Plenary Session Abstracts: Thursday Morning, 26 July Theme ...

Plenary Session Abstracts: Thursday Morning, 26th July Theme: ALLERGY State-of-the-Art Address Epidemiology of human atopic dermatitis H. WILLIAMS University of Nottingham, Nottingham, UK One thing is for certain after working in this area for 20 years: there is no single cause of atopic dermatitis (eczema). It is a complex multi-component disease that relies on genes and environmental interactions. It is also clear that the dichotomisation of eczema into atopic and non-atopic forms has not gotten us very far since most children with eczema in the community are not atopic, and because knowledge of IgE antibodies has not increased our predictive ability by much. The phenotype of eczema is now well described, but what we think of as eczema today may indeed be split into several forms such as diffuse dry types, flexural, adultonset, and discoid types as new discoveries influence our progressive nosology. After years of fruitless searches for genes that determine the eczema phenotype, the field has come alive with the discovery of filaggrin genes which finally explain much of the dryness so characteristic of eczema. But even though those filaggrin mutations are the strongest and most consistent predictor of eczema, they still account for < 20% of disease occurrence. Therefore, there is much more to eczema than genes. I shall present an overview of epidemiological studies that point to a strong role for environmental factors that may be important for childhood eczema. I shall include data to show that eczema is more common in wealthier and smaller families, and share data from the International Study of Asthma and Allergies in Childhood (ISAAC) that show a pattern of eczema across the world that cannot be explained by our current knowledge of possible risk factors. The ISAAC study also shows that symptoms of eczema have increased throughout the world in a relatively short time, a difficult observation to explain in genetic terms alone. I shall summarise data from migrant studies that show that eczema becomes more common when people migrate from low prevalence to high prevalence countries, and I shall touch on the hygiene hypothesis. I will look at some putative exposures in more depth, including house dust mite and hard water. Given the veterinary audience, I cannot of course omit a systematic review of childhood eczema and exposure to pets. I will also mention factors that may cause exacerbation of existing disease, a topic desperately in need of some good science, which is difficult to do. I shall introduce autoimmunity induced by scratching as a possible reason for disease progression and lack of response to allergen reduction. It is important that we do not give up on epidemiology as a means of quantifying disease burden and discovering risk factors that may be amenable to public health manipulation. Disease prevention and early aggressive treatment is so much more sensible than treating sick individuals who present after a long chain of pathological events with expensive toxic drugs that mainly reduce symptoms. Funding: Travel and accommodation costs kindly funded by World Congress of Veterinary Dermatology. Research funded by the UK National Institute for Health Research. Conflict of interest: None declared.

Supporting Review The genomics revolution: Will canine atopic dermatitis be predictable and preventable? T. NUTTALL School of Veterinary Science, University of Liverpool, Neston, Cheshire, UK Atopic dermatitis is a familial condition. Family history is a risk factor for human atopic dermatitis (hAD), and there are well-recognised breed associations in canine atopic dermatitis (cAD). Heritability and linkage studies suggest that AD involves multiple genes and interactions with environment factors. Advances in genomics have given us powerful techniques to study the genetics of AD. Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) allows quantification of gene expression. Microarrays can be used to assess the expression of very large numbers of genes, although the results are prone to false positive associations and require careful analysis. These approaches can identify potential candidate genes, but they are hypothesis-dependent and therefore may miss novel genes. Next-generation sequence-based expression profiling can sequence any active gene to produce a hypothesis-free transcriptome, although this technology is very expensive. Genome-wide association studies are a more widely used hypothesisfree technique to discover disease-associated single nucleotide polymorphisms (SNPs). The recent origin and inbred nature of dog breeds means that linkage disequilibrium extends over long distances; therefore, canine studies require fewer markers and smaller sample sizes to find disease associations. Genomic studies in dogs have implicated over 50 genes in the pathogenesis of cAD, although whether this is associated with cause or effect is not always clear. These genes are involved in innate and adaptive immunity, inflammation, the cell cycle, apoptosis, skin barrier formation, and transcription regulation, and many have also been implicated in hAD (including filaggrin, SPINK5, TSLP, CMA1, TIMP1, S100, PPAR, and others). Findings from genome-wide association studies, however, have been inconsistent. One study found two cAD-associated SNPs in all eight of the studied breeds, with 12 additional SNPs found in some but not all of the breeds. Some cADassociated SNPs were, furthermore, restricted to certain geographical locations. Other studies have failed to detect gene associations with cAD. Possible problems with genomic studies include low case numbers, inappropriate controls, inconsistent diagnosis, incomplete genome coverage (with low penetrance mutations), and environmental factors. Nevertheless, the cAD genotype is likely complex and likely varies between breeds and gene pools, which could explain variations in clinical phenotype and response to treatment. This is analogous to hAD, in which certain genotypes have been associated with particular phenotypes. This complexity and the high prevalence of cAD, means that a screening and breeding programme to eliminate the condition is unlikely to succeed. But despite this, the genomics revolution has huge potential for cAD. These techniques will allow identification of target molecules for novel treatments. In addition, we should be able to genotype atopic dogs and relate this to their phenotypes, which will enable better

2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

Abstracts targeting of treatment options. For example, some dogs may respond well to skin barrier therapy whereas others would benefit more from allergen-specific immunotherapy. Finally, it may be possible to identify atopic genotypes in young dogs and then manage environmental factors to prevent them developing clinical AD. However, we must be careful to avoid misuse of genomic data in diagnosis and by breeders or insurance companies. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 1 Serum anti-Staphylococcus pseudintermedius IgE and IgG antibodies in dogs with atopic dermatitis J. BEXLEY*, T. NUTTALL , B. HAMMERBERGà, J. R. FITZGERALD§ and R. E. W. HALLIWELL§ *Avacta Animal Health, Wetherby, UK; School of Veterinary Science, University of Liverpool, Liverpool, UK; àCollege of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; §Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK Dogs with atopic dermatitis (CAD) are predisposed to colonisation and infection with Staphylococcus pseudintermedius. It is possible that anti-staphylococcal IgE may exacerbate CAD. The aim of this study, therefore, was to compare staphylococcal-specific IgE and IgG levels in dogs with CAD, non-atopic dogs with pyoderma, and dogs without skin disease. Serum antibody levels were measured in four main groups. In Group 1 (n = 68) were dogs diagnosed with CAD according to accepted criteria that were positive to one or more environmental allergens, and that subsequently underwent immunotherapy (the presence and type of pyoderma not always known). In Group 2 (n = 67) were age-matched controls with gastrointestinal disease but no history of skin disease. In Group 3 (n = 82) were dogs with pyoderma confirmed by cytology and isolation of S. pseudintermedius. Of these, 38 had CAD (Group 3a), 13 had atopiclike dermatitis (Group 3b), 12 had demodicosis (Group 3c), eight had a keratoseborrhoiec disorder (Group 3d), six had recurrent idiopathic pyoderma (Group 3e), three had an adverse food reaction (Group 3f), and two had non-recurrent idiopathic pyoderma (Group 3g). In Group 4 (n = 9) were specific-pathogen-free (SPF)derived dogs (Charles River Laboratories, Ballina, Co Mayo, Ireland) maintained under minimal disease conditions. Staphylococcal antigens were derived from a protein-A-negative strain of S. pseudintermedius cultured from a dog with pyoderma. Soluble and cell-wall antigens (using lysostaphlin) were obtained from stationary and exponential growth phases. Detection reagents used were a phosphatase-conjugated monoclonal antibody (mAb) raised against a heat-stable epitope of canine IgE (mAb 5.91) and a phosphatase-conjugated polyclonal goat antidog IgG (gamma-specific; Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Results were expressed as arbitrary units (AUs) by reference to a standard curve, and data were checked for normality by Shapiro-Wilk before analysis by Welch two sample ttest and analysis of variance (ANOVA). Group 1 atopic dogs had a significantly higher mean level of antistaphylococcal IgG (P < 0.0001) than Group 2 control

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dogs, and had significantly higher mean levels of both anti-staphylococcal IgG (P < 0.001) and IgE (P < 0.05) than Group 4 dogs. Although Group 1 atopic dogs had a trend towards higher levels of anti-staphylococcal IgE than the controls, with 25% exceeding five AU compared to just 10.4% of control dogs, differences in mean levels of anti-staphylococcal IgE were not significant (P = 0.08). Levels of mean staphylococcal-specific IgG and IgE were, however, significantly higher in the combined atopic groups (Groups 1 and 3a) than both Group 2 control dogs (P < 0.05) and Group 4 SPF dogs (P < 0.01). Duration of infection was not correlated with levels of anti-staphylococcal antibodies in Group 3a CAD dogs, nor were there any significant differences in mean IgE and IgG antibody levels between Group 3a CAD dogs and dogs with pyoderma unrelated to atopy (Groups 3b through 3g). Thus whilst both atopic and non-atopic control dogs had variable levels of serum anti-staphylococcal antibodies, the finding of significantly higher levels of staphylococcal-specific IgE in atopic dogs is important as a prelude to studies on their antigenic specificity and possible correlations with disease phenotype. Funding: Avacta Animal Health. Conflict of interest: J. Bexley is an employee of Avacta Animal Health, and T. Nuttall and R.E.W. Halliwell serve as consultants. B. Hammerberg and North Carolina University own the rights to the monoclonal antibody employed.

