Proceedings of the European Veterinary Conference ... · 84 Abstracts European Veterinary...

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Proceedings of the European Veterinary Conference

Voorjaarsdagen

Amsterdam, the Netherlands Apr. 22-24, 2010

Next Meeting:

Apr. 27 – 29, 2011 - Amsterdam, the Netherlands

Reprinted in IVIS with the permission of the Conference Organizers http://www.ivis.org

83Abstracts European Veterinary Conference Voorjaarsdagen 2010

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Scientific proceedings: companion animals programme

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T i p s a n d T r i c k s To h e l p g e T T h e b e s T p o s s i b l e r e s u lT s f r o m a s k i n b i o p s y. ( T h e w h aT, w h e r e , w h y a n d h o w a p p r o ac h To s k i n b i o p s y i n p r ac T i c e )Sonya V. BettenayBVSc(Hons); FACVSc (Dermatology); DipECVD; Fachtierarztin in KleintierdermatologieTierdermatologie [email protected]

which skin lesions should be biopsied?Acute severe dermatoses; any skin lesion which appears unusual; lesions which fail to respond to empirical therapy; or where the con-templated disease is either expen-sive or potentially harmful; any sus-pect immune mediated disease;

lesions that are nodular and/or in which neoplasia is considered are all presentations whereby one can con-sider a skin biopsy. Biopsies may also be useful to exclude diagnoses even if one does not necessarily obtain a definitive diagnosis, however one should never take a biopsy looking for a diagnosis of “atopy”.

preparation of the site:With the exception of the excisional biopsy of nodules and the planned sampling of deep tissue for bacterial or fungal culture, no surgical preparation of the site should be employed. Even topical application of alco-hol with air drying may alter the epidermis. The overly-ing hair is clipped and gently removed. If crusts are present it may be less traumatic to use scissors than electric clippers, as the crusts should be sampled “in place” and a note ‘please cut in crusts’ should be added to the request form. Local anaesthesia can be adminis-tered subcutaneously with the needle entry point out-side the biopsy area. Infection as a result of this lack of aseptic preparation is rare.Pretreatment with systemic antibiotics may remove a secondary inflammatory response which can some-times be severe enough to mask the underlying pathol-ogy. Where possible – and this is not always the case where severe ulceration or systemic signs are present – and in particular in cases involving the nasal planum or mucocutaneous junctions, even a 5 day course of anti-biotics may really help in obtaining a diagnosis.

selection of the site:Spend 5 minutes looking for primary lesions and the full range of lesions. Carefully examine the entire ani-

mal for the most representative samples, and try to form a differential diagnosis list prior to biopsy. Sample depigmenting lesions in an area of active depigmenta-tion i.e. a grey colour rather than the final stage of dep-igmentation which is white. Sample alopecia in the centre of the worst affected area, as well as in junctional and normal areas. Sample areas of ulceration via an incisional wedge biopsy; orienting from the ulcer across to the adjacent intact skin. Do not expect a pathologist to be able to describe more than “an ulcer” if no epidermis is sup-plied unless there is an infectious agent or tumour embedded within the deep tissue. Always include a normal sample, if possible from the dorsal trunk, not ventrally. Obtain multiple samples (6 is a good rule of thumb) so that they represent the full spectrum of lesions from normal to worst.

sampling:Handle the biopsy specimen carefully, treat it like tis-sue paper even when excising. The biopsy punch is held at right angles to the surface of the skin and gen-tly placed over the selected lesion. Firm continuous pressure is applied and the punch is rotated in one direction (!) until a sufficent depth has been reached to free the dermis from its underlying attachment. The punch is removed and any blood carefully blotted away. The section of tissue is grasped at the base - which should be the panniculus - and subcutaneous attachments severed. Under no circumstances should the dermis or epidermis be grasped with forceps as this leads to ‘crush artifact’, destroying the cells and archi-tecture of the sample. In the case of a thin sample, it should then be placed - panniculus down - onto a rigid piece of cardboard or broken tongue depressor. This prevents the tissue from curling when placed in the formalin. The volume of for-malin required is approximately 10 times the volume of the sample. Large nodules can be partially* transected @ 1cm intervals to allow adequate penetration of the formalin into the centre of the lesion.* leave the panniculus attached, do not completely section

between “slices”

wedge vs punch:Elliptical excision is indicated with vesicles, suspected cases of panniculitis and when biopsying through the edge of a lesion to also include adjacent normal tissue (such as the above mentioned ulcer example). This allows correct orientation of the lesion by the techni-cian for sectioning.The punch biopsy is quick, relatively atraumatic and usually employed with suspected infectious, inflam-matory and endocrine dermatoses. Disposable biopsy

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Proceedings of the European Veterinary Conference - Voorjaarsdagen, 2010 - Amsterdam, Netherlands

84 Abstracts European Veterinary Conference Voorjaarsdagen 2010

punches are readily available in 4, 6 and 8mm diameter sizes and if in doubt, the Stiefel® brand is to be recom-mended. Punch biopsy instruments must be sharp. Use 6 to 8mm punches except on noses and feet, where smaller punches may be needed.

special dermatologic biopsy techniques:For canine claw disease, there is a special new biopsy technique which spares the toe and which is to be con-sidered when the owners refuse toe amputation as a biopsy technique. This uses a piunch biopsy instru-ment, but be careful, this will only work if the keratin is abnormal. Use of such a punch on a normal claw will probably break the punch. For lesions on the pinna, a technique referred to as “shave biopsy” may help to minimize the potential long term disfigurement which results if the cartilage is also sampled. This technique is not indicated in cases of suspected vasculitis.

