EXAMINATION OF THE MOST COMMON ALLERGENS OF CANINE ATOPIC DERMATITIS - A RETROSPECTIVE STUDY

Canine atopic dermatitis (CAD) is one of the most common skin diseases of dogs. It is estimated that 10-15% of the dogs are showing clinical symptoms of this disease. Canine atopic dermatitis is defi ned as genetically predisposed infl ammatory and pruritic allergic skin disease with characteristic clinical symptoms. It is related to the production of IgE antibodies, mostly directed against external allergens. Allergens that can cause canine atopic dermatitis are quite numerous and depend on the geographical characteristics of research location. Th e aim of the study is to determine the most common allergens that can cause canine atopic dermatitis. Th e research was conducted according to medical records of 100 dogs during a period from the beginning of 2008 to the end of 2012 from the Department for skin diseases of small animals of the Clinic of equine, small animal, poultry and wild animal diseases at the Faculty of Veterinary Medicine, University of Belgrade. According to the clinical documentation, all of the tested dogs had clinically manifested symptoms of CAD. In order to confi rm the diagnosis and detect the causative allergens, intradermal testing has been done. Th is testing has been performed with a standard set of 24 allergens specifi c to the geographical area of the research, produced in the Institute of Virology, Vaccines and Sera “Torlak”. According to the results, the highest percentage of positive responses was established for the following allergens: house dust mites ( Dermatophagoides sp . ) 67%, ragweed ( Ambrosia artemisiifolia ) 61%, household dust 60%, cocks foot ( Dactylis sp.) 59%, mix of weed pollen 57%.


INTRODUCTION
Canine atopic dermatitis (CAD) is one of the most common skin diseases of dogs.It is estimated that 10-15% of dogs are showing clinical symptoms of this disease (Williams, 2001).
CAD is defi ned as genetically predisposed infl ammatory and pruritic allergic skin disease with characteristic clinical symptoms related to the production of IgE antibodies, mostly directed against external allergens (Halliwell, 2006).Th e pathogenesis of CAD is not yet entirely elucidated, but it is considered that a defect in the epidermal barrier allows breakthrough to the external and probably microbial allergens (Marsella 2006).
CAD usually occurs between 3 rd and 6 th month of age.Many studies from diff erent periods and diff erent geographical locations demonstrated that the following dog breeds are predisposed to CAD: West Highland White Terrier, Golden Retriever, Labrador Retriever, Bulldog (English and French), Chinese Sharr-Pei, Dalmatian and Poodle (Picco et al., 2008;Chanthick et al., 2008;Favrot et al., 2010).
Allergens that can cause CAD include house dust mites, pollen of trees, weeds and grasses, house and bed dust, fungal spores, animal and human dandruff , feathers, cigarette smoke, fi lling of the furniture, some bacterial allergens, insects (Chapman et al., 2007 ).
According to some authors, the primary lesion does not exist in CAD (Scott, 1981).Other authors indicated erythematous papula as a primary lesion (Chaberlain, 1974).Secondary lesions are the result of chronic pruritus and subsequent scratching, chronic infl ammation of the skin and competitive secondary pyodermia and malassezia dermatitis.Partial or complete alopecia, reddish discoloration of hair because of the saliva, excoriation, papules, pustules, dry broken hair, hyper pigmentation, scaling and lichenifi cation are noticed on the skin (Scott et al., 2001;Chanthick et al., 2008;Favrot et al., 2010).
Th e aim of this study was to determine the most common allergens that caused CAD in the Ambulance for skin desises of small animals of Department of equine, small animal, poultry and wild animal diseases at the Faculty of Veterinary Medicine, University of Belgrade in the period from the beginning of 2008 to the end of 2012.

MATERIAL AND METHODS
Th e research was conducted by using medical records of 100 dogs during a period from the beginning of 2008 to the end of 2012 in the Ambulance for skin diseases of small animals of Department of equine, small animal, poultry and wild animal diseases at the Faculty of Veterinary Medicine University of Belgrade.According to the documentation, all tested dogs had clinically manifest symptoms of CAD.In all animals, general clinical examination was conducted, as well as specialist dermatological examination.Determination of the presence of fl eas and skin scarifi cation has been performed.
For intradermal application of allergens extracts and the positive and negative controls, 1 ml syringes with a needle dimensions 26 G (0.5 mm) were used.On the previously marked area, 0.05 ml of prepared allergens extracts, as well as positive and negative controls, was injected intradermally.Results of intradermal testing were interpreted aft er 15 minutes.
Aft er this time, the reaction was measured (transparent ruler) and graduated.Th e reaction, which has been of the same size as the negative control has been marked as 0, as a reaction same with the histamine +4.Reactions marked as +2 and higher were considered positive.