Supporting Original Study 2 Characterization of dog filaggrin: Gene structure and protein expression in dog skin S. KANDA*, , T. SASAKI , A. SHIOHAMAà, K. NISHIFUJI*, M. AMAGAI , J. KUDOHà and T. IWASAKI* *Laboratory of Veterinary Internal Medicine, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; àLaboratory of Gene Medicine, Keio University School of Medicine, Tokyo, Japan Filaggrin (FLG) is a key protein for skin barrier formation and hydration. Filaggrin gene mutations cause ichthyosis vulgaris in humans. A strong association between FLG gene mutations and atopic dermatitis has been reported in Irish and other human populations, indicating that skin barrier dysfunction caused by FLG mutations represents a strong predisposing factor for atopic dermatitis in humans. Similar pathogenesis and clinical manifestation have been found in canine atopic dermatitis. However, our understanding of the molecular characteristics and the expression pattern of canine FLG is limited. The aim of this study was to determine the gene structure of canine FLG and the expression pattern of the FLG protein in canine skin for characterization of this protein in dogs. The publicly available canine FLG genomic DNA sequence was further confirmed by sequencing polymerase chain reaction (PCR) fragments amplified from canine genomic DNA. To determine the number of FLG repeat units, we compared two of the same cDNA sequences that code for canine profilaggrin (proFLG) using the self dot matrix analysis to visualize tandemly repeated sequences within canine proFLG. The 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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self dot matrix analysis revealed four units of repeated sequences, which apparently correspond to FLG repeat. We then compared the amino acid sequence of canine proFLG to that of humans and mice to identify conserved regions. This analysis also revealed that the amino acid sequences of the N- and C-termini of proFLG showed similarity among species, whereas those of FLG repeats showed low similarity. Interestingly, predicted FLG linker sequences located between FLG monomers and that induce proteolytic cleavage during proFLG to FLG processing showed some similarities among dogs, humans, and mice, which helped us to predict that canine proFLG has four units of FLG monomers, consisting of three complete units and one incomplete unit. This result was consistent with the number of FLG monomers estimated by the self dot matrix analysis. To further determine the expression pattern of the FLG protein in canine skin, polyclonal anti-dog FLG rabbit antibodies were raised against a synthetic peptide with amino acid sequence in the FLG monomer. FLG expression and localization in canine skin obtained from footpad, dorsal neck, and axilla of healthy beagles were analyzed by western blotting and immunohistochemistry using the anti-dog FLG antibodies. Western blotting detected double bands at 59 and 54 kDa in size, which agreed with predicted sizes of FLG monomers digested the linker regions. Immunohistochemistry using anti-dog FLG antibodies stained the stratum granulosum of the epidermis in the footpad, dorsal neck, and axilla with a granular, cytoplasmic staining pattern. Taken together, these results indicate the unique gene structure of canine proFLG, which includes four units of FLG monomers whose amino acid sequences are virtually not conserved among mammals except for the linker region. We also demonstrated that the anti-dog FLG antibodies made in this study correctly detect the expression of FLG in canine skin and will be useful for screening FLG-deficient dogs with canine atopic dermatitis or ichthyosis. Funding: The Ministry of Education, Culture, Sports, Science and Technology of Japan; and the Ministry of Health, Labour, and Welfare of Japan. Conflict of interest: None declared.

Supporting Original Study 3 The effect of a spot on formulation containing fatty acids and essential oils (Essential 6, Dermoscent, LDCA, France) on dogs with canine atopic dermatitis M. BLASKOVIC*, W. ROSENKRANTZ , A. NEUBERà and R. S. MUELLER* *Clinic of Small Animal Medicine, Centre for Clinical Veterinary Medicine, Ludwig-Maximilian University, Munich, Germany; Animal Dermatology Clinic, Los Angeles, CA, USA; àDerm4Pets Clinic, Buckinghamshire, UK Canine atopic dermatitis is a common disease. In dogs and humans with atopic dermatitis there is evidence for decreased skin barrier function. One treatment methodology to improve the skin barrier function is the use of essential fatty acids. Although studies on the clinical effects of oral fatty acids have been published, rando 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

mised controlled trials evaluating topical fatty acids are rare. This study aimed to evaluate the effects of a spot-on formulation containing essential fatty acids (Essential 6, Dermoscent, LDCA, France) on the clinical signs of canine atopic dermatitis. The study was multicentered, randomised, double-blinded and placebo-controlled. Forty-eight dogs with atopic dermatitis were treated with a spot-on product containing fatty acids or a spoton placebo, applied once weekly directly on the dorsal neck skin surface for 8 weeks. Atopic dermatitis was diagnosed by history and clinical signs and by ruling out differential diagnoses as appropriate. Other therapies such as low-dose glucocorticoids, antihistamines, and/or topical therapy were permitted if they had been administered for more than 12 weeks prior to inclusion and their dose was not altered before and during the trial. Diet changes were not permitted within the 3 months prior to or during the study. Patients with clinical signs or a history of flea-bite hypersensitivity received fipronil spot-on (Merial, Hallbergmoos, Germany) once monthly. Allergen-specific immunotherapy was permitted as long as it began more than 12 months before the study. Before and after the study, Canine Atopic Dermatitis Extent and Severity Index (CADESI) )03 and pruritus scores were determined by veterinarians and owners, respectively. A Wilcoxon matched-pairs signedranks test was used to compare the CADESI and pruritus scores before and after treatment. There was a significant difference between pre- and post-treatment CADESI with a mean of 46 vs. 28 (an improvement of 39%) in the treatment group (P = 0.007), but not the placebo group (P = 0.4928). There was a significant improvement of pruritus scores in the treatment group with a mean of 5.2 vs. 3.9 (an improvement of 25%) (P = 0.0092), but not in the placebo group (P = 0.6807). Subgroups of dogs with severe atopic dermatitis (CADESI > 60 prior to treatment) and mild to moderate atopic dermatitis (CADESI < 60) were evaluated similarly. In the dogs with severe atopic dermatitis there were also improvements in CADESI and pruritus scores in the treatment group (P = 0.0322 and P = 0.0436, respectively), but not with placebo (P = 0.7367 and P = 0.344, respectively). In the mildly symptomatic dogs, CADESI scores decreased in the treatment group (P = 0.042) and increased in the placebo group (P = 0.0078). The pruritus decreased – to a lesser degree – in the treatment group (P = 0.0728), but not in the placebo group (P = 0.7344). Adverse effects were not observed during treatment in any of the dogs. This study indicates that this topical fatty acid preparation is beneficial in alleviating the clinical sings of both mild and severely affected cases of canine atopic dermatitis. Funding: The study was financed by Laboratoire de Dermo-Cosmetique Animale (LDCA) France. Conflict of interest: The study was financed by LDCA France.

Plenary Session Abstracts: Thursday Afternoon, 26th July Theme: IMMUNOLOGY State-of-the-Art Address Antimicrobial peptides and the skin R. GALLO University of California, San Diego, San Diego, CA, USA The innate capacity to produce antimicrobial activity is essential to the immune defense of most living organisms. Various types of molecules with antibiotic activity have been isolated from animals, insects, plants and bacteria, and their use has revolutionized medicine. To this date, more than 1200 types of peptides with antimicrobial activity have been isolated from various cells and tissues, and it appears all living organisms employ these antimicrobial peptides (AMPs) in their host defense. In the last decade, innate AMPs produced by mammals have been shown to be essential for the protection of skin and other organs. Their importance is due to their pleiotropic functions to not only kill microbes but also control host physiological functions such as inflammation, angiogenesis and wound healing. Recent advances in our understanding of the function of AMPs have associated their altered production with various diseases such as psoriasis and atopic dermatitis. In this presentation, I will summarize the history of AMP biology and provide an overview of recent research progress in this field. Funding: National Institutes of Health Veterans Administration. Conflict of interest: None declared.

Supporting Review The impact of immunology research on modern veterinary dermatology V. A. FADOK Gulf Coast Veterinary Dermatology, Houston, TX, USA The immune system is an incredibly complex and wondrous defence mechanism for the body. As we have learned, the immune system alerts the body when it faces something harmful, whether that comes from without or within. The mammalian immune system can be divided roughly into innate and adaptive branches, but these branches are closely intertwined and interdependent. Innate immunity is rapid but nonspecific and lacks memory. It relies on pattern recognition. Adaptive immunity, by contrast, is exquisitely specific and its antigen receptors capable of fine discrimination. However, it is dependent upon signals from the innate immune system to learn to recognise its stimuli, and it reciprocates by enhancing innate immune function. In this presentation, we will focus on three aspects of immunity in which molecular, cellular, and animal research have directly affected our knowledge of veterinary dermatologic diseases. We will start with the dendritic cell, review its biology, and discuss how knowing the dendritic cell has led to insights into histiocytic disorders of the dog and cat. We will then review how our knowledge of T cell subsets has expanded from Th1 and Th2 to a large number of functional subsets that imply more plasticity in the lymphocyte component than we previously thought. In particular,

our understanding about Th2 and regulatory T cells has led to our first inklings about the pathogenesis of atopic dermatitis in dogs. Last, we will discuss the impact of cytokines on veterinary dermatology, emphasising the complexity of these soluble and membrane-bound messengers and how the confirmation of their role in veterinary skin disease must move from expression of RNA to expression of bioactive proteins before conclusions can be drawn. Funding: National Institutes of Health, United States. Conflict of interest: None declared.

