmental trauma or from
certain skin disorders. Repeated epidermal insults from scratching lead to an increase in epidermal turnover and
consequently to epidermal hyperplasia. This in turn leads to inflammation where there is an increased migration of
active Langerhans cells and other inflammatory cells into the epidermis.

The epidermis is an extremely active site of lipid synthesis. Lipids are important in barrier function, stratum
corneum water holding, cohesion and desquamation of corneocytes and control of epidermal proliferation and
differentiation. The three most abundant lipid species in the stratum corneum are cholesterol, ceramides and free
fatty acids. Ceramides are the most important lipid component for the lamellar arrangement of the stratum corneum
and consequently as a barrier function as they allow stretching and bending. Polyunsaturated fatty acids (such as
the omega-6 linoleic acid contained in sunflower oil) are incorporated into the ceramides.

There are differences in the lipid content of skin depending on the anatomic region. Water-soluble antigens (e.g.
plant pollens) are commonly in contact with the lipid-depleted sites. The skin in atopic dermatitis is known to have
a paucity of stratum corneum lipids and is therefore more readily sensitised to hydrophilic antigens. Immunological
abnormalities in the atopic individual also contribute to differences in sensitisation thresholds.

Sebum provides an oily layer over the stratum corneum that helps a little retain moisture. It also helps form a
chemical and physical barrier against potential pathogens. Resident bacteria of the skin and hair follicles produce
lipase which breaks down sebum resulting in fatty acid products that have antibacterial properties.

6
In atopic dermatitis in dogs and humans there have been abnormalities associated with the epidermal barrier.
This leads to three basic problems:

• microfissures form, which then provide a portal of entry to pathogens and allergens
• increase in scale in dry skin presents a greater surface area for bacterial and yeast adhesion, which leads to
secondary infection
• increases in allergic and non-allergic itch (see management for details)

Role of Infections in Atopic Dermatitis

During inflammation, antimicrobial peptides such as defensins and cathelicidins are released by inflammatory cells
and keratinoctytes. In atopic humans there is a lower level of these antimicrobial peptides. This, together with
the fact that there is a greater adherence of staphylococcal bacteria to atopic keratinocytes, localised defects in
barrier function, altered microenvironmental humidity, a TH-2 skewed immune response suppressing an effective
anti-microbial immune response makes atopic individuals more prone to skin infections. Bacterial exotoxins can act
as superantigens and augment the inflammatory response and worsen pruritus. In dogs, hypersensitivity to
Malassezia spp. has also been recognised in recent years. All this leads to more severe pruritus and skin trauma.

Role of Inflammatory Mediators

There are many chemical inflammatory mediators that play a role in the pathogenesis of atopic dermatitis in
triggering inflammation and pruritus and modify the perception of pruritus. It is important to realise that the most
talked about one of these, histamine, is only one of many. There are also several neuropeptides, phosphodiesterases,
eicosanoids, proteases, opioids, kinins, neutrotrophins, endovanniloids and endocannabinoids that all play a role.

Role of Toll-like Receptors

Toll-like receptors (pattern recognition receptors in the immune response to infections) may also play a role in the
pathogenesis of atopic dermatitis. Whether or not there is TLR polymorphism or whether TLR activation and
augmentation of an autoimmune response plays a role, is not yet fully elucidated.

Diagnosis of Atopic Dermatitis

A diagnosis of atopic dermatitis is made based on a history and clinical criteria consistent with AD and ruling out
other differential diagnoses. The skills then required to make this diagnosis are:

a) a thorough understanding of all differential diagnoses and their basic pathogeneses,

b) sound history taking and clinical examination skills
c) sound cytological skills, and importantly
d) a logical diagnostic approach.

History and Clinical Signs

Atopic dermatitis presents with variable clinical signs in both dogs and humans. While the diagnosis may be far
more likely in the West Highland White terrier that chews its paws for the same three months per year every year,
many cases are not so straightforward, and there is not one single historical or clinical feature that, if present,
indicates the presence of AD. Several schemes have been proposed to better define clinical criteria
(see Table 1 next page) and while helpful as a guide none are perfect.

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Table 1: Clinical Criteria Proposed for Diagnosis of Atopic Dermatitis in Dogs

Willemse (1986, 1988) Prelaud (1998)

Major Features (must have at least 3) Major Criteria (must have at least 3)

• Pruritus • Corticosteroid-sensitive pruritus

• Facial and/or digital involvement • Pinnal erythema
• Lichenification of the flexor surface of the tarsal • Bilateral cranial erythematous pododermatitis
joint and/or extensor surface of the carpal joint • Cheilitis
• Chronic or chronic-relapsing dermatitis • First appearance of signs 6 months to 3 years
• Individual or family history of AD and/or breed
predilection

Minor Features (must have at least 3)

• First appearance of signs < 3 years

• Facial erythema and cheilitis
(inflammation of the lips)
• Bacterial conjunctivitis
• Superficial staphylococcal pyoderma
• Hyperhidrosis
• IDT positive
• ASIES positive
• Elevated serum allergen-specific IgGd

Differential Diagnoses and Ruling Them Out

The challenge in the pruritic dog where AD is a consideration is that there are potentially numerous differential
diagnoses which may or may not be concurrent with a diagnosis of AD. Add to this confounding factors such as
environmental exacerbation and secondary infections (Table 2) and the challenge becomes considerable in some
cases. A complete physical examination and history taking are mandatory for this reason in all cases, and some
differential diagnoses may be able to be ruled out by these alone.

8
Table 2: Factors that can cause or contribute to pruritus (not exhaustive)

Primary Disease Modulating Factors Amplifying Factors

• Atopic dermatitis • Dry skin • Bacterial colonisation, folliculitis,
furunculosis
• Flea bite hypersensitivity • Heat • Malassezia dermatitis
• Insect bite allergy • Boredom • Psychogenic / temperament factors
(e.g. Mosquito)

• Food adverse reaction • Anxiety

• Contact allergy / irritation • Stress
• Scabies • Humidity
• Cheyletiella • Maceration

• Demodicosis (rarely pruritic on it’s own,

but common cause for pruritic
secondary infection, can be concurrent
in some AD-susceptible breeds or
following GC therapy)
• Neoplasia (e.g. some cases of mast cell
tumour, epitheliotrophic t-cell
lymphoma)
• Autoimmune disease (e.g. some cases of
pemphigus foliaceous)

Once a list of differential diagnoses fitting with the clinical presentation and history is formulated, specific tests
(Table 3) can be performed to help rule them in or out. If a disease is confirmed then this should be treated
appropriately and the patient then reassessed to see if AD is still a consideration.

Table 3: Tests used to help make a diagnosis of atopic dermatitis.

Test Rationale
Superficial & Deep Skin Scrapings Diagnosis of parasitic skin diseases
Tape Cytolog
Otic Cytology Diagnosis of bacterial or yeast skin infections
Pustule / Papule Cytology Diagnosis of bacterial or yeast ear infections or inflammation
Diagnosis of bacterial, demodex infections; may be supportive of
Trichogram contact dermatitis, pemphigus foliaceous
Diagnosis of demodicosis, dermatophytosis; can help differentiate
Flea Control Trial (>2 weeks) traumatic from non-traumatic hair loss in cases where pruritus is
Scabies Treatment trial (>2-4 weeks) not directly observed
Elimination Diet Trial (>6 weeks, Diagnosis of flea bite hypersensitivity
novel protein) Diagnosis of scabies
Contact Avoidance trials (>10-14 days)
Skin Biopsy Diagnosis of adverse food reaction

Supportive of contact dermatitis

Rarely useful in most cases of pruritic skin disease because

histopathological changes are often non-specific; however it is
important for confirmation of the diagnosis of neoplastic or
autoimmune diseases

It is then only at this stage, once differential diagnoses have been identified and managed and AD clinically
confirmed that consideration should be given to allergy testing.

9
Case Selection for Allergy Testing

If AD is a diagnosis of exclusion where a compatible history and clinical presentation exist, it means that most
patients should be able to be clinically confirmed as suffering atopic dermatitis without ever having had a test for
airborne allergens performed. So what is the role for allergy testing if it is not appropriate or necessary for making
a diagnosis of AD?