submission of biopsy samples:Provide a complete history and physical findings report, with a list of clinical differential diagnoses. Many dermatoses are diagnosed using a combination of the signalment (age, breed, sex), clinical presenta-tion (distribution, type of primary lesions, if present), history (in particular previous response to therapy) and supportive histopathology. The dermatopathologist frequently uses the information submitted with the biopsy sample, to try to prioritise or discard the differ-ent clinical possibilities. Furthermore, if the provided list fails to correlate with the histopathology then a review of the sections, recuts of the tissue or special stains may be ordered immediately. Classical textbook histopathology is certainly seen, but not all patients read the textbooks. Completion of an appropriate skin biopsy request form will greatly improve the chances of establishing a diagnosis in these ‘grey zone’ cases.

h o w s h o u l d i a p p r o ac h T h i s s k i n lu m p ? m a n ag e m e n T c h o i c e s f o r c u Ta n e o u s n o d u l e s , b a s e d o n T h e u s e o f b oT h c y To lo g y a n d h i s To paT h o lo g y, - f r o m a d e r m aTo lo g i s T s’ p e r s p e c T i V eSonya V. BettenayBVSc(Hons); FACVSc (Dermatology); DipECVD; Fachtierarztin in KleintierdermatologieTierdermatologie [email protected]

Skin lumps are a major reason for the presentation of pets. As a clinician in practice, there are a number of typi-cally sub-conscious “first steps” one takes. However it is important to remember that the approach to the diagnosis of a cutaneous nodule must assume one of two things is present: neoplasia or infection.

The most common cause of nodules in the cat is the cat bite abscess and in the dog, a foreign body. The most common cutaneous neoplasms in dogs include the lipoma, mast cell tumour and sebaceous gland ade-noma. The most common tumour group in cats is the basal cell tumour group. All of these typically present as nodules.

Thorough dermatologic and physical examA thorough dermatologic exam must always be under-taken, although it is time consuming, as many concur-rent and early stages of lesions in addition to the nod-ules may be present but obscured by the coat. For example, both dermatophytosis and epitheliotropic cutaneous lymphoma may present as a wide range of lesions:- from diffuse erythema to scale to nodules. In addition, a complete physical examination should be performed with particular attention to respiratory sounds, abdominal and draining lymph node palpa-tion. The possibility of systemic, or concurrent diseases are important diagnostic issues. Although the diagnostic work-up for any nodule should be the same, there is a heightened need for an accurate diagnosis when treating nodules in cats and older dogs, as there is a higher likelihood for malig-nancy. It is important to remember that although cytology is a useful tool and frequently provides answers, histopathology is the definitive diagnostic tool with regard to neoplastic nodules and is required




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to evaluate prognostic features such as metastasis, local spread and tissue architecture.Some nodules are diagnosed by their classical clinical appearance, for example papillomas and sebaceous adenomas, which typically proliferate above the sur-face of the skin. Papillomas often have a pedunculated surface; sebaceous gland tumours are frequently shiny with a yellowish to pinkish hue. The uncommon dilated pore of Winer (a benign follicular tumour of cats) has a characteristic wide-mouthed central pore filled with compact keratin. Keratoacanthomas frequently have a central ‘horn’ or else a hole, where the horn has been lost. Breed predisposition plays a role in some tumors, particularly of dogs and to a lesser degree with some of the more exotic infections (for example mycetomas in Persians and pythiosis in young labradors). Tumors which commonly present with multiple lesions can be both benign and malignant. Tumors which are typi-cally benign in behavior include include the hair follicle tumors and infundibular keratinizing acanthomas (keratoacanthomas). Frequently malignant tumors include epitheliotrophic T-cell lymphoma and mast cell tumors.

cytologyCytology is an extremely useful tool in establishing a likely aetiology and is frequently a decisive factor in the “next step” decision making process. The handling of the tissue and submission of laboratory specimens must be undertaken in such a way as to minimize any possible zoonotic potential and also any local spread of either infection or neoplastic cells.Fine needle cell aspiration is the most commonly used method, although “aspirate” is really a misnomer these days, because the favoured technique does not aspi-rate anything, it is rather a “needle sticking technique” which uses the needle only, with no associated syringe. In this case the needle is inserted into the nodule, par-tially withdrawn, reinserted at a different angle a number of times and then finally withdrawn. This tech-nique minimizes the unintended spread of infectious agents or cells into the surrounding normal tissue and is suitable for all solid tumours. As soon as blood is seen to pool in the needle hub, it should be withdrawn. Blood dilutes the cytological sample and this will inter-fere with the diagnosis. In some cases blood contami-nation cannot be avoided, but it can be kept to a mini-mum. It is important to ensure the mass is firmly fixed between the fingers prior to insertion of the needle. Once removed, the needle is then attached to an air filled syringe and the contents blown onto the glass slide (with needle bevel down!) Subsequent spreading of the cells should be performed using similar tech-niques to those used for making a blood smear so that

the sample is not too thick. Cell features can be identi-fied clearly only when cells have been well spread on a slide. The aim is a so called monolayer. If the smear is too thick, cells may not stain well and are difficult – if not impossible - to identify.Wherever possible, it is best to make duplicate samples and stain one with the “in-house stain” for immediate reference. The other is air dried and can be sent off to the laboratory. The immediate reference slide can then become part of the practice teaching set. Some sam-ples are better sent to the experts – in particular those where neoplasia is a consideration.