Supporting Original Study 4 Evaluation of canine antimicrobial peptides in infected and non-infected chronic atopic skin D. SANTORO, D. BUNICK, T. GRAVES, K. CAMPBELL and M. SEGRE University of Illinois at Urbana-Champaign, Urbana, IL, USA Antimicrobial peptides (AMPs) are small peptides produced by epithelial and immune cells. Their main functions are the defense against pathogenic organisms and the orchestration of innate and adaptive immunity. Among the AMPs, the most studied are beta defensins (BDs) and cathelicidin (Cath). In the past decade, interest in AMPs has increased greatly because many investigators have hypothesized a possible role of AMPs in the high development of pyoderma in atopic dermatitis (AD). Few studies have investigated the expression of AMPs in healthy and atopic canine skin. To date, five BDs (cBD1, cBD1-like, cBD2-like/122, cBD3-like, and cBD103) and one Cath have been isolated from canine skin. Using a canine model of AD, we have shown that increased mRNA expression of cBD1-like, cBD3-like and cCath is present in non-lesional and lesional skin of experimentally-induced AD in beagles when compared with healthy control beagles. We also analyzed AMP protein distribution and found no differences in localization between healthy and atopic beagles. In contrast, other researchers investigating the mRNA expression of cBD103 reported decreased expression in naturallyaffected atopic dogs when compared with healthy controls. The goal of our study was to analyze the mRNA expression of cBD1-like, cBD2-like/122, cBD3-like, cBD103 and cCath in healthy and naturally-affected atopic dogs with and without active skin infection. We also evaluated the distribution of these AMPs in the epidermis using indirect immunofluorescence (IIF). Skin biopsies were taken from 14 healthy (10 males and four females; age: 4 ± 2.9 years) and 11 atopic dogs (four males and 10 females; 5.6 ± 4.1 years). The mRNA levels of cBD1-like, cBD2-like/122, cBD3-like, cBD103 and cCath mRNA were quantified using quantitativereal-time PCR. The protein expression of cBD3-like and cCath was analyzed by inhibition-competitive ELISA, while the distribution of cBD2-like/122, cBD3-like and cCath was detected by IIF. mRNA expression showed a significant difference only for cBD103 [AD 1.9 times more (P = 0.04) than healthy controls]. A higher 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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cBD103 mRNA expression (3.8 times more) was also seen in atopic skin with active skin infection vs. atopic skin without skin infection (P = 0.01). This difference was 4.4 times higher when compared to AD dogs with active skin infection and healthy controls (P = 0.001). In contrast, cBD1-like mRNA expression level was significantly lower (0.48 times) in AD dogs with active skin infection compared with AD dogs without skin infection (P = 0.04). No significant differences between healthy and atopic groups were seen for cBD3-like or cCath protein expression. cBD2-like/122 and cBD103 protein levels were not quantified due to a lack of antibodies suitable for ELISA-based quantitation. Finally, no discernable changes were observed in the distribution of AMPs in canine skin. These results show that naturallyaffected atopic dogs have a higher expression of certain AMPs than previously reported in humans and experimentally-induced atopic beagles. Additionally, cBD103 mRNA expression was greater, while cBD1-like mRNA was lower in dogs with active infections. Further testing of protein expression will require development of usable antibodies. Funding: The study was funded by the American College of Veterinary Dermatology. Conflict of interest: None declared.

Supporting Original Study 5 IL-31: Its role in canine pruritus and prevalence in naturally occurring canine atopic dermatitis A. GONZALES, W. HUMPHREY, J. TEEL, G. FICI, J. MESSAMORE, C. CHIO, J. SHELLY, M. ALEO, E. COSCARELLI, T. FLECK, B. GALVAN, J. CURRY, D. MANN, D. WHEELER, S. COSGROVE, P. AEED, P. CLARE, G. BAMMERT, S. DUNHAM, S. MAHABIR, G. BAINBRIDGE, T. FULLER, O. MARTINON, K. GREENWOOD and R. MCCALL Veterinary Medicine Research and Development, Pfizer Animal Health, Kalamazoo, MI, USA Cytokines are secreted signalling proteins that play a key role in cell-to-cell communication; however, their dysregulation can contribute to a variety of chronic diseases. Interleukin (IL)-31 is a recently identified member of the gp130/IL-6 cytokine family that is produced by a variety of human cell types including Th2 lymphocytes and CLA+ skin homing T cells. When over-expressed in transgenic mice, IL-31 induces severe pruritus, alopecia, and skin lesions. In humans, IL-31 serum levels correlate with severity of atopic dermatitis in adults and children. As with humans, atopic dermatitis in dogs is believed to be a multifactorial disease determined by a combination of genetic and environmental factors affecting the skin barrier, immune system, and neurological responses. Because of the complexity of this disease, there remains a need for novel therapeutic approaches that provide safer and more effective control of atopic dermatitis throughout the lifetime of the animal. To facilitate improvements in the treatment and management of canine atopic dermatitis, investments in expanding our understanding of the pathobiology of the disease must be made. Therefore, investigational studies were performed to evaluate the role of IL-31 in canine pruritus and its prevalence in dogs diagnosed with atopic dermatitis. Recombinant canine IL-31 was generated by polymerase 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

chain reaction (PCR) amplification of canine IL-31 (cIL31) sequences using cDNA prepared from canine testis tissue. PCR products were then cloned into expression constructs that were transfected into mammalian cells for protein production followed by purification. To evaluate the biological function of purified cIL-31, the canine DH82 histiocytoma line was stimulated with cIL-31 in vitro, and activation of signal transduction pathways were evaluated using western blotting and immunoassay techniques. The role of IL-31 in canine pruritus was evaluated in a laboratory model using purpose-bred beagles. Beagles were administered cIL-31 (10–40 lg) via several routes (intravenous, subcutaneous, intradermal), and pruritic behaviour was observed/quantitated via video monitoring and use of a categorical scoring system. Finally, quantitative immunoassay techniques were employed to measure serum levels of IL-31 in samples obtained from non-diseased client-owned dogs, purposebred beagles, and client-owned dogs diagnosed with naturally occurring atopic dermatitis. Purified cIL-31 protein was able to activate JAK/STAT and MAPK signal transduction pathways in the canine DH82 histiocytoma cell line in a dose-dependent manner. Injection of cIL-31 into beagles caused transient episodes of pruritic behaviour such as licking, scratching, headshaking and body-rubbing, regardless of administered route. When quantitated over a 2 h period, dogs receiving cIL-31 exhibited a significant increase in pruritic behaviour compared to placebo-administered controls. Finally, cIL-31 levels were detectable in over 50% of dogs with naturally occurring atopic dermatitis (‡ 13 pg/ mL) but were below limits of quantitation (< 13 pg/mL) in normal, non-diseased animals. These findings suggest that cIL-31 is functionally active against canine cells and can induce pruritus in dogs. Moreover, cIL-31 was detectable in the majority of dogs with atopic dermatitis, suggesting an important role for this cytokine in pruritic skin conditions such as atopic dermatitis in this species. Funding: Pfizer Animal Health, Kalamazoo, Michigan. Conflict of interest: All authors are employees and shareholders of Pfizer Incorporated.

Supporting Original Study 6 Expression of thymic stromal lymphopoietin in canine atopic dermatitis J. KLUKOWSKA-RO¨TZLER*, L. CHERVET , E. J. MU¨LLER , P. ROOSJEà, E. MARTI§ and J. JANDA§ *Institute for Genetics, Vetsuisse Faculty, University of Bern, Bern, Switzerland; Institute for Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland; à Division of Clinical Dermatology, Vetsuisse Faculty, University of Bern, Bern, Switzerland; §Division of Experimental Clinical Research, Vetsuisse Faculty, University of Bern, Bern, Switzerland Canine atopic dermatitis is a highly pruritic allergic dermatitis caused by reactions to environmental allergens, especially house dust mites. T helper 2 (Th2) differentiation and allergen-specific IgE antibodies have been implicated in the pathogenesis of canine atopic dermatitis, but the mechanisms involved in the induction of Th2 responses are not known. In mice and humans, thymic stromal lymphopoietin (TSLP) has been shown to induce Th2 differentiation and allergic dermatitis. The