The only reasons that allergy testing needs to be performed in atopic animals are:
• to identify airborne allergens that may be avoidable
• to identify airborne allergens for inclusion in allergen-specific immunotherapy (aka ASIT, allergy vaccine)
• where an owner ‘just wants to know’ for their peace of mind

This means that if symptomatic therapy for pruritus and / or infections is to be the only therapy employed in a
clinically confirmed AD patient where the owner has no interest in the fine details of triggering allergens, then any
form of allergy testing in that patient is redundant. Furthermore, even if allergens may be able to be identified on
allergy testing, if a patient or client is not an appropriate case for avoidance or ASIT, then again any form of allergy
testing in that patient is redundant because these cases will need to be managed symptomatically only. However, if a
client and patient are good candidates for possible avoidance strategies and / or immunotherapy, then allergy
testing is strongly recommended, as this can lead to treatment options that can minimise or eliminate drug usage
(and concurrent adverse drug reactions) while still maintaining patient comfort.

Case Selection for Avoidance Strategies

Avoidance of airborne allergens is difficult and not practical in many cases. However there are some specific cases
where avoidance of the allergens in atopic dermatitis may be possible as a sole therapy, and in these cases allergy
testing is required to confirm the allergen that needs to be avoided. These cases are described in more detail below
in the management section.

Case Selection for Allergen-Specific Immunotherapy

ASIT is a useful tool in the management of many cases of Table 4: Advantages and disadvantages of ASIT
AD (Table 4). However, not all cases are suited to this mode
of therapy. Advantages Disadvantages

There are two critical stages during the work-up of an • Less frequent • Owner fear of
atopic dog where the appropriateness of ASIT needs to be administration than giving injections
considered. symptomatic therapy • Possible poor
• No long term side patient tolerance
1. Prior to allergy testing. ASIT (and therefore allergy effects reported of injections
testing) is indicated for a patient when a • Acceptance of • Risk of
clinical diagnosis of atopy is made, where suspected injections compared anaphylaxis
allergens are unavoidable, and where symptomatic to oral medication • Initial up front
therapy is ineffective or associated with unacceptable • Often more cost cost
side effects (e.g. glucocorticoids). Importantly, there effective especially • Client education,
are client factors as well that must be satisfied: the medium to large patient follow
ability to either give injections or get them done, the breeds up and support
ability for regular rechecks, the ability to understand • Preventative, not essential to the
the process, the ability to support the up front costs reactive treatment success of ASIT
of testing and vaccine, and the ability to communicate
effectively

2. After allergy testing. ASIT is strongly indicated where the allergens identified in testing match with the
presence of the allergens in the environment and the seasonality of the dog, and where avoidance is impossible.
If these criteria (esp. the first two) cannot be fulfilled, the appropriateness of ASIT needs to be reconsidered
because the response is more likely to be suboptimal.

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Options for Allergy Testing

The major options currently commercially available for diagnosis of relevant allergens in AD are the intradermal test,
or allergen-specific IgE serology. Where allergy testing is elected for, it is important for the clinician to understand
the principles, benefits and limitations of the tests that are available to be able the make the right recommendations
for the patient and client. It is also important to realise that no allergy test is completely sensitive and specific and
clinically normal animals can have positive reactions and clinically diagnosed atopics can have negative reactions
on testing.

Intradermal Testing

How it works. A panel of 40-70 suspect allergens relevant for the geographic area of the patient are injected into
the dermis under sedation. Where allergen-specific IgE is present on tissue mast cells in the epidermis, the
allergen leads to cross-linkage of FcRI molecules, which induces release of preformed granules (containin
histamine and other enzymes) and immediate production of lipid mediators (inc. prostaglandin D2, leukotriene C4,
D4, E4, platelet activating factor) and some pro-inflammatory cytokines. The subsequent wheal and flare reaction
though is almost totally histamine mediated and H1 receptor agonists (i.e. antihistamines) can block the reaction
almost completely.

Histamine binds to the venular endothelial cells leading to endothelial cell synthesis and release of mediators that
cause vascular smooth muscle relaxation and a red injection site from local accumulation of RBCs. Endothelial cells
subsequently retract leading to plasma extravasation and swelling of the injection site (the “wheal”). Finally the
blood vessels at the margins of the wheal dilate (augmented by the nervous system) and become engorged causing
the characteristic red rim (the “flare”). This immediate phase reaction occurs within 5- 30 minutes of injection. A
‘positive’ reaction is one that shows erythema, induration and a diameter greater than halfway between the negative
(saline) and positive (histamine) controls. Late phase reactions may also be seen 4-24 hours later, and these result
from the inflammatory cell infiltrate caused by the chemotactic factors from mast cells.

Pros and Cons. To date this is still considered the ‘gold standard’ test for identification of relevant allergens in dogs
with AD as it assesses the afferent (i.e. IgE) as well as the efferent (i.e. mast cells, mediators and response of the
blood vessels) pathways for a type I hypersensitivity.

The test though is not without limitations. Performing and reading such test requires a high level of skill and
storage and handling of allergens must adhere to strict guidelines to maintain potency. Tests must be performed at
least once weekly to fortnightly to be economically viable, and also to maintain skill in interpreting in administering
and reading the test. Drug withdrawal times must be observed in most cases and chronic skin changes can make
interpretation impossible. Patients must be sedated and an area clipped for the test. False positive reactions can
occasionally occur but recent studies on the correct ‘threshold’ concentrations of allergens to use have reduced
this problem. Clinically normal animals can show rare positive reactions also and it has been considered that these
may suggest sub-clinical hypersensitivity, or be a harbinger of future allergies.

There is a rare risk of anaphylaxis with IDT, and the patient will need to be able cope with light sedation for the test.

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Table 5: Limitations of Intradermal Testing

False Positive Reactions False Negative Reactions

• Improper technique (poor test site selection / • Improper technique (insufficient volume, air or SC
preparation, injections too close, volume too injection, late reading time)
big, traumatic injections) • Insufficient allergen (poorly manufactured,
• Irritant test allergens (excessive concentration, non-standardised or old extract, solution too dilute)
contamination, harbinger of future allergy?) • Drug interference (glucocorticoids & progestagens,
• Irritable skin (infections, nonspecific mast cell high dose CyA, ACP, opiates, antihistamines,
degranulation) immunosuppressive drugs, adrenergic compounds
• Urticaria Bronchodilators Theophylline, long term EFAs,
• Dermatographism immunotherapy)
• Host Factors (pigmentation, stress, reproductive
status, age, chronic skin changes, mast cell
exhaustion, hypothyroidism, hyperadrenocorticism,
low IgE producer, lack of tissue IgE)
• Incorrect antigen selection or relevant antigens not
available for testing
• Test performed at wrong time (i.e. seasonal
variation)
• Monovalent epitopes

Allergen-Specific IgE Serology

How it works. Traditional ELISA testing for allergen-specific IgE begins with binding of a fixed amount of allergen
to a plastic microtitre well (though some techniques start with the allergen in a liquid phase). The serum containing
the IgE is then applied to the plate, and antibodies in the serum bind to the allergen. A concentrated solution of
non-interacting protein (a ‘blocker’) such as casein is used to block non-specific adsorption of other proteins to the
plate. The excess serum is then rinsed away and the IgE detection reagent is added. This detection reagent may
be a polyclonal or monoclonal anti-IgE antibody, or the recombinant high-affinity IgE receptor (FcεRI). The detection
reagent binds to the IgE that has already bound to allergen on the plate. The specificity of binding to IgE is critical
because antibodies other than IgE will also have bound to the allergen in most cases. The plate is washed again
to remove any unbound detection reagent. After this wash, only the detection reagent-antigen complexes remain
attached to the well. Additional steps allow the quantification of bound detection reagent by use of a chromogenic
or fluorogenic signal. The amount of colour should correlate with the amount of IgE bound in the well. The results
are then compared with ranges based on historical positive and negative controls.