deep tissue cultures and biopsiesWhen an infectious aetiology is likely, deep tissue cul-tures may be indicated and are obtained using both aseptic technique and additionally cutting off the sur-face layers. For a solitary neoplastic nodule, wide surgi-cal excision is the biopsy technique of choice, as this technique provides the entire specimen for histologic examination, allows margins to be examined and also effects a local cure in many cases. However as wide sur-gical margins are recommended for some neoplasms and as this may actually be cosmetic surgery in many cases, cytology can be used to determine whether wide excision is really needed. In addition, the cyto-logic identification of tumor cells allows discussion regarding possible outcomes before any general anesthesia and surgery is undertaken. In some cases, where cytology has established the presence of a tumour, but not the severity 8for example mast cell tumour, but not the grade) it may be better to simply obtain a punch biopsy sample, using local anesthesia from the centre of the nodule to determine the grade of the tumor. In that particular case, it may lead to the referral of the patient directly to a surgical oncologist / or oncology unit with radiation therapy possibilities.




a l l e r g y T e s T i n g i n p r ac T i c e – i s i T w o r T h i T ?Ralf S. MuellerProfessor of Veterinary Dermatology, DipACVD, FACVSc, DipECVDLudwig Maximilian University, [email protected]

When one thinks of allergy testing, it is typically in the context of intrader-mal tests or serum tests for allergen-specific IgE to diagnose atopic der-matitis. However, I will also discuss elimination diets to diagnose adverse food reactions in this lecture.

Atopic dermatitis is a fairly common cause of skin disease in the dog and increasingly diag-nosed in the cat as well. Serum tests for allergen-spe-cific IgE and skin testing have all been advocated as diagnostic aids, but neither is a good way to diagnose atopic dermatitis!!! It should be diagnosed based on the history and clinical signs as well as diagnostic trials to rule out diseases with similar clinical presentation. There are only three reasons to identify the offending allergens in an atopic animal:

Curiosity• Some owners will simply want to know what their •dog is allergic to without any effect on treatment of the condition.Avoidance• This is initially the most common reason mentioned •by owners in support of further testing. However, it is extremely rare that we can avoid allergens that are airborne.

• Immunotherapy Allergy shots or immunotherapy is the major reason

for us to recommend identifying the offending allergens in an atopic animal. This treatment aims at ‘getting the immune system used to the allergens’ and hopefully eliminating or at least decreasing the pruritus and/or predisposition to infection. This is a very safe and fairly easy treatment, that may replace concurrent therapy or at least decrease the dose needed to control clinical signs. Approximately 60% of the patients show a good to excellent response, 20% improve moderately and 20% do not show any improvement at all.

skin testing Skin testing is still the test of choice for atopic dermati-tis in animals. The allergens used in the test should be

chosen according to their importance in the respective environment. A positive skin test result indicates only the presence of specific IgE on mast cells in this patient and has to be interpreted in light of the patients history and environment. If one cannot perform at least 1-2 skin tests per week, evaluation of weak reactions may not be very reliable. In addition, allergens have a very short half life (approximately 2-6 weeks), once diluted to the concentration used for skin testing. Thus, skin testing is financially viable usually only if at least 1-2 skin tests per week allow efficient usage of the diluted allergens. False positive reactions are fairly uncommon with the exception of dust and storage mites which should be tested at lower concentrations than pollens. Ideally antihistamines should be discontinued 2 weeks prior to skin testing, any injectable glucocorticoids 6 weeks prior, glucocorticoid tablets 4 weeks prior and any topical steroid-containing creams, drops or oint-ments (such as ear or eye drops) 2 weeks prior to skin testing. One possibility in a dog with atopic signs and a negative skin test is that the offending allergen was not included in the test kit.

allergen-specific serum igeThese tests are convenient and particularly the Fcε receptor based test is very reproducible and reliable. There is increasing evidence that glucocorticoid admin-istration influences serum testing as well thus with-drawing those is recommended where possible. Some-times, grouped allergen testing is utilised for financial reasons and due to the high cross-reactivity in human medicine. If group testing is positive, then one, some or all of the allergens in that group may have contrib-uted to the hypersensitivity and thus the clinical signs. If all the allergens in that group are included in the allergy shots, immunotherapy with nonrelevant aller-gens may lead to development of new allergies in addi-tion to treating the present ones. Thus, immunotherapy based on testing for grouped allergens is strongly discour-aged! There is some evidence in human medicine, that antibody production may be local (in skin disease pro-duced by plasma cells in the dermis) and blood and tis-sue levels of antibodies may not correlate for that rea-son. Some dogs do not produce IgE despite the classical clinical signs. Serum IgE concentrations thus may not be elevated in every atopic dog.

elimination dietAn elimination diet for canine patients consists of a pro-tein source and a carbohydrate source previously not fed. These are home-cooked and usually fed in a ratio of three parts of the carbohydrates and one part of the protein. Possible proteins are deer, rabbit, buffalo, shark, salmon, horse or kangaroo, but any protein




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source previously not fed is suitable. Similarly, carbo-hydrate sources may be potato, sweet potato, kidney beans, tofu or yam. The protein part can be increased, but should not fall below 25%. Nothing else is included in the diet. In cats, only the protein source is fed, as most cats are not extremely eager to eat rice, potatoes or beans. Depending on the diet, jerky of the used meat, potato chips or little pieces of fried or grilled meat are all possibilities to allow the feeding of snacks without adding proteins potentially causing pruritus and masking the success of the eliminiation diet. An elimination diet should be started gradually, initially just added to the normal food, to increase the chance of acceptance. Spices such as salt or garlic may increase palatability. Warming the food in the oven or micro-wave may also increase acceptance. If home-cooked diets are not possible, commercial diets should be rec-ommended and chosen based on the same principles.After a period of 8 weeks or longer the patient is reeval-uated. If the animal is much improved, a rechallenge with the previously fed diet is performed. If there is no improvement after 8 weeks or if clinical signs do not return after a few days or at the most 2 weeks on the old diet, food adverse reaction is ruled out.

i n - h o u s e T e s T i n g i n s m a l l a n i m a l d e r m aTo lo g yRalf S. MuellerProfessor of Veterinary Dermatology, DipACVD, FACVSc, DipECVDLudwig Maximilian University, [email protected]

There are basic tests in which every practice interested in dermatology should become proficient and which can be performed and evaluated quickly in house, these are covered in this lecture.

cytology

A. General Comments We sample any chronic and pruritic skin condition

and all cases with otitis externa. Secondary micro-bial infections of the skin usually are bacteria or yeast organisms. These can be identified on cyto-logic preparations and appropriate therapy initi-ated. Bacterial cultures should always be interpreted

in the light of a cytologic evaluation, with multiple types of organisms growing on culture we need to know which one of them is most numerous in the lesions and thus most important to treat!