Abstracts aim of the present study was to identify the canine TSLP homolog and characterize expression of canine TSLP in the skin of dogs affected by canine atopic dermatitis. To clone the canine TSLP cDNA, sequences homologous with human TSLP were identified in the canine genome using BLASTN. We cloned and sequenced a partial coding sequence from an RNA sample isolated from canine keratinocytes, and we used quantitative real-time polymerase chain reaction (RT-PCR) to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsies taken from lesional and nonlesional skin of 12 atopic-dermatitis-affected dogs and from six healthy control dogs. Our results showed that the nucleotide sequence of canine TSLP is organized in four exons and shows homology with other species (human, 70% identity; equine, 73% identity). Deduced amino acid sequence shows 60.8% and 59.9% identity with human and equine TSLP proteins, respectively. Although we sequenced only the partial coding sequence of canine TSLP, it includes complete sequence coding for the mature cytokine and will allow cloning and production of recombinant TSLP protein needed for further functional studies or for the production of canine-TSLPspecific antibodies. Quantitative RT-PCR showed higher TSLP expression in both lesional and non-lesional skin of

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atopic-dermatitis-affected dogs compared to healthy control dogs (P < 0.05), whereas no significant difference in TSLP expression was found between lesional and nonlesional samples of atopic-dermatitis-affected dogs. Since keratinocytes have been proposed as potential sources of TSLP, we sought to determine whether and under which conditions canine keratinocytes express TSLP. Stimulation of primary canine keratinocytes for 18 h with allergen (Dermatophagoides farinae extract) and with the toll-like receptor (TLR) ligands lipopolysaccharide (LPS; TLR4) and polyI:C (TLR3) resulted in increased expression of TSLP, whereas stimulation with Pam3Cys (TLR2) or imiquimod (TLR7) did not increase TSLP expression. In conclusion, the detection of increased TSLP expression in the skin of dogs with atopic dermatitis suggests that TSLP may play an important role in the pathogenesis of canine atopic dermatitis. Keratinocytes were identified as potential sources of TSLP, as they responded to stimulation with allergen extract with increased expression of TSLP. Further studies should elucidate the function of TSLP in this disease and explore the potential of TSLP as a therapy target. Funding: Self-funded (Vetsuisse Faculty, University of Bern). Conflict of interest: None declared.


Plenary Session Abstracts: Friday Morning, 27th July Theme: SKIN BIOLOGY State-of-the-Art Address Skin wars: Episode I – Rampart of horny layer in mammalian skin K. NISHIFUJI Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan Skin is an anatomical and physiological barrier between environment and organism. The barrier function of the skin consists mainly of the horny layer, which resides in the outermost layer of the epidermis and is composed of ‘bricks’ represented by flattened, protein-enriched corneocytes, and ‘mortar’ represented by intercellular lipidenriched layers. Due to this ‘bricks and mortar’ structure, the horny layer in the mammalian skin can be compared to a ‘rampart’, which encloses ‘citizens’ of water and solutes essential for physiological homeostasis and prevents the ‘castle’ (the host) from physical, chemical and biological ‘assaults’. Filaggrin monomers are generated by proteolytic processing of their precursor protein, profilaggrin, during cornification, and they aggregate keratin intermediate filaments into tight bundles in corneocytes. In addition, the filaggrin monomers crosslink to the cornified cell envelope, in which structural proteins are polymerised by transglutaminases and deposited just beneath the plasma membrane to provide rigidity to the corneocytes. Meanwhile, three major intercellular lipids – ceramides, free fatty acids, and cholesterols – constitute intercellular lipid lamellae, which may prevent transepidermal water loss and invasion of the harmful agents through the horny layer. Ceramides (CERs) are the dominant lipids in the horny layer in humans. In humans and dogs, there are at least 11 classes of free ceramides in the horny layer of the skin. In addition, some classes of ceramides are known to bind covalently to proteins in the cornified cell envelope and interdigitate with the intercellular lipid lamellae. It has been reported that reduced expression of the key components in the horny layer, such as filaggrin, transglutaminase-1, and intercellular lipids, cause a defect of the cutaneous barrier function in some diseases status and mouse models. For example, loss-of-function mutations in the gene encoding profilaggrin are recognised in humans with ichthyosis vulgaris and atopic dermatitis, as well as in flaky-tail mice in which topical application of an allergen provokes systemic immune responses including allergen-specific IgE antibody production. In addition, a mutation in a gene encoding transglutaminase-1 in a mouse model causes early neonatal death due to excessive water loss. Mutations in a gene encoding transglutaminase-1 are also associated with extensive scaling and an increased risk of cutaneous infection in humans with lamellar ichthyosis. Deficiency of essential fatty acids causes disturbed function of the cutaneous permeability barrier in humans, rats, and mice. ATPbinding cassette transporter 12 is crucial for cytoplasmic transportation of lipid precursors to the plasma membrane of keratinocytes, and its gene mutations cause severe dehydration and sepsis in humans with harlequin ichthyosis. Moreover, the quantities of total free extractable CERs, and the CER[EOS], CER[EOP], and CER[NP], which are exclusively expressed in the horny


layer, are decreased in humans and dogs with atopic dermatitis. Taken together, this evidence supports the hypothesis that the structural integrity of the horny layer is crucial in maintaining the optimal function of the permeability barrier and preventing invasion of ‘aggressors’ through the ‘rampart’ of the mammalian skin. Funding: Ministry of Education, Culture, Sports, Science and Technology of Japan; and Kao Corporation. Conflict of interest: None declared.

Supporting Review Fixing the skin barrier – Past, present, and future: Humans and dogs compared R. MARSELLA Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA Skin barrier dysfunction is a key player in the pathogenesis of human atopic dermatitis (AD). The impairment has been described at both lipid (e.g. ceramides deficiencies) and protein levels (e.g. filaggrin). Numerous genetic mutations have been described in the literature, but the most confirmed risk factor in humans seems to be the loss of function mutations in the C-terminal portion of filaggrin. Skin impairment is also secondary and linked to the negative effects of T helper 2 cytokines, which interfere with filaggrin expression, as well as caspase 14, which plays an important role in filaggrin metabolism. Proteases, both endogenous and exogenous, are very important for barrier dysfunction as well as inflammation and pruritus. Great efforts have been devoted to identifying ways to correct the barrier dysfunction to decrease the risk for allergic sensitisation in early cases as well as to decrease pruritus and inflammation even in chronic cases where sensitisation has already occurred. Numerous studies in humans have shown the efficacy of emollients and moisturisers to promote skin barrier amelioration, and daily application of moisturisers has constituted for years the mainstay of therapy for patients with AD. Products used typically contain a combination of ceramides, fatty acids, and other natural ingredients such as glycyrrhetinic acid that can decrease inflammation, and proteases. Evidence is also building regarding the presence of skin barrier impairment in canine AD. Several studies have highlighted decreased ceramides in the upper layers of the epidermis of atopic dogs when compared to normal dogs as well as decreased expression of filaggrin when using an antibody directed at the Cterminal portion of the protein, suggesting that a subset of canine AD patients may have a similar mutation as reported in humans. Despite this preliminary research, mutations in the filaggrin gene have not been identified yet in dogs, and studies have emphasized variability due to geography and breeds. Several products have been made available with the proposed mechanism of improving the skin barrier. Although some positive results have been found for some of these preparations containing fatty acids and essential oils, definitive evidence to support the use of other products is still lacking. An

Abstracts alternative approach to decreasing skin barrier dysfunction is to decrease inflammation, which would reasonably lead to an improvement of the skin barrier. A preliminary study to evaluate the effect of prednisone vs. ciclosporin in dogs with AD failed to report a significant improvement of transepidermal water loss, and, more importantly, thus far several studies have failed to report a significant correlation between transepidermal water loss and severity of clinical signs. Thus, the question of whether the improvement of the skin barrier in dogs directly translates into improvement of clinical signs is still open. It is possible that multiple strategies (e.g. correction of lipid deficiency and decrease of proteases) must be adopted concurrently and that the modality of application should be different (e.g. application after water exposure and under occlusion to achieve maximal penetration) to induce marked clinical improvement. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 7 An ichthyosis mutation in the golden retriever breed revealed a new ichthyosis gene in humans: Implications for practitioners E. GUAGUERE*, A. GRALL , A. THOMASà, E BOURRAT§, I. HAUSSER–, L. LAGOUTTE , F. DEGORCE-RUBIALES**, C. HITTE , E. BENSIGNOR , S. PLANCHAIS , J. FONTAINEàà, M. LE GALLO , D. PIN§§, F. GALIBERT , J. FISCHER–– and C. ANDRE *Clinique Ve´te´rinaire Saint Bernard, Lomme; Centre National de la Recherche Scientifique, University of Rennes, Rennes; àAntagene, Animal Genetics Laboratory, Limonest; §Hoˆpital St Louis, Paris, France; –University Clinic Heidelberg and Electron Microscopy Core Facility, University of Heidelberg, Heidelberg, Germany; **Laboratoire d’Anatomie Pathologique Ve´te´rinaire du Sud-Ouest, Toulouse; Clinique Ve´te´rinaire de la Boulais, Cesson-Se´vigne´, France; ààClinique Ve´te´rinaire, Bruxelles, Belgium; §§VetAgro Sup Ecole Ve´te´rinaire de Lyon, Lyon, France; ––Institute for Human Genetics, Freiburg, Germany Ichthyoses encompass a heterogeneous group of genodermatoses characterized by abnormal desquamation over the entire body due to defects in the terminal differentiation of keratinocytes and desquamation in the upper layer of the epidermis. Even though in humans more than 30 genes have been identified, including seven genes for autosomal recessive congenital ichthyoses, the genetic causes of several forms remain unknown. Strikingly, several purebred dogs are also affected by specific forms of ichthyoses, and several genes have been identified. The Norfolk terrier presents a mild recessive epidermolytic hyperkeratosis, due to a splice-site mutation in the keratin 10 gene; the Jack Russell terrier presents a severe non epidermolytic ichthyosis, due to a LINE 1 insertion in the transglutaminase 1 (TGM1) gene; the American bulldog presents an ichthyosis form, due to an Ichthyin mutation. Several other dog breeds are affected by particular forms of ichthyosis with unknown genetic causes and these may provide opportunities to identify novel genes. In the golden retriever an autosomal recessive form of ichthyosis, resembling human autosomal recessive congenital ichthyoses, has recently been