Pros and Cons. This test assays the presence of allergen-specific IgE in the serum, but this is not always indicative
of clinical allergy. IgE serology tests historically have frequently resulted in large numbers of positive results because
of cross reactions with IgG and the fact that this can occur in clinically normal (non-atopic) dogs supports these as
false positive reactions. Advances in technology have meant that initially monoclonal antibody and now Fcε receptor
based tests have become available both utilizing variously solid phase and liquid phases, both of which have
increased the positive predictive value compared with the older polyclonal tests.

ASIES are subject to some of the limitations of IDT, and in addition also laboratory error. They are however less
subject to many host factors and not at all subject to issues associated with preparation, administration or
interpretation of IDTs. A problem specific to all ASIES tests is that of large numbers of false positive reactions to
irrelevant botanical carbohydrate epitopes in pollen allergic dogs. This has been reported to occur in a small number
(~5-10%) of cases. Furthermore, the majority of ASIES test for fewer allergens than IDT, meaning a greater chance of
missing possible allergens for inclusion in ASIT, which may affect the outcome of therapy.

Clinicians must therefore have a solid understanding of the various serology tests, their specificity, sensitivity,
methodologies and understand their short-comings and benefits if they are to be able to correctly use these tests.

12
Table 6: Limitations of allergen-specific IgE serology

False Positive Reactions False Negative Reactions

• IgG binding to allergens • Reacting phase of allergen (liquid vs solid)
• Laboratory error • Non-standardised allergen
• Laboratory cut-offs • Loss of IgE in transit (4h at 56oC)
• IgE to non-biologically active botanical • Laboratory error
carbohydrate epitopes • Laboratory cut-offs
• Drug interference (glucocorticoids & progestagens,
high dose CyA, immunotherapy)
• Host Factors (age, low IgE producer,
• low serum allergen specific IgE)
• Incorrect antigen selection or relevant antigens not
available for testing
• Test performed at wrong time
(i.e. seasonal variation)

Controversies in Allergy Testing

Why do some dogs with clinical AD have negative allergy tests?.

Up to 16-20% of dogs clinically diagnosed with AD can have negative IDTs, and in one study about half of these also
had negative ASIES13. Most had no definable problems with the testing. The cause for these cases where there is no
identification of causative allergens and no obvious reason for failure of the test has not been well defined but there
are three major possibilities.

• The dog is allergic to allergens not in the tests

• The dog has low levels of IgE
• The dog has ‘atopy-like disease’ (ALD). In humans a proportion of cases of atopic dermatitis are described as
‘intrinsic’ and having no allergen-specific IgE. While clinically indistinct from ‘extrinsic’ IgE mediated cases,
there have been subtle differences in the pathogenesis reported. Analogous cases may exist in dogs.

What’s wrong with allergen mixes in testing?

Allergens used in most testing are crude antigens that likely contain numerous epitopes. Some of these (but
probably not all) are likely shared with other plants within the same genus but the problem is that this has not
been proven in dogs. So while allergen mixes of similar species of plant may appear harmless, it can lead to dilution
of individual epitopes leading to false negative reactions. Furthermore, inclusion of irrelevant allergens in ASIT
may lead to subsequent sensitisation. Allergen mixes are thus best avoided.

13
Why is there a lack of correlation between IDT and ASIES?

It is not surprising that there is not complete correlation between IDT and ASIES for at least two reasons. Firstly, the
tests are not measuring the same thing (tissue-bound IgE vs. serum IgE), and secondly the tests are not standardised
compared with each other (i.e. the cut-offs for positive and negative reactions are not comparable)

Is IDT or ASIES better at identifying allergens for ASIT in seasonal allergic dogs?

This question was addressed recently in a study by Rosser14. While IDT has been previously considered the ‘gold
standard’ for allergen identification, in reality the only relevant standard is the history and clinical signs of the
patient. If any allergy test results do not match the seasonality of the patient then the test results are wrong not the
patient!

This study was conducted in Michigan, where there are distinct pollination periods for trees, grasses and weeds.
Twenty-nine seasonally atopic dogs underwent IDT and ASIES (FcεRI-based) to try and determine relevant antigens
for ASIT and results were compared with the seasonal histories of the patients. Combining IDT and ASIES results
correlated well in 27 of 29 dogs (93%) for all groups of allergens. These findings strongly supported the
simultaneous use of both IDT and ASIES for the selection of aeroallergens for ASIT in dogs with atopic dermatitis.

Does outcome of immunotherapy depend on the test used?

Numerous studies have been performed in the last 20-30 years to try and answer this question with the findings
suggesting at least a 50% improvement in response to ASIT in 50-100% of dogs irrespective of the allergy test used
to select allergen for the vaccine. However, the bulk of studies were open and uncontrolled, and methodologies
were not comparable. Interestingly two reports (one anecdotal and one unpublished) suggested similar to Rosser14
that better results may be had using a combination of IDT with ASIES. Further studies to confirm these and Rosser’s
findings are needed.

What are the effects of glucocorticoids and cyclosporin on allergy testing?

Glucocorticoids (GCs) have numerous potential effects on allergy testing. In vitro, rat and mouse studies show a
decrease in allergen-specific IgE with GC use, though a paradoxical increase in total IgE may be seen. Furthermore
GCs also decrease mast cell cytokine and histamine production, mast cell IgE receptors, and blood vessel
permeability and neurogenic oedema. GCs do not interfere though with mast cell degranulation.

Recommendations: These changes will interfere with both ASIES and IDT and withdrawal periods should be
observed for best results (see below). Note though that response to GCs is individualistic and the amount of IgE
suppression can vary considerably between individuals.

The effect of cyclosporin A (CyA) on allergy testing is less straightforward. Laboratory and human clinical studies
have shown that large doses (~25mg/kg/day) suppress IgE production but lower doses (5-10mg/kg/day) have either
little impact or possibly increase IgE production. This is supported by a case report in humans where good clinical
response was noted but allergen-specific IgE remained essentially unchanged. In vitro CyA can also decrease mast
cell degranulation but this is not supported in clinical studies in humans and dogs where positive prick or IDT have
been noted following 6-16 weeks of therapy at 5mg/kg/day with little change from initial testing.

Recommendations: CyA therefore can likely be used at standard doses with minimal interference on IDT for up to
16 weeks. CyA may not interfere with ASIES but this is yet to be substantiated in the dog.

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Clinical Decision Making: IDT vs. ASIES

Having made the decision to perform allergy testing, the clinician is faced with the options of what test to choose.

When to perform allergen specific IgE serology with a view to in-house ASIT or avoidance.

• When IDT is not accessible

• When sedation or clipping is not possible
• Where the AD is uncomplicated and the only symptom is non-lesional pruritus
NOTE:
• Testing should not be performed in dogs <5-6 months of age
• Drug withdrawal must be observed
- 3-8 weeks off prednisolone
- 3-4 months off methylprednisolone acetate, progestagens
- 2 weeks off topical glucocorticoids, antihistamines, and clomipramine (latter up to 4 weeks)
• Test < 8-30 days after end of the patient’s pollen season (and preferably the middle to end of pollen season)
• Testing should not be performed unless the managing veterinarian is experienced with
immunotherapy / atopic management
• Apoquel® and Cytopoint® are fine to continue using as they will not affect the test

When to refer for further workup or IDT.

• When the managing veterinarian is not experienced with immunotherapy / atopic management
• When referral is accessible
• When AD is not able to be confirmed clinically
• When the ASIES test result does not correlate with clinical history of the dog
• Where there are significant skin lesions, as these dogs will have lots of non IgE mechanisms involved and are less
likely to respond to “recipe book” straightforward immunotherapy
• When patient is ‘not quite right’ – either looks wrong or is not responding to medications as it should if
straightforward AD was present

Management of the atopic patient.

Atopic dermatitis (AD) is a common diagnosis in the dog, but our ideas of what an atopic dog is have changed over
the years. Twenty years ago we thought atopic dermatitis was an IgE mediated disease due to inhaled allergens.
Ten years ago we thought atopic dermatitis was an IgE mediated disease due to percutaneously absorbed allergens.
Now we realise it is a far more complex disease and that more than one pathophysiological pathway can create the
“atopic” clinical picture. Barrier dysfunction, irritable skin, microbial proteases, IgE and non-IgE mechanisms with
genetic and environmental modification all combine to create the atopic dog. It appears that we have to re-define
our definition of canine AD: it is not “just an allergy”.