B. Methods• Rubbing, or impressing a slide onto a skin

surface. • Insertingacottonbudintheears.• Adirect impression techniquewithclearsticky

tape collects the debris from the surface of the skin. The tape is impressed onto the skin (sticky side down) and then laid (also sticky side down) onto a drop of methylene blue or a drop of the blue stain of DiffQuick on a slide (especially use-ful for Malassezia).

• Aspiration fromnodulesorabscesses.Aa22gneedle is inserted and redirected through the same insertion point several times in different parts of the nodule. The needle is then with-drawn and needle contents are blown onto a slide. The smear is air dried.

Stain• ModifiedWright’sstainsarecommonlyused.

C. Evaluation• Aretheorganismsyeasts(mostlikelyMalassezia

canis) or cocci (most likely Staphylococcus)?Rodsin the ear are most likely Pseudomonas or Proteus.

• Inflammatory cellswith intracellular organismsare pathognomonic for a clinically relevant infection.

skin scrapings

A. Superficial skin scrapings Superficial skin scrapings are used to detect Sar-

coptes or Cheyletiella. Mineral oil should be put on affected skin, scraped off with a scalpel blade and evaluated on a slide under a cover slip. 50% of sca-bies cases may be negative on several scrapings. One mite or egg is diagnostic. It is important to scrape over a large area and in hairy dogs clipping may be necessary. It is important not to remove the surface scale or crust. Sarcoptes mites are extremely superficially located within the epidermis and may be dislodged with such cleansing. We use scissors to remove the hair.

B. Deep skin scrapings Deep skin scrapings are performed to detect Demo-

dex mites which live in the hair follicle. It is useful to squeeze the skin prior to the scraping to push the mites out from the depths of the follicles. A blade covered with mineral oil should be used in the




direction of hair growth until capillary bleeding is observed. More than 1 mite is diagnostic.

When evaluating Demodex scrapings it is impor-tant to assess and to note the site of scraping, the relative numbers of adults, larvae/nymphs and eggs per field. In subsequent monthly revisits assess-ment of therapeutic response relies on the com-parison of such numbers.

wood’s light examinationIn 50% of all infections with the dermatophyte Micro-sporum canis, greenish fluorescence of tryptophan metabolites is seen. This fluorescence runs along the hair shafts (rather than fluorescing individual occa-sional scales that will be seen in normal animals and humans as well). Drugs, soaps and bacteria such as Pseudomonas may fluoresce as well but are usually not associated with the hair shafts. It may be helpful to warm up the lamp for 5 minutes before use.

fungal cultureHairs and scale from the edge of a lesion should be •taken. Alternatively, the McKenzie tooth brush method may be used, where the hair is brushed with a toothbrush and scale and loose hairs gently imprinted onto the agar. Sabouraud agar is the most common agar for fungal •cultures, however, in practice dermatophyte test medium is commonly used. This is a Sabouraud agar with added ingredients to inhibit overgrowth with saprophytes and bacteria as well as a colour indica-tor. The culture agar should be incubated at 20 - 25oC, or in a warm, dark corner with the lids not screwed down tight. PH change (and subsequently colour change) which occurs as the colony is grow-ing is indicative of dermatophytes. It is imperative that the colour change be observed coincidentally with the development of the colony as colour changes will also occur in association with mature (large) saprophyte colonies. Always check the colony under the microscope. •Clear sticky tape is impressed gently onto the culture (sticky side down) and then laid onto a drop of meth-ylene blue (also sticky side down) on a microscope slide. Microsporum canis has a white, woolly colony with a •yellowish reverse pigment and spindle-shaped mac-roconidia with knobs at the ends and typically greater than six internal compartments.

Microsporum gypseum has granular, beige cultures with yellowish reverse pigment and thin-walled macroconidia with less than six internal compart-ments.

Trichophyton mentagrophytes colonies characteris-tically have very few, cigar-shaped macroconidia and small, round microconidia.

TrichogramA forceps is used to forcefully pluck hairs in the •affected area. The hairs are then placed onto a slide and evaluated under low power. If the animal is pruritic and licks the hair off (or if the •hair shafts are damaged due to dermatophytes), the tips of the hairs are broken off. If the hair falls out for other reasons, the tips are tapered. If demodicosis is present you may find them on •trichogramms. This is particularly useful when sites close to the eyes are affected or the lesions are very painful. However, only a positive result is diagnostic. If dermatophytosis is present, you may see fungal •spores efface the clear shape of the hair shaft. How-ever, a negative result does not rule out dermatophytosis.

references1. BettenaySV,MuellerRS.Skinscrapingsandskinbiopsies In:

Ettinger S. J.,Feldman E. C., eds. Textbook of Veterinary Inter-nal Medicine. Philadelphia: W.B. Saunders 2005 388-391.