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diagnosed with a high incidence. We first characterized the disease occurring in the golden retriever breed and collected cases and controls. While collection and clinical examinations are still going on a recently published work reported the identification of a new ichthyosis gene, PNPLA1. A genome-wide association study on 40 unrelated golden retriever dogs, using the canine 49.000 SNPs (single nucleotide polymorphisms) array (Affymetrix v2), followed by statistical analyses and candidate gene sequencing, enabled the identification of the causal mutation in the PNPLA1 gene (patatin-like phospholipase domain-containing protein). Although this gene had been annotated in the canine and human genomes its function was unknown. Screening for alterations in the human ortholog gene in 10 autosomal recessive congenital ichthyoses families, for which no genetic cause had been identified thus far, allowed the identification of two recessive mutations in the PNPLA1 protein in two families. This work enabled the identification, in humans, of an eighth gene for autosomal recessive congenital ichthyoses, and revealed the function of this as-yetunknown skin specific lipase in the lipid metabolism of the skin barrier. For veterinary medicine and breeding practices, a genetic test has been developed. Its use on geographically distinct golden retriever populations allowed us to estimate the mutation frequencies in different countries and the correlation between the mutated state of PNPLA1 and the development of the disease in the golden retriever breed. These findings illustrate the importance of the discovery of relevant homologous human and canine genetic diseases, whose causes can be tracked in dog breeds more easily than in humans. Indeed, due to the selection and breeding practices applied to purebred dogs, the dog constitutes an unique species for unravelling phenotype/genotype relationships and providing new insights into human genetic diseases. This work paves the way for the identification of rare gene variants in humans that may be responsible for other keratinisation and epidermal barrier defects. Funding: The study was mainly supported by the Centre National de la Recherche Scientifique, University of Rennes 1, and by the European Commission LUPA project on canine genetics (FP7-LUPA, GA-201370). Conflict of interest: An international patent has been deposited on behalf of the Centre National de la Recherche Scientifique and Rennes 1 University.

Supporting Original Study 8 Canine hair follicle keratinocytes enriched with bulge cells constitute epidermal structure in vitro in a 3D skin equivalent model: The implications for regenerative therapy for canine epidermis T. KOBAYASHI*, , K. ENOMOTO , Y. H. WANG , J. S. YOON , R. OKAMURA , K. IDE , M. OHYAMA*, T. NISHIYAMA , T. IWASAKI and K. NISHIFUJI *Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan In human medicine, living human skin equivalents, which are developed by tissue engineering technologies, are 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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Abstracts

readily available for repairing large skin defects caused by burns, chronic wounds, or surgical scars. However, such technologies are not yet available in veterinary medicine. In human and mouse skin, an epithelial stem cell population is located in the bulge area of hair follicles. Recently, we reported that canine hair follicle keratinocytes enriched with bulge cells up-regulated some key bulge-stem-cell markers, and yielded more highly proliferative colonies than other keratinocyte subsets, including epidermal keratinocytes. The aim of this study was to determine whether the canine hair follicle keratinocytes enriched with bulge cells can construct a canine living skin equivalent that represents the phenotypes of interfollicular epidermis in vitro. The middle portion of canine hair follicles, in which bulge cell markers such as keratin 15 (K15), CD34, and follistatin are expressed, were microdissected from healthy dog skin. The bulgecell-enriched keratinocytes were isolated from the microdissected hair follicle and cultured in vitro on a bovine type-I collagen scaffold that contained primary canine fibroblasts. After 10–14 days of culture exposed to the air, seeded keratinocytes formed thin cell sheets covering the collagen gel matrix. The cell sheets were subjected to histological and immunohistochemical analyses. Histologically, the cell sheets represented an epidermal structure consisting of 4–5 living cell layers (mean epidermal thickness: 56.9 ± 13.9 lm) covered with an orthokeratotic horny layer. Basophilic cytoplasmic granules resembling keratohyalin granules were recognized in the uppermost layer of living cells, just beneath the horny layer. Immunohistochemical analysis demonstrated that the cells in the basal and suprabasal layers were positive for K14 and K10, respectively. Cytoplasmic staining of filaggrin and loricrin occurred in the uppermost living cell layer. Conversely, immunostaining for K15, CD34, or follistatin were not recognized at all in the tissue sections, indicating that the canine hair follicle bulge cells underwent normal differentiation and cornification processes of the epidermis and constructed a canine skin equivalent in vitro. Next, we assessed whether the keratinocytes in the canine skin equivalent expressed cell adhesion molecules, as seen in authentic canine epidermis. Two major desmosomal cell–cell adhesion molecules, desmoglein (Dsg) 1 and Dsg3, were expressed in the suprabasal and basal layers of the skin equivalent, respectively. Claudins, which are the major transmembrane constituents of tight junctions, were expressed on the cell surface of keratinocytes in the entire epidermis of the skin equivalent. Hemidesmosome-composing proteins such as a6 integrin and collagen XVII, as well as laminins that are an integral part of the structural scaffolding of the epidermis, were detected in the basement membrane zone of the skin equivalent. Taken together, these findings indicate that the canine hair follicle keratinocytes enriched with bulge cells constitute a three-dimensional skin equivalent model that mimics the phenotype of interfollicular epidermis and expresses some key proteins critical for maintenance of the epidermal barrier function. Our findings also indicate that bulge-stem-cell-enriched populations in canine hair follicle keratinocytes represent promising material for regenerative therapy of canine epidermis. Funding: Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest: None declared.


Supporting Original Study 9 Profiling of the skin surface lipids in shih tzu dogs and their alteration in seborrhoea J. S. YOON, S. ISHIOROSHI, K. IDE, K. NISHIFUJI and T. IWASAKI Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan Seborrhoea is a clinical condition characterized by greasy and scaly skin caused by excessive lipid production and abnormal keratinization. However, understanding of the skin surface lipid profiles is extremely limited. Shih tzu dogs commonly have greasy skin and occasionally exhibit seborrhoea. The purposes of this study were to profile the skin surface lipids in shih tzu dogs and to determine whether the lipid composition was altered in shih tzu dogs with seborrhoea. High-performance thin-layer chromatography (HPTLC) was performed to profile lipids in the sebaceous-gland-enriched fraction and the stratum-corneum-enriched fraction from shih tzu dogs. Lipids in the sebaceous-gland-enriched fraction were collected from a lesion of sebaceous gland hyperplasia, and lipids in the stratum-corneum-enriched fraction were collected by tape stripping following acetone treatment to remove the sebum covering the surface of the stratum corneum. We found that lipids in the sebaceous-glandenriched fraction consisted mainly of cholesterol esters (18.3%), wax esters (19.2%), and triglycerides (45.2%), as well as small proportions of cholesterol (8.5%) and free fatty acids (5.3%). In contrast, lipids in the stratum-corneum-enriched fraction consisted mainly of ceramides (CERs; 65.6%) and small proportions of cholesterol (7.7%) and free fatty acids (11.6%). We next analyzed the lipid profiles in an acetone-extracted fraction containing the sebaceous gland exudates and the stratum-corneum-enriched fraction, and compared this lipid profiles in shih tzu dogs with seborrhoea (n = 7) with that of age-matched control shih tzu dogs (n = 7). All seborrhoeic dogs had greasy and scaly skin, whereas all control dogs did not appear to exhibit these conditions upon clinical examination. In the acetone-extracted fraction, relative proportions of wax esters (19.3 ± 4.5%) and triglycerides (14.2 ± 2.6%) in seborrhoeic dogs were significantly higher than those in controls (wax esters, 14.3 ± 2.8% and triglycerides, 10.9 ± 1.8%; unpaired Student’s t-test, P < 0.05). Conversely, the proportions of free fatty acids (8.5 ± 1.7%) and total ceramides (29.3 ± 4.7%) in seborrhoeic dogs were significantly lower than those in control dogs (free fatty acids, 13.1 ± 2.7%; total ceramides, 36.4 ± 4.5%; P < 0.05). Further, we investigated whether the profiles of ceramide classes in the stratum-corneum-enriched fraction were altered in seborrhoeic shih tzu dogs. The quantities of CER[EOS] (1.1 ± 0.3 lg), CER[EOP] (0.7 ± 0.3 lg), CER[EOH] (1.1 ± 0.3 lg), and the mixture of CER[AS] and CER[NH] (1.7 ± 0.3 lg) per 1 mg of the stratum corneum in seborrhoeic dogs were significantly lower than those in controls (CER[EOS]; 1.6 ± 0.4 lg, CER[EOP];1.1 ± 0.3 lg, CER[EOH]; 1.7 ± 0.3 lg, CER[AS] and CER[NH]; 3.2 ± 0.8 lg; unpaired Student’s t-test, P < 0.05). Interestingly, two unknown ceramide fractions, which accounted for 20% of the total ceramides, were recognized exclusively in seborrhoeic dogs. Taken together, these findings suggest

Abstracts that increased levels of wax esters and triglycerides (two major components of sebaceous gland lipids) and altered ceramide profiles are present in the skin of shih tzu dogs with seborrhoea, indicating that these changes may be

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associated with excessive lipid production and abnormal keratinization in dogs with seborrhoea. Funding: Self-funded. Conflict of interest: None declared.