The major clinical symptom in atopic dermatitis is pruritus. This can impact not only on the quality of the dog’s life
but also on the enjoyment of owning a dog. If we are to successfully manage dogs with atopy we need to address
ALL the mechanisms of itch including itch due to:

• barrier dysfunction (itch from dry skin, irritants, environmental proteases),

• infections (IgE mechanisms, compliment cascade, microbial proteases), and
• allergy (IgE and non IgE mechanisms and allergen dependent and independent upregulation)

Pruritus then becomes a complex interplay of numerous neurochemical mediators. We must also be cognisant that
the contribution of barrier dysfunction, infections and allergy will be variable among our patients and that barrier
dysfunction may be acquired due to inflammation. Identifying what mechanism(s) are involved in each individual
patient so as to formulate a treatment rationale addressing each aspect of these is the art of dermatology.

Even though the signal to make you feel itchy usually starts in the skin the signal must be carried by afferent nerve
fibres from the skin to the spine, then up the spine to the brain. Ultimately it is the brain that itches not the skin itself.
This means that not only is the generation of the pruritic signal complex but also spinal and central processing will
affect the quality of the itch and the patient’s temperament will affect the response to that itch.

15
The pruritic response (lick, scratch, rub, roll, chew etc) will vary with the intensity of the itch and the temperament
of the patient and be modified by other competing environmental signals. Pain fibres and itch fibres compete for the
same pathways in the brain and so the more painful the pruritic behaviours the longer the itch relief will be.

If pruritus is the symptom that we are trying to control then we need to try and establish an objective baseline
measure of itch so we can assess efficacy of our treatments. This is remarkably hard to achieve as our clients tend
to be very subjective. As a general rule, a comfortable level of itch is a dog who exhibits pruritic behaviour of low
intensity, terminates that behaviour spontaneously after 15 to 60 seconds, gets relief and therefore does not need
to repeat that behaviour, at the same site, for several hours. This level of itch does not require intervention. Where
the itch signal is strong enough for a dog to interrupt their walk, play, eating or sleeping behaviour to scratch OR
where the pruritic episode is intense and includes vocalisation or needs to be physically interrupted to terminate, OR
is prolonged > 60 seconds OR frequently repeated, then intervention is required.

Unfortunately there is no one treatment that addresses all the mechanisms of itch. So if we are to achieve our goals
of having a comfortable patient while minimising steroid reliance and hopefully avoiding the need for life-long,
non-specific immunosuppressive therapies, then treatment is always going to be a multifaceted attack and also
needs to consider the patient’s temperament.

1. Improving the epidermal barrier

Epidermal barrier dysfunction in lesional and non-lesional skin is well recognised in people with atopic eczema.
Changes including reduced lipid (ceramide) levels and elevated chymotryptic enzyme activity (leading to premature
breakdown of corneodesmosomes that hold the stratum corneum together to create the barrier) result in reduced
barrier function. This is further reduced by soaps/shampoos (increasing skin pH increases chymotryptic enzyme
activity) and microbial and environmental proteases (inc. dust mite proteins). In dogs, abnormalities in essential fatty
acids contributing to the skin barrier and in the intercellular lipid lamellae have been identified.

The outcome of poor epidermal barrier function is ALLERGIC itch associated with increased percutaneous
allergen absorption, and NON-ALLERGIC itch associated with liberation of inflammatory cytokines from the
epidermis (irritable skin) PLUS itch associated with dry skin PLUS itch associated with environmental proteases
PLUS itch due to increased microbial proteases. Improving the epidermal barrier can lessen the severity of the
atopic disease.

Moisturising the skin.

A. Hydrotherapy

Moisture can be returned to the skin by bathing in water. A long rinse cycle when shampooing helps hydrate the
stratum corneum. This will rapidly dehydrate unless moisturises are applied post rinsing to hinder evaporation. The
mechanical action of washing can be useful for removal of allergens/irritants from the skin, reduction of microbial
adhesion sites and reducing the itch due to cooling of the skin.

B Topical Moisturising Agents

Formulations may be shampoos, rinses, lotions, sprays, creams, emulsions and ointments. Each formulation has
advantages and disadvantages and selection depends on the type of condition being treated, the surface area
involved and the presence of hair.

Shampoos. Shampoos are generally drying because of their detergent action but some formulations have
sustained-release microvesicles that contain active ingredients. These vesicles have an outer lipid membrane that
will bind to hair and skin and breakdown to release their contents having a moisturising effect. Ingredients in
shampoos that may help moisturise are fatty acids, lipids, urea, glycerin, colloidal oatmeal and chitosanide
e.g. Epi-soothe Spherulites® (Virbac) which incorporate oatmeal and chitosanide; Allergroom S® (Virbac) with
spherulites that contain glycerine, lactic acid, urea and chitosanide. Shampoos should not be relied upon as the
sole moisturising treatment except in very mild cases. It should also be remembered that even “non-irritant”
shampoos may irritate skin with a dysfunctional epidermal barrier. Not all dogs are improved by shampooing!

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Lotions, creams, and ointments. Lotions are liquids with the active ingredients dissolved or suspended. If mixed in
an alcohol base, they are drying (e.g. Dalacin T). Moisturising lotions often contain propylene glycol and water but
may also deliver therapeutic agents. An example is Resisoothe Lotion® (Virbac) which incorporates oatmeal. Lotions
have a longer action than shampoos but shorter duration then creams and ointments. For lichenified skin they are
probably inadequate. Alpha keri lotion (fractionated lanolin and mineral oil) is very useful for application after
shampooing. It is easily applied to large areas.

Dry, lichenified skin is best treated with Hydraderm (sorbolene cream and hygroscopic agent glycolic acid). As the
water evaporates, it leaves an oily film on the skin and glycolic acid will draw water from the dermis and trap it in
the stratum corneum.

Ointments and oily creams are more occlusive, which facilitates hydration of stratum corneum and increases
penetration of the incorporated active ingredient. Their disadvantage is that they occlude the pilosebaceous
orifice and may produce a folliculitis and are too greasy to apply effectively on hairy skin.

Fatty Acids. Fatty acid (FA) additives in topical preparations can decrease transepidermal water loss. The skin also
incorporates some circulating lipids such as plant sterols, essential fatty acids, polyunsaturated fatty acids and
arachidonic acid. An example is Megaderm® (Virbac) which contains omega 3 and 6. Dietary supplementation
of omega 6 fatty acids may assist barrier repair although appropriate studies to validate this are currently lacking.
There is evidence though that the addition of both omega 3 and omega 6 fatty acids to the diet may reduce steroid
reliance in atopic dogs. Omega 3 fatty acids become incorporated in the T-cell TCRs and may modulate T-cells
directly. The action of fatty acids may be more than the presumed down regulation of pro- inflammatory
eicosanoids. Interestingly, inhibition of PGD2 by fatty acids may have a negative effect on barrier repair. Maximum
benefit from fatty acid supplementation in most studies has taken 8 to 10 weeks. The best reported ‘effective’ doses
of omega 3 as a sole therapy range from 66-85mg/kg combined docosahexanoic and eicosapentanoic acids or up to
126 mg/kg α-linolenic acid.

Future treatments for barrier repair.

Ceramide moisturisers that mimic the ceramide, cholesterol and free fatty acid levels of human skin are already
available and showing great promise in reducing steroid reliance in human atopic dermatitis and should be useful
for dogs. Phytosphingosine shampoos/sprays and spot on treatments are marketed in Europe for the treatment of
barrier dysfunction in dogs. Although our experience with these products is limited they do appear to be promising

2. Controlling infections

Both human and canine atopic patients show a tendency to more commonly develop secondary infections.
Dogs commonly develop secondary bacterial (particularly S. intermedius) and Malassezia (particularly
M. pachydermatis) infections. Microbial colonisation may exacerbate the inflammation via numerous mechanisms
including IgE responses to bacterial and fungal proteins, T-cell activation by bacterial superantigens, protease
liberation and microbial PAMPs (pathogen associated molecular patterns) activating the immunological response
via TLRs (toll-like receptors).