2. Mueller RS. Dermatology for the Small Animal Practitioner.Jackson: Teton New Media, 2000.

paT h o g e n e s i s o f aTo p i c d e r m aT i T i s i n h u m a n s a n d d o g sRalf S. MuellerProfessor of Veterinary Dermatology, DipACVD, FACVSc, DipECVDLudwig Maximilian University, [email protected]

Atopic dermatitis first has been described in 1911 in human medi-cine and at this time was considered a rare disease. In the last decade, the prevalence of atopic dermatitis in humans has dramatically increased in industrialized countries and is now considered to be higher than 30%. Similarly, allergic canine

patients seem to have increased in small animal practice. Initially, an increased production of allergen-specific IgE due to an alteration of the immune system was postulated as the cause for atopic dermatitis in humans.




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Increased IgE concentrations were found in atopic patients, allergen-specific IgE was demonstrated in those patients and the concentrations of IgE correlated to the severity of the disease. Although in the dog, total IgE concentrations of atopic individuals were not significantly higher than normal dogs, allergen-spe-cific IgE was demonstrated as well. Intradermal testing as well as testing for serum allergen-specfic IgE is widely used for the diagnosis of offending allergens in veterinary medicine. However, IgE concentrations do not decrease with clinical improvement in humans. Furthermore, serum allergen-specific IgE can be docu-mented in normal dogs. Although IgE plays a role in the disease, it has moved from centre stage to being one of many players on the scene and certainly not the ultimate cause. Subsequently, T cells were the focus of allergy research and an exaggerated Th2 response was considered crucial in the pathogenesis of atopic dis-ease. Although it is well accepted that one of the early changes in atopic dermatitis is an exaggerated Th2 response, such a response is also seen in nonallergic humans with endoparasites. Furthermore, in chronic lesions of atopic dermatitis, an increased Th1 response could be demonstrated in humans and dogs. More recently, the hygiene hypothesis has received much attention in human medicine, where a lack of exposure to Toll-like receptor ligands may change the reactivity of antigen-presenting dendritic cells and T cells. A number of epidemiologic studies have shown an inverse correlation between the development of aller-gic disease and life style, environmental endotoxin concentrations and concentrations of other allergens. The involvement of intestinal flora has also been dis-cussed. Decreased concentrations of lacto- and bifido-bacteria have been documented in allergic infants and these changes were suspected to influence the prim-ing of the immune system in early age. A defective barrier function of the epidermis with sub-sequent increased penetration of environmental aller-gens was long suspected as a relevant factor in the pathogenesis. Recently, this received renewed atten-tion. An increase in transepidermal water loss and enzymes degrading components of the stratum cor-neum and a decrease in ceramides was documented in human atopic dermatitis. More recently, loss of func-tion variants of the gene encoding filaggrin have been identified as a major predisposing factor for atopic der-matitis. In dogs, abnormalities in essential fatty acids contributing to the skin barrier and in the intercellular lipid lamellae have been identified. Very recently, decreased filaggrin expression has also been demon-strated in atopic beagles compared to healthy controls. These changes may lead to an increased penetration of antigens into the skin and subsequent binding to IgE

on the surface of antigen presenting cells (APC) such as Langerhans cells.Activated dendritic cells then contribute to the genera-tion of allergen-specific CD4+ T helper (TH) cells. Once generated, effector TH2 cells produce IL-4, IL-5, IL-9 and IL-13, cytokines with several regulatory and effector functions. Among other functions these cytokines induce the production of allergen-specific IgE by B cells and development and recruitment of eosinophils. The degranulation of basophils and mast cells by IgE-mediated cross-linking of receptors is the key event in type I hypersensitivity. Although in humans TH2 cells are initially involved in the development of allergic dis-eases, TH1 cells may play an important role in the chronic and effector phase of allergic disease or decrease allergic inflammation depending on disease type and stage of inflammation. Distinct TH1 and TH2 subpopulations of T cells counter-regulate each other and play an important role in distinct diseases. In the dog,aTH2responsewithoverproductionofIL-4mRNAin lesional infiltrated lymphocytes and low expression ofIFN-γmRNAinperipheralbloodmonocytes(PBMC)has been reported. A subset that is increasingly the topic of interest are regulatory T cells (Treg) that in humans have specific surface markers. These cells pro-duce inhibiting cytokines such as TGF-β and/or IL-10. In allergic humans, those Treg cells as well as their cytokines are decreased and increase with successful allergen-specific immunotherapy. Similarly, a recent report has described an increase of IL-10 after success-ful allergen-specific immunotherapy in atopic dogs.In late and chronic stages of the disease, autoimmunity may play a role and explain the difficulty to control clinical signs with anti-allergic therapies. Autoantibod-ies have been described in humans. In dogs, a recent study failed to detect such antibodies. The role of autoimmunity in late stage atopic dermatitis needs more study.Summarizing these puzzling and sometimes contrast-ing reports, it seems that atopic dermatitis is caused by a complex interplay of genetic factors (affecting the barrier function, the immune system or both) and exposure to various antigens that lead to increased contact of some environmental antigens with the immune system. This results initially in an increased Th2 response to those antigens. Subsequently, an addi-tional exaggerated Th1 response in chronic lesions occurs. With time the immune response may also be directed against self antigens and become a self-per-petuating process.




e V i d e n c e - b a s e d T r e aT m e n T o f c a n i n e aTo p i c d e r m aT i T i sRalf S. MuellerProfessor of Veterinary Dermatology, DipACVD, FACVSc, DipECVDLudwig Maximilian University, [email protected]

Canine atopic dermatitis (CAD) is one of the more common skin dis-eases in small animal practice and in many respects similar to the fre-quently diagnosed atopic dermatitis in humans. A number of drugs have been reported as successful treat-ment for atopic dermatitis, but in contrast to the human side, many

treatment modalities have not been evaluated care-fullywithrandomizedcontrolledtrials(RCT)inthevet-erinary field.