Plenary Session Abstracts: Friday Afternoon, 27th July Theme: THERAPY State-of-the-Art Address Stem cell therapy in veterinary dermatology R. HARMAN Vet-Stem, Inc., Poway, CA, USA Adult stem cells come from many sources and have the capacity to differentiate into many cell types, including those of the skin. The most commonly studied and clinically relevant stem cells are those commonly termed mesenchymal stem cells (MSC), which are easily isolated from the bone marrow and adipose tissue. Perhaps more relevant than differentiation, MSCs are now known to produce a wide array of growth factors and cytokines that modulate and stimulate the tissue repair and regeneration process. The ease of collection, propagation and use of these MSCs has spawned a global research effort to apply them in therapy of traumatic, ischemic and immunemediated skin conditions. In both traumatic and ischemic skin damage, MSCs have been applied in creation of exvivo tissue-engineered skin and in direct injection into the damaged tissue. For immune-mediated diseases such as atopic dermatitis and pemphigus, the focus has been on systemic administration of stem cells to modulate the immune system, particularly the T cell populations. The earliest clinical work in regenerative medicine has been with autologous stem cell sources such as adipose and bone marrow. Autologous stem cells can be used with minimal risk to the patient and with minimal regulatory intervention. More recently, researchers and commercial companies have focused on allogeneic or donor stem cell products that can be produced in a manner to allow for immediate ‘off-the-shelf’ access. These products require full FDA approval in the United States. The primary approach for immune-mediated diseases is to use the MSC to down-regulate production of inflammatory cytokines and to cause the reactive T cells to become apoptotic and to block T cell activation. Cells are generally given intravenously and often in multiple doses. Multiple sclerosis, rheumatoid arthritis, lupus and other relevant diseases have been successfully treated in human clinical trials. MSCs have been shown to produce immunomodulatory cytokines such as TGF-b, HGF, and PGE2 in response to inflammation and injury. Finally, many in-vivo studies have shown that therapy with MSCs can stimulate resident local cells such as keratinocytes and precursors to proliferate, migrate, and repair skin injury and disease. Stem cells can be applied by direct injection or in conjunction with biomaterials to increase residence time at the local site or to induce lineage directed differentiation. The discovery of the MSC in adipose tissue was made at the University of Pittsburgh by the plastic surgery group and this has spawned a global effort to utilize these cells in therapy of a wide range of diseases of the skin. Reconstructive surgery, scar blocking and resolution, and skin regeneration all have been shown possible in human and animal studies. In the next decade we will see further translation into clinical protocols and approval of multiple veterinary cell therapy products for treating dermatological conditions. Regenerative medicine is now beginning to deliver on the promise of safe and effective treatments for these difficult and debilitating diseases. Funding: Self funded. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

Conflict of interest: Author is employee and shareholder of Vet-Stem, Inc.

Supporting Review A systematic review of randomized controlled trials for prevention or treatment of atopic dermatitis in dogs: 2008–2011 update T. OLIVRY and P. BIZIKOVA Department of Clinical Sciences and Center for Comparative Medicine and Translational Research,College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA The management of canine atopic dermatitis (AD) is multifaceted and relies, principally, on the use of immunomodulating interventions to reduce pruritus and skin lesions. A systematic review that analyzed results of randomized controlled trials (RCTs) published until 2007 concluded that oral and topical glucocorticoids, topical and oral calcineurin inhibitors and injectable interferons and allergen-specific immunotherapy appeared effective for treatment of AD in dogs. The objective of this updated review is to critically analyze recent clinical trials reporting the efficacy and safety of interventions for canine AD. We performed a systematic review of RCTs published, presented or completed between 2008 and 2011 and that had all enrolled dogs with AD. The study search was done using electronic databases, reviewing published meeting abstracts and sending queries to professional email lists. Trials reporting the efficacy of interventions aimed at treating, preventing or reducing glucocorticoid usage in atopic dogs were selected. In all, 21 RCTs were included. In this review, we found additional moderate quality evidence of efficacy and safety of oral glucocorticoids and ciclosporin for treatment of canine AD. There was additional moderate quality evidence of the efficacy of a novel topical glucocorticoid spray containing hydrocortisone aceponate. Furthermore, low quality evidence was found for the efficacy and safety of injectable recombinant interferons, a new budesonide leave-on conditioner, a novel ciclosporin topical nano-emulsion as well as oral fexofenadine. There is low quality evidence of efficacy of oral masitinib with a need of monitoring for protein-losing nephropathy. We also uncovered low quality evidence of efficacy of a novel commercial diet as a glucocorticoidsparing intervention. Furthermore, there was low quality evidence of efficacy of the novel glucocorticoid spray as a flare retarding measure. Finally, very low quality evidence was found for the efficacy, or lack thereof, of several other interventions. In conclusion, results of this updated systematic review on interventions for canine AD suggest that topical or oral glucocorticoids and oral ciclosporin remain the interventions with highest evidence for efficacy and relative safety to treat dogs with AD. Funding: Self-funded. Conflict of interest: From 2008 to 2011, Thierry Olivry has participated in NC State University-approved consulting activities (C) for, has received lecturing honorar-

Abstracts ium (LH) or obtained research funding (RF) from the following companies whose products are discussed herein: AB-Science (France; C), Novartis Animal Health (Switzerland; C, LH, RF) and Virbac (France; C, RF). Petra Bizikova has received research funding from Virbac (France) via NC State University and she does not report other relevant conflict of interest.

Supporting Original Study 10 The effect of ketoconazole on whole blood and skin ciclosporin concentrations in canines L. GRAY, A. HILLIER, L. COLE and P. RAJALA-SCHULTZ The Ohio State University, Columbus, OH, USA Ciclosporin (Atopica; Novartis Animal Health) is approved for treatment of canine atopic dermatitis. Ciclosporin is metabolized in the liver by cytochrome P450 enzymes, a process inhibited by ketoconazole. Thus, concurrent administration of ciclosporin and ketoconazole potentially reduces the dose of ciclosporin required to maintain therapeutic blood and tissue concentrations in canine atopic dermatitis. This study aimed to determine skin and blood ciclosporin concentrations when it was administered alone at recommended and subtherapeutic doses, and when administered at subtherapeutic doses concurrently with ketoconazole. We hypothesized that ciclosporin skin and blood concentrations at ciclosporin’s recommended dose alone (5 mg/kg/ day) would not differ significantly from subtherapeutic ciclosporin dosing (2.5 mg/kg/day) concurrently with ketoconazole (2.5 mg/kg/day or 5.0 mg/kg/day). In a randomized cross-over study design, six healthy research hounds received each of the following four treatments once daily for 7 days followed by a 14-day washout: ciclosporin 5.0 mg/kg/day [Treatment 1 (T1)]; ciclosporin 2.5 mg/kg/day (T2); ciclosporin 2.5 mg/kg/day + ketoconazole 5.0 mg/kg/day (T3); and ciclosporin 2.5 mg/ kg/day + ketoconazole 2.5 mg/kg/day (T4). After the first, fourth, and seventh dose of ciclosporin or ciclosporin/ketoconazole for each treatment, a skin sample (8mm-punch biopsy) was collected at 4 h (peak) and 24 h (trough) after dosing to determine skin concentrations of ciclosporin, and a blood sample was collected at 1.4 h (peak) and 24 h (trough) after dosing to determine whole blood concentrations. Ciclosporin concentrations were quantified by high-performance liquid chromatography tandem mass spectrometry. Data were analyzed using repeated measures approach with PROC MIXED in SAS (SAS Institute Inc.). Pairwise comparisons between days and treatments were performed by obtaining least squares means and Tukey–Kramer adjustment for multiple comparisons. Correlation between skin and blood ciclosporin concentrations was assessed using Spearman correlation coefficients. Results showed that mean blood ciclosporin concentration in T1 (307.5 ng/mL) was not significantly different from T2 (169.41 ng/mL, P = 0.287) or T4 (417.74 ng/mL, P = 0.136), but was significantly less than T3 (644.83 ng/mL, P = 0.0002). Mean skin ciclosporin concentration in T1 (0.6 ng/mg) was significantly greater than T2 (0.262 ng/mg, P = 0.05), not significantly different from that in T4 (0.697 ng/mg, P = 0.895), and significantly less than in T3 (1.236 ng/mg, P = 0.0006). Correlation between blood and skin ciclosporin concentrations was moderate

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across treatment groups (r = 0.6679). There was no significant difference in the mean peak blood ciclosporin concentration after the first dose compared to the seventh dose in any treatment group. There was a significant increase in mean peak and trough concentrations of ciclosporin in skin from the first dose to the seventh dose in all treatment groups [e.g. T1 mean peak after seventh dose (1.06 ng/mg) was significantly greater than mean peak after first dose (0.272 ng/mg) (P = 0.0001)]. In conclusion, as there was no difference in mean blood or skin ciclosporin concentrations when ciclosporin was dosed at 5.0 mg/kg once daily (T1) compared to ciclosporin at 2.5 mg/kg once daily with 2.5 mg/kg of ketoconazole once daily (T4), it is anticipated that similar clinical outcomes would occur with either dosing regime. Ciclosporin accumulates in the skin over the course of seven doses. Funding: Canine Research Funds; College of Veterinary Medicine, The Ohio State University. Conflict of interest: A. Hillier is a consultant and speaker for Novartis.