Clinical experience indicates that controlling microbial colonisation and active infection in some atopic dogs can
dramatically reduce the intensity of the atopic lesion and reduce pruritus and reduce steroid reliance. Management
of infection falls into two main parts.

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Control of current infections

The immediate priority with infection is in controlling the current infection and assessing the contribution of
infection to the overall clinical picture. Total pruritus in a dog is the result of pruritus due to infection AND pruritus
due to allergy AND pruritus from barrier dysfunction. Controlling the infection with antimicrobials will allow you to
assess how important pruritus from infection is to your atopic patient (remember this will vary among atopics). If
infection is a major contributor to the pruritus then preventing relapses will be a priority to successful management.

a. Cytology and clinical evaluation is needed to determine the nature of the infection and the depth of the
infection. Depth of the infection determines whether systemic or topical treatment is needed and
the duration of treatment
b. Antimicrobials with good antistaphylococcal activity (cephalexin 22mg/kg bid, clindamycin 5.5mg/kg
bid and the fluoroquinolones) are good empirical choices for superficial infection (i.e. folliculitis).
Antimicrobials should be continued for a minimum of 3 weeks (7 days beyond clinical resolution)
but improvement should be noted within 4 to 7 days and 14 days is usually adequate length of time
to evaluate the response (or lack thereof) in the pruritus.
c. For deep infections where other bacteria may be an issue antibiotics that have anaerobic activity should be
considered (e.g. clindamycin) or adding metronidazole 20mg/kg sid to the cephalexin or fluoroquinolone
regimen. Minimum duration of treatment in deep infections is at least 4 to 8 weeks (2 weeks beyond clinical
resolution). Due to the duration of treatment needed, deep tissue culture (NOT swabs of draining sinuses)
should be considered in some cases prior to treatment and always performed if there is a failure of initial
empirical treatment.
d. Surface bacterial infections (bacterial overgrowth) can be dry and exfoliative or moist and exudative. The
latter are often in intertriginous areas and associated with marked self-trauma. The exfoliative lesions can
do poorly with systemic antibiotics and topical treatments e.g. Dalacin T® (clindamycin 1% lotion) can be
more effective for these. Dalacin T is drying due to alcohol in the vehicle and should not be used more than
twice daily or for longer than 7 to 14 days, and should be discontinued if irritancy occurs. Exudative lesions
require antibiotic and steroid combinations e.g. Fuciderm® and should resolve rapidly with twice daily
application. Fuciderm is a carbomer gel and is preferred for exudative lesions over lotions or ointments that
can be macerating.
e. Malassezia overgrowth where generalized or associated with lichenified skin should be treated with
systemic azoles such as ketoconazole 5mg/kg sid-bid or itraconazole 5mg/kg sid. Both should be given with
food and discontinued if anorexia, hyporexia or vomiting occurs. Azoles should not be given to pregnant
animals and can interact with other medications. Clinicians should familiarise themselves with the drug
interactions and potential side effects with these drugs if not using these routinely. Mild and
localised Malassezia overgrowth may respond to shampoo therapy (azoles or 3-4% chlorhexidine),
miconazole (Micotopic®) or clotrimazole lotions (Canesten®), or 2% acetic acid / 2% boric acid
(Malacetic®) wipes.

Maintenance, minimisation of recurrent infections

a. If there is a good response to antimicrobials then prevention of recurrence of microbial overgrowth or

infection is vital. For recurrent superficial bacterial infections pulsed antibiotics (weekend dosing) can be
useful together with the use of leave on chlorhexidine lotions. For surface bacterial infections leave on chlor
hexidine lotions and use of chlorhexidine based shampoos can be effective.
b. For Malassezia overgrowth regular use of antifungal shampoos together with treatment of regionalised
areas (e.g. interdigital areas, lip folds) with Malacetic Wipes® can be effective in short haired dogs.
For dogs with hairy feet the Malacetic otic® applied with a cotton ball is a better option to penetrate the
haircoat. Maintenance frequency will vary with the patient from daily to twice weekly for regional treatment
and weekly to fortnightly for shampoos.

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3. Controlling the allergy

Avoidance

Avoidance is not likely to be effective for airborne allergens like pollen as these allergens are widely dispersed in
both indoor and outdoor environments. Climatic conditions including wind velocity, humidity and variables such
as pollution levels and environmental activity (including vehicular movements) can all affect pollen dispersion.
We have no control over these factors. Avoidance may be possible though for dogs with only dust mite allergies.

Based on studies in dogs and humans, it is be possible to control dust mite allergies with aggressive control of dust
mites in the environment, or total avoidance of going indoors at all. However, this is not always practical in the
domestic situation. It could be expected though, that if exposure of allergic pets to dust mites can be reduced at
least partly, then response to other therapies, including ASIT, is likely to be better. Strategies include:

• complete exclusion from human bedrooms as dust mite counts are high in bedrooms.
• pets sleeping at night in a non carpeted room to try and reduce nightly dust mite exposure.
• pets sleeping on a suspension bed with a synthetic material (e.g. ‘shade cloth’ type with aluminium frame) as
they do not accumulate mites.
• pet bedding using cotton sheets only. Rugs and blankets can harbour dust mites, and even the dead bodies of
the mites can still trigger the allergies in most dogs. Mite bodies are more easily washed from sheets.
• weekly hot wash (> 55oC) of pet bedding. This kills the dust mites.
• if > 55oC is not possible with modern hot water systems then eucalyptus oil in a load of washing at a 0.4% rate,
with a 60 minute soak followed by a cool rinse kills 99% of mites.
• weekly vacuuming of the home with a vacuum cleaner with a fine particle filter (e.g. HEPA filers or double
bagged vacuum cleaners). Dust mites pass through the filters of average vacuum cleaners. There is no benefit to
vacuuming more frequently.
• steam cleaning of the carpets and cleaning the ducted heating at the commencement of the process may also
be advantageous.

Avoidance may also be useful for dogs with certain types of “grass allergy”. Clients will sometimes observe that the
dogs flare quickly after contact with grass. The distribution affected correlates with contact areas so the distribution
may vary in dogs depending on coat type. Although well recognised, the actual pathomechanism is poorly
understood and probably involves IgE mediated, DTH and irritant contact pathways. Avoidance may be useful
in these cases e.g. environmental isolation versus lycra suits.

Allergen specific immunotherapy (ASIT)

The first reported successful use of ASIT in the dog was in 1941. Currently, this intervention is one of the mainstays
of the management of AD, and there have been numerous studies examining this mode of therapy in the last 38
years. However, a wide variety of non-standardised trials and a lack of focus of case selection criteria have left us
none the wiser as to the true efficacy nor optimal protocols of ASIT.

Mechanism of action. The induction of peripheral T cell tolerance appears to be the most essential step in ASIT.
Initial T-cell anergy is followed by T-cell re-activation with Th-2 to Th-1 subversion together with IgG interference of
CD23 mediated T-cell activation. These changes are initiated by multiple suppressor factors including IL-10 and/or
TGF-β produced by ASIT activated allergen-specific regulatory T (Treg) cells. Treg cells also directly or indirectly
influence effector cells of allergic inflammation, such as mast cells, basophils and eosinophils

Goals of ASIT. The goals of ASIT are to have a comfortable dog that requires LESS steroid medications to be
comfortable. ASIT will sometimes allow a cessation of oral medications, and very rarely may put an animal into long
term remission (<1% of cases). These outcomes are extremely important to convey to clients prior to allergy testing
and commencement of ASIT.

It is important to remember that immunotherapy does not address innate barrier dysfunction or all the mechanisms
that lead to bacterial and yeast colonisation in atopic dogs. Realistically if ASIT is successful we expect the typical
dog to show a 60 to 80% reduction in medication reliance during the first 12 months on immunotherapy.