glucocorticoidstopical 0.015% triamcinolone acetonide as well as 0.0584% hydrocortisone aceponate were evaluated in RCTs.Oralglucocortocoidstypicallywereevaluatedasstandard-of-carecontrolsinRCTsforcomparisonwithother interventions and methylprednisolone, pred-nisone and prednisolone at 0.4-1 mg/kg/day were most commonly used. Adverse effects included poly-phagia, PU/PD, weight gain and/or intermittend GI signs. There is good evidence for high efficacy of oral and topical glucocorticoids and low harm of short term treatment. The benefit of long term treatment with glucocorticoids must be weighed against the risk of adverse drug effects impacting on health and quality of life.

calcineurin inhibitorsseveral large blinded trials have been conducted treat-ing several hundred dogs with cyclosporine at 5 mg/kg/day. It was found to be equally effective as gluco-corticoids in reducing lesion scores and owner-assessed pruritus. Vomiting and diarrhea were the most com-monly seen adverse effects but were mostly mild to moderate. Papillomatous eruptions and gingival hyperplasia were also seen occasionally. The trials pro-vided good evidence for high efficacy of oral cyclosporine in canine atopic dermatitis. However, the long term safety of cyclosporine in dogs is not known beyond the safety studies of cyclosporine treatment for one year in Beagle dogs.

Tacrolimus is a topical calcineurin inhibitor evaluated in several studies. At a concentration of 0.1%, it decreased clinician –assessed lesion scores and owner-assessed pruritus scores of localized atopic dermatitis after 4-6 weeks of once to twice daily therapy. The only adverse effect seen was mild and transient irritation at the site of application. These trials provide good evi-dence for medium efficacy of 1% tacrolimus in CAD.

histamine receptor antagonistsMost studies evaluating first-generation, sedating anti-histamines were neither blinded nor randomized or controlled. They provide conflicting evidence of bene-fit for chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, promethazine and trimeprazine for treat-ment of canine AD. However, adverse effects are rare and consist most commonly of drowsiness. One blindedRCTprovidedfairevidenceofmediumefficacyof a combination of hydroxyzine and chlorpheniramine. This combination product is the first choice treatment with type 1 histamine receptor antagonists in our clinic. Second generation low-sedation antihistamines such as loratidine and astemizole did not show efficacy in several studies, terfenadine showed conflicting results. Oxatomide did show medium efficacy in several trials with 20-50% of patients exhibiting a satisfactory con-trol of pruritus. Adverse effects were only mild and transient, most commonly increased appetite.

polyunsaturated fatty acidsThere are a number of studies evaluating polyunsatu-rated fatty acids (PUFA) for the treatment of canine atopic dermatitis. There is good evidence for their moderate efficacy. Between 25 and 40% of patients responded favorably in the treatment groups. There is also evidence that concurrent administration of PUFAs decreases the requirements for glucocortocoids in atopic patients and that a combination of antihista-mines and PUFAs is more effective than either therapy alone. Adverse effects of PUFA therapy were mild and typically consisted of diarrhea.

phospodiesterase inhibitorsPapaverine, arophylline and pentoxifylline were all evaluated. Papaverine did not lead to satisfactory con-trol of pruritus in an open, nonblinded, non-ran-domised trial. In a randomized, controlled trial, aro-phylline at 1 mg/kg twice daily showed a fair efficacy. However, vomiting was common and often severe. Due to either inefficacy or an unacceptable rate of adverse effects, papaverine and arophylline thus can-not be recommended for the treatment of canine atopic dermatitis. Pentoxifylline at 10 mg/kg twice




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daily for 4 weeks was found to be moderately effective. Clinical adverse effects were not noticed in this trial.

leukotriene inhibitorsleukotriene inhibitors have been evaluated for the treatment of canine atopic dermatitis in 4 trials. Zafirlu-kast caused vomition in some dogs, adverse effects were not seen with Zileutin. The trials provided good evidence for no or very low efficacy of leukotriene inhibitors for CAD.

prostaglandin analoguesin both trials evaluating the efficacy of the PGE1 analog misoprostol the clinician-assessed lesional scores and the owner-graded pruritus values decreased with treatment. Adverse drug effects were mild and inter-mittent vomiting or diarrhea. These studies provide fair evidence of medium efficacy of the prostaglandin analog misoprostol (5 µg kg-1 three times daily for up to six weeks) to treat dogs with AD. Aspirin was evalu-ated in an open clinical trial and showed no efficacy.

Topical antipruritic drugsan open four week trial provided limited evidence of medium efficacy of pramoxine rinses. Another RCTcompared the efficacy of whirl pool therapy with an antiprurituc shampoo, conventional shampoo therapy and whirlpooling in water. Dogs improved significantly more with conventional shampooing and with sham-poo therapy in the whirl pool compared to the control group.

allergen-specific immunotherapyalthough there are a number of studies evaluating the effect of ASIT on canine atopic dermatitis, most studies were retrospective and thus neither blinded nor con-trolled. The reported efficacy of conventional ASIT is greater than that seen with antihistamines or fatty acid supplementation, and adverse effects occur much less frequently than with glucocorticoid administration. Most of these studies report excellent efficacy in approximately 20% of the patients and good response inanadditional25-45%.RushASITischaracterizedbya dramatically reduced time interval between injec-tions of increasing amounts of allergen extract, with a consequent decrease in the induction period from weeks to months to one day with injections adminis-tered every 30 minutes subcutaneously. A double-blindedRCTcomparingconventionalwithrushimmu-notherapy found significant improvement in pruritus, medication and clinical scores after 12 months of immunotherapy. Maintenance therapy occurs with an individually determined concentration at a certain fre-quency, typically every 1-4 weeks. At this point, the

numerous studies can be considered fair evidence for moderate efficacy of ASIT in the treatment of CAD.