Supporting Original Study 11 Antiseptic susceptibilities for Staphylococcus pseudintermedius isolated from canine superficial pyoderma N. MURAYAMA*, M. NAGATA*, Y. TERADA*, M. OKUAKI*, N. TAKEMURA , H. NAKAMINAMI and N. NOGUCHI *ASC Dermatology Service, Tokyo, Japan; Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan Canine superficial pyoderma is generally treated with systemic antimicrobials, but these are not sufficient in dogs infected with meticillin-resistant Staphylococcus pseudintermedius. Our previous studies demonstrated that high-concentration chlorhexidine was effective in 60–70% of the dogs, but not in all. In humans, multidrug efflux pump genes detected in S. aureus isolates confer resistance to antiseptic agents including chlorhexidine, although there are no studies on antiseptic susceptibilities for S. pseudintermedius regarding multidrug efflux pump genes. The aim of this study was to access antiseptic susceptibilities of both meticillin-resistant and meticillinsusceptible S. pseudintermedius isolated from canine superficial pyoderma in terms of multidrug efflux pump genes. One hundred dogs including 23 initial and 77 recurrent cases of superficial pyoderma were evaluated at the ASC Dermatology Service, Tokyo, Japan, from May 2009 to September 2010. Bacterial isolates were taken from skin lesions compatible with superficial pyoderma. S. pseudintermedius and the mecA gene were identified using polymerase chain reaction (PCR) analysis. Minimum inhibitory concentrations (MICs) of 19 antimicrobials including amoxicillin, amoxicillin/clavulanic acid, oxacillin, cefalexin, imipenem, fosfomycin, norfloxacin, enrofloxacin, levofloxacin, erythromycin, lincomycin, gentamicin, arbekacin, minocycline, vancomycin, linezolid, chloramphenicol, sulfamethoxazole-trimethoprim, and fusidic acid were determined for all isolates by a standard agar plate dilution method. Minimum inhibitory concentrations of five antiseptic agents including chlorhexidine acetate, chlorhexidine gluconate, acriflavine, ethidium bromide, and benzalkonium chloride were determined for all isolates by a standard agar plate 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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Abstracts

dilution method. Multidrug efflux pump genes including qacA, qacB, and smr were identified by PCR. Of all cases, 51% (51/100) had mecA-positive S. pseudintermedius, of which 48% (11/23) were from initial and 52% (40/77) from recurrent cases. mecA-negative S. pseudintermedius isolates were susceptible to amoxicillin (85.7% of cases), amoxicillin/clavulanic acid (100%), oxacillin (87.8%), cefalexin (87.8%), imipenem (100%), fosfomycin (100%), norfloxacin (73.5%), enrofloxacin (81.6%), levofloxacin (75.5%), erythromycin (75.5%), gentamicin (91.8%), arbekacin (100%), minocycline (98.0%), vancomycin (100%), linezolid (100%), chloramphenicol (91.8%), and sulfamethoxazole-trimethoprim (46.9%). In contrast, mecA-positive S. pseudintermedius isolates were susceptible to fosfomycin (92.2% of cases), norfloxacin (9.8%), enrofloxacin (9.8%), levofloxacin (9.8%), erythromycin (11.8%), gentamicin (43.1%), arbekacin (100%), minocycline (96.1%), vancomycin (100%), linezolid (100%), chloramphenicol (52.9%), and sulfamethoxazole-trimethoprim (5.9%). The MIC90 of chlorhexidine acetate, chlorhexidine gluconate, acriflavine, ethidium bromide, and benzalkonium chloride were 1, 1, 2, 0.5, and 2 lg/dL, respectively. The MICs for all isolates were effective concentrations in contrast with previous studies for S. aureus, and there were no significant differences in MICs between mecApositive and -negative S. pseudintermedius isolates. Multidrug efflux pump genes including qacA, qacB, and smr were analyzed, but not detected in all isolates. Among the dogs, 21 cases including eight with meticillin-resistant and 13 meticillin-susceptible S. pseudintermedius isolates were treated with only 2% chlorhexidine, and its effectiveness did not vary among the dogs. This in vitro study indicated that chlorhexidine was fully effective for meticillin-susceptible as well as meticillin-resistant S. pseudintermedius. Future investigations of the ineffectiveness of chlorhexidine in dogs with superficial pyoderma, particularly cutaneous milieu analysis, are warranted. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 12 New approach to treat pythiosis: In vitro and in vivo assays with photodynamic therapy L. PIRES*, S. M. BOSCO , N. F. SILVA JUNIORà and C. KURACHI* *Physics Institute of Sao Carlos, University of Sao Paulo, Sao Carlos, Sao Paulo, Brazil; Instituto de Biocieˆncias de Botucatu, Universidade Estadual Paulista ‘‘Ju´lio de Mesquita Filho’’, Botucatu, Sao Paulo, Brazil; àEscola de Engenharia de Sao Carlos, University of Sao Paulo, Sao Carlos, Sao Paulo, Brazil Pythiosis is a life-threatening disease caused by the fungus-like organism Pythium insidiosum. Since this pathogen is not a true fungus, anti-fungal drugs do not affect it because of the lack of ergosterol, the main target of these drugs. The conventional treatment for pythiosis


is surgery; however, depending on the lesion extension and site, this approach can be unfeasible. Even when the surgery can be performed, recurrence is frequent. Photodynamic therapy is a technique that uses the interaction of a photosensitizer, light, and molecular oxygen to cause cell death. In this study, we evaluated the effect of photodynamic therapy on in vitro growth of the pathogen and in an in vivo model of pythiosis. For in vitro studies, we used two photosensitizers, a haematoderivative porphyrin (Photogem, Moscow, Russia) and a chlorin (Photodithazine, Moscow, Russia), and three different light doses for two P. insidiosum isolates, a human and an equine isolate. Amphotericin B (Eurofarma, Sao Paulo, Brazil) was also evaluated. Cultures were imaged at 24, 48, and 168 h after treatment, and the growth area was measured using ImageJ software (National Institutes of Health, USA). The growth area was compared to the control group, and the inhibition rate was then calculated. The cellular morphological aspects were evaluated using scanning electron microscopy. Ten rabbits were inoculated with 20 000 zoospores of the equine isolate of P. insidiosum, and the lesions were treated with two different protocols. The photosensitizers were administrated intravenously, at concentrations of 1.0 mg/kg for Photodithazine and 1.5 mg/kg for Photogem. The light-drug interval used was 4 h. The presence of the photosensitizer in the lesions was monitored by fluorescence spectroscopy. Photodynamic therapy irradiation was performed using a laser emitting at 660 nm for Photodithazine and 630 nm for Photogem, and a fluence of 200 J/cm2. The animals were clinically evaluated daily and the lesion size measured. Histopathological analysis was performed using the Grocott technique to identify the pathogen. In vitro assays showed high inhibition rates for photodynamic therapy when compared to Amphotericin B. For Photogem assays, the inhibition rate was over 70%, reaching 100% for 10 mg/ mL and 30 J/cm2. This finding was supported by scanning electron microscopy, which showed changes in hyphae morphology and the leakage of amorphous material. For Photodithazine, the inhibition rate was over 99% for all parameters evaluated. Scanning electron microscopy showed hyphae rupture and loss of its integrity. In in vivo assays, Photodithazine concentrated more in lesions than did Photogem. Therefore, the inhibition obtained using Photodithazine as the photosensitizer was higher than the one obtained for Photogem. After only one session of photodynamic therapy with Photodithazine, experimental lesions of up to 1 cm2 regressed completely. In vitro and in vivo studies showed that photodynamic therapy was effective in the inactivation of P. insidiosum for the protocols used. Photodithazine showed better results than Photogem in both assays, proving to be the best choice for the treatment of pythiosis. Photodynamic therapy appears to be an innovative and promising approach in the treatment of this life-threatening disease. Funding: Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo and the National Counsel of Technological and Scientific Development, Fundaçaõ para o Desenvolvimento da Universidade Estadual Paulista. Conflict of interest: None declared.