19
Efficacy of ASIT. Efficacy of ASIT has been examined in many nonstandardised studies and significant response
has been reported in 52-100% of dogs. The truth is likely to lie somewhere in the middle of this range. Many dogs
that fail ASIT are due to poor selection of the patient, poor owner compliance, poor management of concurrent
problems or an inability to identify the important allergens.

Time to effectiveness. Response times vary from 2 months to 12 months but study comparisons are difficult due to
lack of standardisation of response assessment criteria. If there is no change with ASIT after 9-12 months the patient
should be re-evaluated for unaddressed concurrent problems. If none are found then consideration should be given
to repeat allergen testing and modification of vaccine if there is evidence that important allergens have been
missed. If this is not the case the vaccine should be discontinued.

Predictive factors for efficacy. Standard ASIT involves subcutaneous administration of native allergens. The key
though is accurate selection of allergens. The differences between intradermal skin testing and serum IgE testing
have already been discussed. Regardless of the test used, immunotherapy is unlikely to be successful unless the
patient is a good candidate for immunotherapy (disease, patient and client factors), that the allergens are selected
based on knowledge of the patient’s environment, plant pollination periods and seasonality of disease. The best
number of allergens to use is not known but one report suggested ASIT with more allergens (11-20) in dogs with >10
reactions increased the response rate.

Experience of the managing veterinarian also has a significant impact on the case. Nuttal et al 1998 described cases
managed by the dermatology practice had a significantly better response to ASIT than externally managed cases
(95% versus 60%). This is likely a result of a better understanding of the disease, management and also just
increased communication time with the clients. In our practice we spend several hours every day talking to clients
on the telephone in addition to longer than standard face to face consultations. Compliance was also better for
referral managed patients. Similar outcomes have been shown in studies of human allergy patients where
compliance is poorer by patients managed by the referring practice (11% versus 34% for non- compliance rate).

Inherent patient factors are less clear, but some studies have variously suggested that dogs that had clinical signs
for 3-5 years or more before commencing ASIT were less likely to respond, that dogs over 5 years of age were less
likely to respond, and that certain breeds may be more (Golden retrievers, Shar Peis, Australian Shepherds) or less
(WHWT, Cairn and Yorkshire terriers, Bichon Frises) likely to respond. More studies are required to clarify these.

ASIT protocols. There is no “recipe book” for ASIT and manipulation of allergy vaccine protocols to get the best
results requires experience. The allergen dose may play a critical role to outcome with individual cases requiring
sometimes more or less than the ‘standard’ dose. A detailed examination of protocols available for ASIT are
beyond the scope of these notes and the reader is referred to Scott et al 20012 and 22nd Proceedings of the North
American Veterinary Dermatology Forum p.55 for more on this subject.

Assessment of Efficacy. Assessment of effectiveness of ASIT is determined by improvement in clinical signs

(pruritus, infection recurrence), and / or reduction or elimination of maintenance medications such as
glucocorticoids and antibiotics. It is important to document the level of drug reliance at the start of
immunotherapy so that we have an objective measure of ASIT response. It is also important to give the owners
realistic expectations to the outcome of ASIT such as target levels for drug reliance, and also to inform them at what
point do we make a determination as to whether this is a satisfactory outcome. We typically compare both drug
dependence and clinical signs 12 months after starting to minimize the possible confounding factors of seasonal
variation. Many clients struggle with the concept that atopic dermatitis is a life-long disease and that management
will likewise for most cases be life-long. The real benefits of drug minimisation with ASIT are the long-term health
(and often financial) benefits.

20
Duration of ASIT. Persistence of benefit after discontinuation has not been evaluated in a controlled study so results
vary from 4% to 35%. Such information needs to be reevaluated in a controlled trial and so ASIT should be
considered life ling until such data is published.

The future of ASIT.

Immunotherapy is not a static “science”. Altering ASIT to maximise the beneficial immunological response to
administered allergens is the future focus of ASIT. CpG motifs (from bacterial DNA) conjugated to antigens can
enhance Th1 maturation, and suppress Th2 responses and inhibit IgE production. Epitope mapping and cloning of
allergens allows for production of specific antigenic epitopes that can be modified to maintain T cell epitopes but
not to allow IgE binding. This may overcome the greatest risk of immunotherapy, namely anaphylaxis. Anaphylaxis
is a real risk in humans on ASIT and a minor risk for dogs however it is the PERCEPTION of risk that will deter some
clients from pursuing ASIT.

Non - allergen specific treatments (Anti-IgE immunotherapy)

IgE plays a pivotal role in allergic disease. Anti-IgE therapy can be both passive (vaccination with antibodies that
target the variable portion of allergen specific IgE or the Fc portion of all IgE) or it can be active (immunisation to
induce antibody production against IgE in either an allergen specific or allergen non-specific, targeting Fcε,
manner). Down regulating IgE responses without cross-linking IgE (and hence triggering possible anaphylaxis) is the
aim.

Passive immunisation with high dose monoclonal antibodies (mAb) directed against Fcε epitope Cε3 (to minimise
the risk of anaphylaxis) can reduce serum IgE levels, inhibit IgE binding to mast cells and basophils by FcER1 and
other effector cells and dendritic cells via FcεRII, and inhibit IgE production and induce apoptosis in IgE expressing B
cells. Immune complexes to mAb Cε3 are small and do not activate compliment or accumulate in tissues.
However, with passive therapies there is no sustained response after treatment is discontinued.

Bacteriophage immunization expressing Cε3 may lead to active anti-Cε3 therapies in the future. Safety studies are
pending. It should be noted that anti-IgE therapy is only recommended currently as an adjunctive therapy in human
asthma as response can be variable. Similarly anti-IgE vaccines for dogs are eagerly awaited to be used TOGETHER
with ASIT but it is likely to be less effective then ASIT if used as a monotherapy. Remember the main mechanism of
action of ASIT (and of AD itself) is via allergen specific T-cells.

Non - allergen specific treatments (pharmacotherapy)

Pharmacotherapy is usually required together with ASIT, at least in the short to medium term, and may still be
needed long term. Clients must also be made aware of HOW medications work and why some of the safer
medications (e.g. antihistamines) are less likely to be effective. Remember that histamine is a small player in the
big overall picture of pruritus and there are a paucity of pharmacokinetic studies done in dogs for the
antihistamines we use.

Of the drugs that have good evidence for their use in atopic dermatitis we have only glucocorticoids and
cyclosporin while there is moderate evidence for the use of pentoxifylline and misoprostol and little objective
support for the use of antihistamines. The side effects of glucocorticoids are problematic where supraphysiological
levels (> 0.1 to 0.2mg/kg) are given frequently and cyclosporin causes broad spectrum suppression of T-cell functions
making it less ideal for life long therapy and probably not compatible with ASIT.

Recently there has been a re-birth of interest in topical therapies for dogs with regional atopic lesions where the coat
type allows for topical application. Topical glucocorticoids can be effective and offer more targeted treatment
with les systemic side effects. Local side effects of cutaneous thinning and comedone formation can be
problematic especially in thin skin areas like the axillae and inguinal folds. Currently Gentocin spray® (Schering
Plough) is the only registered topical steroid spray for animals available in Australia however there are others in the
USA and Europe that will likely be available in Australia in the future. For some axillary and inguinal regions topical
pimecrolimus can be effective. It is a topical immunomodulator that causes localised cutaneous immunosuppression
is a similar mode to cyclosporin without the atrophy of glucocorticoids.

21
Conclusion

When starting ASIT great care should be taken to improve barrier function with moisturisers, fatty acid
supplementation and appropriate shampoo protocols, control recurrent infection if present with systemic and
topical antimicrobial strategies if required, then control the atopic itch with allergen/irritant reduction strategies,
topical therapies if possible and where the itch is generalised, systemic prednisolone. A minimum effective
prednisolone dose is determined (prednisolone reliance) and this is continued but should gradually be reduced as the
ASIT takes effect. Dogs with high prednisolone reliance at the outset of ASIT will often also receive antihistamines or
occasionally pentoxifylline for steroid sparing effect. Once prednisolone reliance is determined this is a useful
objective guide to ASIT response - the better the response to ASIT the lower the ongoing prednisolone reliance.
Immunosuppressive therapies (cyclosporin, azathioprine, chlorambucil) are used where ASIT is not possible or does
not effectively reduce the prednisolone reliance to an acceptable level within 12 months of compliant ASIT.
Immunosuppressive therapy is only undertaken with owner informed consent of known and unknown risks and
adherence to appropriate monitoring protocols.