d i ag n o s i s o f i m m u n e - m e d i aT e d s k i n d i s e a s eRalf S. MuellerProfessor of Veterinary Dermatology, DipACVD, FACVSc, DipECVDLudwig Maximilian University, [email protected]

Immune-mediated skin diseases come in many varieties with varied clinical signs. There are some clinical or historical clues that make these diseases more likely and should lead to a more aggressive diagnostic work-up early on in the disease. An immune-mediated disease should be considered when

historically the disease had an acute onset and the •patient rapidly deteriorates.mucous membranes or mucocutaneous junctions •are affectedskin lesions are only part of the disease and other •organ systems seem to be involved (joints, kidney, etc.).the planum nasale is affected.•

Cytology may be useful in the diagnosis of diseases of the pemphigus complex. An impression smear is most commonly used. The slide is gently pressed against an eroded, exsudative or ulcerated area (if non it present, one can carefully peel off a crust and sample the eroded surface underneath) and stained with Dif-fQuick. In many patients with pemphigus, acantholytic keratinocytes will be identified. These cells stain blue to purple, are round and have a central nucleus. They are not diagnostic for pemphigus (as occasionally they can be found in pyodermas as well), but indicate a need for a biopsy and a probability of pemphigus. If an immune-mediated disease is on the list of differential diagnoses, a biopsy is always indicated.

There are a number of immune-mediated skin diseases but most are rare. However, dogs and cats with discoid lupus erythematosus or pemphigus foliaceus are pre-sented infrequently but regularly in small animal prac-tice and we will focus predominantly on these two diseases.




The pemphigus complex includes several subtypes in all of which the immune system for various and often unknown reasons starts to produce antibodies against parts of the desmosomes holding adjacent keratinoc-ytes together. These (auto-) antibodies are called pem-phigus antibodies.

The antibodies in the different subtypes are directed against different antigens of the desmosomes expressed in different layers of the epidermis. Thus, the location of the forming vesicles within the epider-mis and the resultant clinical signs vary. Once the anti-body is bound to the part of the desmosome which forms the antigen, the complex is “swallowed” by the cell and elicits intracellular reactions leading to the release of plasminogen activator. The subsequent plas-minogen activation results in the production of plas-min, a protease that destroys the desmosomes and leads to acantholysis, where keratinocytes lose their intercellular bridges and “round up”. These acantho-lytic cells are located as single cells in vesicles formed by the destruction of the intercellular connections.

In the case of pemphigus vulgaris the blisters are located suprabasally. Such deep vesicles rapidly develop into ulcers once burst. Pemphigus foliaceus and erythematosus develop a split within the spinosal layer or subcorneally. Thus, the blisters are more super-ficial and erosions and crusting predominate. Drug reactions can trigger pemphigus, thus a history of recent drug application should be taken. Some animals will exhibit a worsening of clinical signs upon sun exposure as well.

Clinical signsLesions of pemphigus vulgaris are characteristically seen in the oral cavity (80% of the cases have oral ulcers or vesicles at the time of diagnosis), other mucous membranes and the groin and axillae. Lymphadenopa-thy, anorexia, lethargy and fever as well as secondary pyoderma (and sepsis) are frequently present. These pets are sick, look disgusting and feel miserable. pem-phigus foliaceus is one of the most common immune-mediated skin diseases. The mean age in a bigger study was 4.2 years. Akitas, Chows and Dobermans are some of the more commonly affected breeds.

Depigmentation of the planum nasale and/or crusty lesions of the dorsal muzzle, periocular areas and pin-nae are often the first signs. Feet, foot pads and groin are affected commonly. Generalisation may occur after weeks to months. Pruritus, pain and lethargy may be seen in some cases. Paronychia (with often a caseous, brownish material accumulating in the nail fold) and

nipple involvement is frequent in cats. One of the clas-sical clinical clues for pemphigus foliaceus is crusting on the inside and nonhaired center of the pinnae. pemphigus erythematosus is clinically indistinguish-able from early pemphigus foliaceus with facial involve-ment only.

DiagnosisBiopsy is the diagnostic test of choice. It is usually per-formed after skin scrapings have been negative, no evidence of fungi or bacteria has been found on cytol-ogy or antibiotic therapy improved the patient, but pustules and crusts without microorganisms are still evident on reexamination.

Multiple biopsies should be taken (I usually take four to five samples) and you must choose your sites carefully. Ideally you want to take intact pustules. The second best lesion is a papule. And last, you may include some really crusty lesions with the thick crust still attached to the biopsy. On a classic biopsy intraepidermal or sub-corneal pustules are filled with neutrophils, acantho-lytic cells and occasionally eosinophils.

discoid lupus erythematosus (DLE) is one of the more frequently encountered autoimmune skin dis-eases. The exact pathogenesis of DLE is not elucidated in dogs and cats. However, a subset of dogs with clini-cal signs definitely deteriorates when exposed to UV light and it is safe to assume that UV rays play a role in at least some of the patients with DLE. Breed predispo-sitions exist for Collies, Shetland sheepdogs, German shorthaired pointers, Siberian Huskies and Brittany Spaniels.

Clinical signsDiscoid lupus erythematosus in early stages is charac-terized by depigmentation, erythema and scaling of the nose. The rough cobblestone-like architecture of the planum nasale changes into a smooth surface. Depigmentation of the skin around the eyes and the lips is observed less commonly. Erosions, ulceration and crusting are changes occurring later and may extend from the planum nasale and nostrils up to the dorsal and lateral part of the haired muzzle. These changes may also be encountered around the eyes and on the pinnae.

DiagnosisBiopsy of nonulcerated lesions is the diagnostic option of choice. Ideally, a greying area is chosen because the depigmenting is actively going on and the histopatho-logical findings are most diagnostic. For discoid lupus erythematosus two to three punch biopsies are taken.