RESULTS
Table 1 displays the percentage of dogs, clinically suspected for CAD, which showed positive reactions to intradermal test on individual allergens.All tested dogs were multi-sensitive to intradermal testing.In the majority of dogs, between 6 and 11 positive reactions were recorded.

DISCUSSION
In the examined dogs, the highest number of positive reactions has been registered to the following allergens: house dust mites (Dermatophagoides sp.), ragweed (A.artemisiifolia), cock's foot (Dactylis sp.), weed pollen and house dust mixture (Table 1).
A total of 24 allergens were used, and all dogs were multi-sensitive.In the majority of dogs, between 6 and 11 positive reactions were recorded.One dog had 23 positive reactions.Also, the results in the literature confi rm that the majority of the tested dogs had been multi-sensitive (Zur et al., 2002), which indicates the complexity of the CAD pathogenesis.
According to our study, house dust mites (Dermatophagoides sp.) were the most common allergen that caused positive reaction in dogs in intradermal testing (67%).However, literature data reported strong response in dogs (47-80%) to that allergen at the same concentrations of 250 PNU / ml that we used (Zur et al., 2002;Tarpataki et al., 2006;Chanthick et al., 2008).House dust mites belong to the family Arachnidae and they live freely in the epidermal debris of humans, animals, yeasts, and household food remains (Spieksma, 1990).House dust is a complex allergen, which consists of house dust mites, dandruff , mold spores, insect feces, bacteria, fi brous material from plants and animals, food debris and many other substances.Th ere are diff erent opinions about whether house dust mite allergen should be included in the intradermal testing because of its complex structure and strong irritating eff ect that may lead to false-positive reactions.Based on the results of intradermal testing, positive reaction to this allergen occurs in 39.4-75.5% of tested dogs (Willemse and Van den Brom 1983, Zur et al., 2002).In our study, reaction to house dust has been identifi ed in 60% of cases, and these data are consistent with the literature reports.In our study, we used concentrations of 250 PNU / ml, as recommended by other allergists (Reedy et al, 1997.;Sousa and Halliwell, 2001).
Weeds are annuals that grow wild and do not have any agricultural or decorative importance.Th e pollination of weeds takes place from the second half of July to November.According to data obtained by intradermal testing, weed pollen can cause a positive reaction in 2.7-45.5% of suspected atopic individuals (Willemse and Van den Brom 1983, Zur et al., 2002;Tarpataki et al., 2006;Chanthick et al., 2008).In our survey, the weeds were identifi ed as an important allergen in our environment (57%), especially ragweed (61%).Th is information is not surprising considering the fact that the pollen of these plants manifests strong allergenic potential (Reedy et al , 1997.).Th is plant is most widespread in Eastern and Central Europe, Hungary, Serbia, Croatia, Slovakia and the Czech Republic (D' Ammato et al., 1991).Also, the data from our country indicate prevalence of these plants all over Serbia, especially in Vojvodina, Mačva, Podrinje, Šumadija and in the valleys of large rivers (Janjic et al., 2007).In veterinary medicine, there are no published data on the percentage of positive reactions to this allergen (Milicic Matic et al., 2010).All American authors agree that ragweed (group of weeds) is the most important allergen in dogs and point out its strong allergenic properties (Zur et al., 2002;Tarpataki et al., 2006;Chanthick et al., 2008).
Tree pollen is one of the important allergens and, according to the literature data, 12-35% of dogs showed a positive reaction to these allergens (Willemse antigens and Van den Brom 1983, Zur et al., 2002;Tarpataki et al., 2006;Chanthick et al., 2008).According to our results, the single most important pollen allergen were willow tree (55%) and poplar (54%).Grass pollen is considered responsible for 10-30% of all allergies in humans.In dogs, these data are diff erent and mostly depend of the geographical location (Willemse and Van den Brom 1983, Zur et al., 2002;Tarpataki et al., 2006;Chanthick et al., 2008).In studies conducted so far, the intradermal testing was performed using the mixture of herb extracts, as well four of single allergens extracts: cock's foot (Dactylis sp), ryegrass (Lolium perene), timothy grass (Phleum pratense) and meadow fescue (Festuca pratensis).In our study, grass pollen was present in a slightly higher percentage (56%) as compared to the already published results.

CONCLUSION
Th e results of the conducted tests showed that most of the dogs with clinical signs of atopic dermatitis were multi-sensitive or demonstrated more than 2 positive reactions to the intradermal testing.Th e most common allergens that cause clinical symptoms of this disease were house dust mites, following ragweed, house dust, cock's foot, weed pollen mixture, mixed grass pollen, willow and poplar.

Table 1 :
Percentage of dogs, which showed positive reaction to intradermal testing on certain allergens