Further reading

Atopic Dermatitis

1. Olivry (ed.) (2001). The ACVD task force on canine atopic dermatitis (XV): fundamental concepts in clinical
diagnosis Vet Immunol Immunopathol 81 pp143-387
2. Scott DW, Miller Jr WH, Griffin CE. (2001) Muller and Kirk’s Small Animal Dermatology, 6th edn.
WB Saunders Company, 2001

Pathophysiology

3. Aiba S. Dendritic cells: importance in allergy. Allergol Int. 2007 Sep;56(3):201-8. Epub 2007 Aug 1.
4. Elkord E.Role of regulatory T cells in allergy: implications for therapeutic strategy. Inflamm Allergy Drug
Targets. 2006 Dec;5(4):211-7
5. Freedberg IM, Eisen AZ, Wolf K, et al editors. Dermatology in General Medicine 5th edn. McGraw Hill Inc
publishing, New York, 1999.
6. LinksAllam JP, Novak N.The pathophysiology of atopic eczema.Clin Exp Dermatol. 2006 Jan;31(1):89-93
7. LinksHomey B, Meller S, Savinko T, Alenius H, Lauerma A.Modulation of chemokines by staphylococcal
superantigen in atopic dermatitis.Chem Immunol Allergy. 2007;93:181-94.
8. LinksMarone G, Rossi FW, Detoraki A, Granata F, Marone G, Genovese A, Spadaro G.Role of
superallergens in allergic disorders.Chem Immunol Allergy. 2007;93:195-213.
9. Pivarcsi A, Homey B.Chemokine networks in atopic dermatitis: traffic signals of disease.Curr Allergy Asthma
Rep. 2005 Jul;5(4):284-90
10. Romagnani S.The increased prevalence of allergy and the hygiene hypothesis: missing immune deviation,
reduced immune suppression, or both? Immunology. 2004 Jul;112(3):352-63
11. Wittmann M, Werfel T.Interaction of keratinocytes with infiltrating lymphocytes in allergic eczematous skin
diseases.Curr Opin Allergy Clin Immunol. 2006 Oct;6(5):329-34.
12. Wittmann M, Werfel T.Interaction of keratinocytes with infiltrating lymphocytes in allergic eczematous skin
diseases.Curr Opin Allergy Clin Immunol. 2006 Oct;6(5):329-34

Allergy Testing

13. Foster et al (2003) Comparison of intradermal and serum testing for allergen-specific IgE using a
FceRIa-based assay in atopic dogs in the UK. Vet Imm Immunopathol 93(1-2) 51-60
14. Rosser, E. J. (2004) Comparison of the results of intradermal testing and aeroallergen-specific IgE serum
testing in dogs with warm weather, seasonal atopic dermatitis. Veterinary Dermatology 15 (s1), 3-4
15. Munro et al (1991) Cyclosporin A in atopic dermatitis: therapeutic response is dissociated from effects on
allergic reactions. Br J Dermatol. 124(1):43-8.

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16. Akdis CA et al (1997) Glucocorticoids inhibit human antigen-specific and enhance total IgE and IgG4 pro
duction due to differential effects on T and B cells in vitro. Eur J Immunol 27(9):2351-7
17. Sepp & Fritsch (1995) Can cyclosporin A induce permanent remission of atopic dermatitis? Br J Dermatol.
28(2):213-6
18. Puigneró V et al (1995) Effects of cyclosporine and dexamethasone on IgE antibody response in mice, and
on passive cutaneous anaphylaxis in the rat. Int Arch Allergy Immunol. 108(2):142-7
19. Proceedings of the 5th World Congress of Veterinary Dermatology, Vienna
20. Thoday K et al (ed) (2002) Advances in Veterinary Dermatology Volume 4, Blackwell Science 21.Abbas AK et
al (2000) Cell and Molecular Immunology 4th edition. W.B. Saunders

Management

22. Bell A: Mechanisms of human ASIT: an overview and update. Proc Dermatology Chapter Meeting, ACVSc
Science Week 2006.
23. Biedermann, T. 2006. Dissecting the role of infections in atopic dermatitis. Acta Derm Venerol. 86: 99-109
24. Hara J, Higuchi K, Okamoto R, Kawashima M, Imokawa G. Highexpression of sphingomyelin deacylase is an
important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis. J Invest
Dermatol 2000 115 406-413.
25. Inman AO, Olivry T, Dunston SM, Monteiro-Riviere NA, Gatto H. Electron microscopic observations of
stratum corneum intercellular lipids in normal and atopic dogs. Vet Pathol 2001 38 720-723
26. Kietzman M. Et al Effects of PDE4-inhibitors in a model of allergic dermatitis Fourth World congress of
Veterinary Dermatology, 2000
27. Macheleidt O, Kaiser HW, Sandhoff K. Deficiency of epidermal proteinbound omega-hydroxyceramides in
atopic dermatitis. J Invest Dermatol 2002 119 166-173
28. Ong PY, Ohtake T, Brandt C et al. Endogenous antimicrobial peptides and skin infections in atopic
dermatitis. New England Journal of Medicine 2002. 347: 1151-1160
29. Palmer CN, Irvine AD, Terron-Kwiatkowski A, et al. Common loss-offunctionvariants of the epidermal barrier
protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 2006 38 441-446
30. Rolinck-Werninghaus, Hamelmann C, Keil T et al: The co-seasonal application of anti-IgE after preseasonal
specific immunotherapy decreases ocular and nasal symptom scores and rescue medication use in grass
pollen allergic children. Allergy 2004: 59: 973–979
31. Scott D.Antihistamines in the management of allergic pruritus in dogs and cats. JSAP 40 1999
32. Taugbol O, Baddaky-Taugbol B, Saarem K. The fatty acid profile of subcutaneous fat and blood plasma in
pruritic dogs and dogs without skin problems. Can J Vet Res 1998 62 275-278
33. Werner Y, Lindberg M. Transepidermal water loss in dry and clinically normal skin in patients with atopic
dermatitis. Acta Derm Venereol 1985 65 102-105.
34. 22nd Proceedings of the North American Veterinary Dermatology Forum, pp 49-64

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Why are accurate results important in
IgE testing ?
To show the implications of accurate test results in
the treatment decision process, a study using sera
from atopic laboratory beagles sensitised to a single
allergen has been performed.
Methodology:

High IgE responder beagles were bred and maintained in an indoor facility. Puppies were sensitized to house dust
mites, a grass pollen, or a weed pollen, by an injection of allergen extract, given once per month for 6 months, the
first administration starting within 10 days of birth. Sera were obtained from these dogs at 6 month of age or later.
In addition, sera were obtained from laboratory beagles, which had been experimentally sensitized to flea bite and
showed classic symptoms of flea allergy dermatitis (FAD) upon flea infestation.
Serum from barrier raised specific pathogen-free (SPF)
laboratory dogs was purchased from a commercial
supplier. Sera from dogs sensitized to only a single
allergen were aliquoted, coded and submitted in a blinded
fashion to the Heska® Veterinary Diagnostic Laboratory,
and to other commercial testing laboratories. After serum
collection, the dogs were intradermally skin tested with
a panel of 12 allergens by a Board-certified veterinary
dermatologist who did not know the dog’s sensitization
status. In vitro test results for IgE to the allergen group
mites, fleas, grass pollens and weed pollens were
compared. A result was considered “false” if it was not in
agreement with the known sensitization status of the dog.

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FLEA-ALLERGIC DOG: IgE to flea only*

Test results

Potential treatment consequences based on test results

ALLERCEPT Commercial test-1, Commercial test-2,

IDST
test MoAB based PoAB based
Allergens tested 12 48 52 45
Correct results 1 1 0 0
False results 0 0 26 40
Flea allergen
YES YES NO NO
detection
Recommended
immunotherapy NONE NONE YES YES
treatment

Potential allergens to
include in
immunotherapy

Green = correct
Red = false

* This study was made using sera from laboratory beagles sensitized only to flea bite.