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I typically choose 6mm punches for the nose and 8mm for the dorsal muzzle in most dogs and 4mm punches for the nose and 6mm for the dorsal muzzle in cats and toy breeds. For cutaneous changes on the trunk or legs I always use 8mm punches or excisional biopsies.

T r e aT m e n T o f i m m u n e - m e d i aT e d s k i n d i s e a s eRalf S. MuellerProfessor of Veterinary Dermatology, DipACVD, FACVSc, DipECVDLudwig Maximilian University, [email protected]

First we will cover the more benign ways of regulating the immune system:

Vitamin E• is an antioxidant and as free radicals are thought to play a role in many types of immune-mediated inflammation and vita-min E successfully deals with these radicals, it has been used in

a number of inflammatory diseases. It is adminis-tered at 400 to 800 Units daily.

Scott and Miller reported essential fatty acid supple-•mentation with omega 3/omega 6 fatty acids as an alternative to vitamin E for dogs with DLE. I use it often as adjunctive therapy.A combination of • tetracycline and niacinamide (vita-min B3) at 250 (if the patient is <15kg) -500mg (if the dog is >15kg) q 8 hours has been reported to treat a number of immune-mediated diseases. We have reasonable success with that combination in about half of our patients with discoid lupus erythemato-sus or pemphigus foliaceus. If this combination works, we usually try to replace it with doxycycline at 5-10 mg/kg once to twice daily for convenience reasons. Topical steroids• may be used in dogs with nonulcer-ated skin and focal disease. The drugs are absorbed extremely quickly and if the dog does not lick the area for 5-10 minutes, sufficient absorption has occurred. Feeding or walking the dog directly after application may be beneficial. The disadvantage with most topical steroids is the thinning of skin seen after chronic use with rapid bleeding in these areas. Tacrolimus• is a newer drug absorbed through intact epithelium. In dogs, we use it most commonly for the treatment of localized immune-mediated dis-

ease, particularly discoid or cutaneous lupus ery-thematosus, and for the treatment of anal and peri-anal fistulae. It needs to be applied in very small amounts only once to twice daily.

Now we come to the more extreme suppression of the immune system:Before you think about immunosuppressive therapy you must be sure about your diagnosis. If the animal has an infectious disease (fungal, bacterial or parasitic), it can rapidly deteriorate and even die. There is no place for trial therapy in immune-mediated disease (!) (except may be in the case of a patient facing eutha-nasia otherwise). Another problem with immunosuppressive therapy is the fact that is impossible to give you a good general purpose recipe. Every dog or cat reacts differently to each of the drugs mentioned below.

GlucocorticoidsAll of my patients with uncomplicated pemphigus •receive prednisolone as the first agent. This drug is inexpensive, relatively safe, has a fast onset of action and is easy to monitor. Thus expensive laboratory monitoring is usually not necessary.Induction dose is 1 - 2 mg/kg twice daily. After two •weeks, the patient needs to be reexamined. If signifi-cant to full remission is not achieved within that time, it is unlikely that the animal will do well on glu-cocorticoids alone and other drugs have to be added. About 30 - 40% of the patients with pemphi-gus will respond to glucocorticoid treatment. Once remission is achieved, the dose is tapered •down slowly (over about 10-20 weeks) to a minimal maintenance dose. The lower the dose the slower the tapering should be. Semi-yearly to yearly monitoring with urine cultures •are performed, as occult urinary tract infections were present in 40% of canine patients treated with long-term corticosteroids independent of the dose.

AzathioprineAzathioprine competes with purin in the synthesis •of nucleic acids leading to nonfunctional nucleic acid strands that prevent the proliferation of divid-ing cell populations. It also inhibits T lymphocyte-dependent antibody synthesis and cyclo-oxygenase (and thus production of proinflammatory prostaglandins).The use of azathioprine in cats is not recom-•mended. The canine dose is 50 mg/m2 or 2 mg/kg once daily •initially. Once remission is achieved, you can give the medication every other day for 4-6 weeks and then




taper further. As with all cytotoxic drugs frequently used in veterinary dermatology, there is a lag period of up to several weeks. For this reason azathioprine is commonly used in conjunction with prednisolone for the first weeks or months. Vomiting and diarrhoea can often be avoided by giv-•ing the drug with food or lowering the dose. Hepa-totoxicosis can be severe and of acute onset (within the first 10 days of treatment) in individual patients. Initial chemistry screens may be performed before and after 1, 2 and 4 weeks of therapy. Easier to over-look, but as serious is the bone marrow suppression which occurs in some patients on treatment. I evalu-ate complete blood counts (including platelet counts) for leukopenia, anemia and thrombocytope-nia before treatment, after week 1, 2, 4, 8, 12 and every 3 months thereafter. Another possible side effect is an increased suscepti-•bility to infections due to the immunosuppression. If a patient previously in remission suddenly shows clinical signs of disease, evaluate the possibility of demodicosis, bacterial or fungal infections. Not always is a relapse due to insufficient immuno-suppression!

ChlorambucilChlorambucil is an alkylating agent, which forms •covalent bonds with nucleic acids thus crosslinking or breaking DNA strands. It suppresses antibody production.Chlorambucil is used alone or more commonly in •conjunction with other drugs. It is one of the safest cytotoxic drugs, but its lag period is longer than that of azathioprine (up to 8 weeks). It is given at 0.1-0.2 mg/kg daily until the patient is in •remission. Then it is administered every other day and tapered in a similar fashion to azathioprine.Side effects are gastrointestinal upset and bone •marrow suppression. Monitoring is similar to azathi-oprine. Hepatotoxicosis is not of major concern. Sei-zures can occur rarely due to chlorambucil therapy.



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