25
MITE-ALLERGIC DOG: IgE to mites only*

Test results

Potential treatment consequences based on test results

ALLERCEPT Commercial test-1, Commercial test-2,

IDST
test MoAB based PoAB based
Allergens tested 12 48 52 45
Correct results 2 2 2 2
False results 0 0 19 12
Mite allergen
YES YES YES YES
detection
Recommended
immunotherapy YES YES YES YES
treatment

Potential allergens to
include in
immunotherapy

Green = correct
Red = false

* This study was made using sera from atopic laboratory beagles sensitized to a single allergen.

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GRASS-ALLERGIC DOG: IgE to grasses (Meadow fescue) only*

Test results

Potential treatment consequences based on test results

ALLERCEPT Commercial test-1, Commercial test-2,

IDST
test MoAB based PoAB based
Allergens tested 12 48 52 45
Correct results
(cross-reacting 1 (1) 1 (5) 0 (3) 1 (3)
grasses)
False results 1 0 19 26
Grass allergen
YES YES YES YES
detection
Recommended
immunotherapy YES YES YES YES
treatment
Potential allergens to
include in
immunotherapy

Green = correct

Light-green =
cross- reacting grass

Red = false

* This study was made using sera from atopic laboratory beagles sensitized to a single allergen.

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WEED-ALLERGIC DOG: IgE to weeds (Yellow Dock/Sorrel) only*

Test results

Potential treatment consequences based on test results

ALLERCEPT Commercial test-1, Commercial test-2,

IDST
test MoAB based PoAB based
Allergens tested 12 48 52 45
Correct results 1 2 2 2
False results 0 5 25 35
Weed allergen
YES YES YES YES
detection
Recommended
immunotherapy YES YES YES YES
treatment

Potential allergens to
include in
immunotherapy

Green = correct

Red = false

* This study was made using sera from atopic laboratory beagles sensitized to a single allergen

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Leading Veterinary Reference Laboratories Worldwide use the Heska® ALLERCEPT® Allergy program

Europe
VetMed Labor Axiom Veterinary Laboratories Laboklin
Mörikestrasse 28/3 The Manor House Brunnel Road, Prinzregentstrasse 3
71636 Ludwigsburg Newton Abbot 97688 Bad Kissingen
Germany Devon TQ12 4PB, Germany
www.vetmedlabor.de UK www.laboklin.de
www.axiomvetlab.com
Dr. Baddaky Dr. Baddaky Europe AB Vet-Allergy
Vestmarkaveien 16 Hantverksgatan 1 Skalcentret, Skalhuse 13
2230 Skotterud 67321 Charlottenberg 9240 Nibe
Norway Sweden Denmark
www.drbaddaky.no www.drbaddaky.com [email protected]
Synlab Labor In vitro Univet s.l.
Leitershofer Starsse 25 Rennweg 95/Ecke Facultad de veterinaria
86157 Augsburg 1152 Vienna Campus de Bellaterra
Germany Austria 08193 Bellaterra
www.synlab-vet.de www.invitro.com Spain
www.univet.es
Médivetlab Utrecht University Vetlab Ltd.
1 rue Pierre Brossolette Faculty of Vet. Medecine Väinölänkatu 11
8700 Limoges Dept. Clin. Sci. Comp. Animals 33500 Tampere
France Yaleaan 8; 3584 Utrecht CM Finland
[email protected] The Netherlands www.vetlab.fi
North America
Heska Co. Vita-Tech
3760 Rocky Mountain Ave 1345 Denison Street Markham
Loveland, CO 80538 ON, L3R 5V2
USA Canada
www.heska.com www.vita-tech.com
Asia
Falco Biosystems Ltd. Hitachi Chemical Co., Ltd. Saloon Ltd.
346, Shimizu-cho, Nijoagaru 13-1 Higaschi-cho-4-chome 272-7 Horinouchimachi,
Kawaramachi-dori, Nakagyo-ku Hitachi-shi Takatsujidori
Japan Japan Shimogyo-Ku
www.falco.co.jp www.hitachi-chem.co.jp Kyoto
Japan
www.saloon.co.jp
Africa & Oceania
Vetdiagnostix Gribbles Veterinary
P.O. Box 13624 1868 Dandenong road
3202 Cascades Clayton VIC 3168
South Africa Australia
www.vetdiagnostix.co.za www.gribbles.com.au

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Submission of Samples for Allercept® IgE Test

1. Serum sample required (min 1mL) collected in a plain collection tube. Mix the tube thoroughly by gentle
inversion approximately 5 to 6 times. Samples should be stored at 2-8oC before delivery. Deliver to the
laboratory as soon as possible.

2. In cases whereby the sample(s) cannot be dispatched to Gribbles Veterinary i.e. collected over the weekend
or during public holidays like Easter, Christmas, remove the clot from the sample by either centrifugation or
standing samples until the serum separates and pour off the serum into a blood tube or plain tube NOT a blood
collection tube. Store both serum and clot at 2-8oC until delivery day.

3. It is recommended that you use permanent marker pen to label all sample containers with client/animal identity.
This is best done on the body of the container and not on the lid. Please detail date collected.

4. A completed submission form must accompany each case submission. Phone 1300 307 190 to order these forms.
For companion animals the small animal submission form is required. It is essential that the forms be filled out
correctly and completely. A full but concise history helps us give you the best information from the laboratory
results.

5. Package the individual specimens into biohazard bags containing a separate section for the submission form.
Samples are to be transported in an esky packed with an absorbent impact proof protective layer and clearly
labelled Gribbles Veterinary Pathology. The use of protective bubble wrap and coolant pads is recommended for
specimens, especially those travelling some distance.

6. Gribbles Veterinary operates a courier service in Victoria, NSW and South Australia. In regional areas contract
couriers are utilised. Please contact Gribbles Veterinary Pathology Help Desk on 1300 307 190 for further
information.

When to perform allergen specific IgE serology with a view to in-house ASIT or avoidance.

• When IDT is not accessible

• When sedation or clipping is not possible
• Where the AD is uncomplicated and the only symptom is non-lesional pruritus

NOTE:
• Testing should not be performed in dogs <5-6 months of age
• DRUG WITHDRAWAL MUST BE OBSERVED
- 3-8 weeks off prednisolone
- 3-4 months off methylprednisolone acetate, progestagens
- 2 weeks off topical glucocorticoids, antihistamines, and clomipramine (latter up to 4 weeks)
• Test < 8-30 days after end of the patient’s pollen season (and preferably the middle to end of pollen season)
• Testing should not be performed unless the managing veterinarian is experienced with
immunotherapy / atopic management
• Apoquel® and Cytopoint® are fine to continue using as they will not affect the test

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The Heska ALLERCEPT® Allergy IgE test Specific for Australia

WEEDS TREES GRASSES MITES/INSECTS MOULDS

Curled dock Elm Paspalum Flea saliva Alternaria

Couch
Sheep sorrel Maple Ant Aspergillus
(bermuda grass)
Bent grass
Red root amaranth Grey birch Cockroach Cladosporium
(red top)

Phalaris Dermatophagoides
Lamb’s tongue Red oak Penicillium
(canary grass) pteronyssinus

Fat hen Silver poplar Sweet vernal Dermatophagoides farinae

Kentucky
Perennila ragweed Liquidamber Tyrophagus putrescentiae
blue grass
Mugwort Plane tree Orchard grass
Mustard week Privet Broome grass
Dandelion Peppercorn Timothy grass
Daisy Cypress Perennial grass
Black widow Yorkshire fog
Pine mix Johnson grass
Eucalyptus
Melaleuca
Casuarina
Wattle

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 1868 Dandenong Road, Heska AG
Clayton, Vic 3168 Grand’Places 16,
Australia Switzerland

Phone: 1300 307 190 Phone: +41 26 347 2140

Fax: 03 9538 6741 Fax: +41 26 347 2141
www.griblesvets.com www.heska.com

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