Plenary Session Abstracts: Saturday Morning, 28th July Theme: INFECTIOUS DISEASES State-of-the-Art Address The skin microbiome in health and disease J. S. WEESE University of Guelph, Guelph, ON, Canada As an important defensive barrier exposed to the external world with nearly constant microbiological, environmental and physical challenges, the skin plays a critical role in prevention of disease. While typically effective, this barrier can be compromised by combinations of host, pathogen and environmental factors with subsequent development of clinical disease. Subclinical alternation of the skin and its protective abilities also undoubtedly occurs, yet it is a less recognizable (but potentially important) outcome. Various factors help the skin maintain its role as a protective mechanism, and these are often difficult to evaluate. One aspect that is receiving increasing attention is the resident and transient bacterial population (microbiome) that is present on the skin and in the ear. This vast and often under-estimated microbial population has been characterized in humans, revealing incredible complexity, variability and diversity. As methods to assess microbiomes have improved, we now know that the canine skin microbiome can consist of a wide range of diverse microorganisms and a hundred or more different species per skin site, with differences in composition between skin sites and between animals. A role of the skin microbiome as a source of opportunistic pathogens is beyond doubt and has been known for years; however, the role of the skin microbiome in health and disease is still poorly understood. An understanding of the normal skin microbiome is a critical first step but evaluation of factors that modify this bacterial population is critical for proper understanding of disease, and manipulation of the microbiome may represent a new frontier in infectious disease prevention and treatment. The role of the skin microbiome in health and disease also likely involves more than opportunistic bacterial infections. The skin also has abundant exposure to antigens and plays an important role in the host immune response. The skin microbiome and immune system should not be considered as completely separate entities, as they presumably work in concert and are influenced by each other. Additionally, the influence of bacterial microbiomes of other sites (namely the gastrointestinal tract) on skin disease is also attracting attention based on the potential for modulation of systemic or local immune responses by gastrointestinal microorganisms, with subsequent impacts on the development of inflammatory or allergic diseases. The traditional approach to elimination of skin infections was to attempt to eliminate the offending agent. While rather crude, this approach has typically been effective, although the widespread dissemination of organisms such as methicillin-resistant staphylococci is certainly compromising the effectiveness of traditional antimicrobial-based approaches. Increasing treatment failures with opportunistic bacterial infections, difficult to control dermatophyte outbreaks and various other issues indicate a need for a broader understanding and more comprehensive approach to the management of skin disease. While application of specific new measures may still be elusive, developing knowledge of the composition and interaction of skin microbiome and

local and systemic immune responses may be harnessed in the near future to improve prevention and treatment of infections. Funding: Self-funded. Conflict of interest: None declared.

Supporting Review Update on Mycobacteria spp. affecting the skin and subcutis of dogs, cats, and wildlife R. MALIK Centre for Veterinary Education, University of Sydney, Sydney, NSW, Australia Human mycobacterial diseases were originally categorised as (i) tuberculosis, (ii) leprosy, and (iii) other conditions (formerly called atypical mycobacterial diseases). With the advent of molecular biology and advances in the treatment of immune-mediated and neoplastic diseases, the spectrum of ‘other’ diseases has broadened considerably, and the term ‘atypical’ no longer applies. The understanding of tuberculosis has been extended by Gunn-Moore and collaborators, who showed that cats in Europe and the United Kingdom can develop tuberculosis due to either M. bovis or M. microti. Molecular tests can distinguish between these members of the M. tuberculosis complex. These infections in cats result from injuries from sylvatic hosts. Although cutaneous manifestations can be prominent, these infections may also involve the lungs or alimentary tract. Lung disease is interstitial rather than cavitary, which poses minimal risk of disease transmission to owners. Treatment regimens using three different drugs (e.g. clarithromycin, rifampicin and a fluoroquinolone) can cure such patients. Mycobacteria bovis also produces disease in New Zealand cats and dogs, with cats sometimes having cutaneous features. Canine leproid granulomas are increasingly seen, likely due to heightened awareness and greater availability of polymerase chain reaction (PCR) testing at reference laboratories. Despite very different geographies, the disease is remarkably ‘syndromic’ the world over. In all cases subjected to PCR testing, the same 16S rRNA sequence (suggestive of a fastidious slow-growing saprophytic mycobacteria) has been amplified. Although evidence is anecdotal, topical silver sulphadiazine seems to be a reasonably effective therapy for most cases, although systemic therapy and/or surgery are still sometimes required. Feline leprosy is becoming a complex entity, with a number of specific mycobacterial species capable of producing variants of this ‘syndrome’. Mycobacterium lepraemurium (the murine leprosy bacillus), the novel New South Wales/Queensland East Coast species (in Australia and New Zealand), M. visibilis (in the USA), Mycobacterium sp Tarwin (in a localised part of Victoria, Australia) and M. avium can cause localised to generalised pyogranulomatous lesions affecting principally the subcutis. Some species seem to be more associated with tuberculous pathology, while others are more likely to be seen with lepromatous histology (a pattern consistent with immune deficiency). Treatment regimens using ‘triple therapy’, sometimes combined with surgery, usually have favourable outcomes. Mycobacteria 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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Abstracts

ulcerans is closely related to the swimming pool bacillus M. marinum, although the acquisition of a plasmid coding for a toxin causing skin necrosis accounts for the characteristic nature of lesions. In rural Victoria, Australia, M. ulcerans has recently been reported to cause severe ulcerative disease in dogs, one cat, two horses, one alpaca, and a variety of native animals. Animals are a sentinel for human disease. Finally, the licensing of pradofloxacin in Europe has provided a safe, affordable, and palatable drug suitable for treating a variety of mycobacterial infections. It possesses the best intrinsic activity against mycobacteria of all the veterinary fluoroquinolones. This drug should be given with at least one additional agent to avoid resistance. Rifampicin and clofazimine, however, remain the two ‘big guns’ for treating non-cultivatable slow growing mycobacteria. Funding: Bayer, to support studies of the efficacy of pradofloxacin against rapidly growing mycobacteria. Conflict of interest: R Malik is a consultant to Pfizer Animal Health for the Australian Infectious Diseases Advisory Panel’s initiative related to antimicrobial guidelines.

Supporting Original Study 13 Prevalence of and risk factors for infection with meticillin-resistant Staphylococcus spp. in dogs with pyoderma N. G. ECKHOLM, C. A. OUTERBRIDGE, S. D. WHITE and J. E. SYKES University of California, Davis; Davis, CA, USA Recently, skin infections caused by meticillin-resistant Staphylococcus spp. (MRS), particularly meticillin-resistant Staphylococcus pseudintermedius (MRSP) have become more widespread in dogs. Meticillin-resistant staphylococci possess the mecA gene, which encodes penicillin-binding protein 2a and confers resistance to all beta-lactam antimicrobial drugs. Only a few studies of the prevalence and risk factors associated with MRSP infections have been reported. We hypothesized that the prevalence of MRS infections in dogs seen at a tertiary care facility is higher than in dogs seen at a primary care facility. Dogs with clinical and cytologic evidence of superficial pyoderma were examined at a primary and a tertiary care facility (veterinary medical teaching hospital) in northern California to compare the prevalence of MRS infections and identify associated risk factors. A questionnaire was used to obtain information about signalment, history of antimicrobial drug administration, underlying systemic disease, previous hospitalizations or surgeries, bathing, contact with other antimicrobialdrug-treated pets, and contact with humans with a history of antimicrobial drug treatment, immunocompromised humans, school-aged athletes, healthcare workers, or MRSA-infected humans. Aerobic bacterial culture and antibiotic susceptibility was performed, meticillin resistance was confirmed by mecA gene polymerase chain reaction (PCR), and a panbacterial PCR assay was used for quality control. Cycle threshold (Ct) values < 40 were positive. Dogs at the teaching hospital were treated with antimicrobial drugs for 30 days and the underlying cause of pyoderma managed. Follow-up culture and susceptibility was performed monthly for dogs with persistent pyoderma that returned for re-evaluation. Univariate analysis of risk factors for meticillin resistance 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

was performed. Of dogs seen at the teaching hospital, 89 staphylococci were isolated from 80 dogs on visit 1, and of dogs seen at the primary care facility, 33 isolates were obtained from 33 dogs. The prevalence of meticillin resistance was 34/89 (38.2%) and 9/33 (27.3%), respectively (P = 0.29). Thirty-one dogs at the teaching hospital had one or more follow-up cultures performed; antimicrobial drug susceptibility changed unpredictably. Multidrug resistance was identified in 41/53 (77.3%) MRS isolates from all teaching hospital visits and in 5/9 MRS isolates from the primary care facility. The mecA gene PCR was performed on 126 isolates, which included 39 MRS and 87 meticillin-susceptible staphylococci. The panbacterial PCR assay was positive for all isolates (Ct values 14.11–23.86). All except five of the MRS isolates tested positive for the mecA gene. One meticillin-susceptible S. pseudintermedius isolate tested positive (Ct value 20.72). Thus, the sensitivity and specificity of the mecA gene assay relative to phenotypic testing was 85% and 99%, respectively. Treatment with antimicrobial drugs in the past year was a risk factor for MRS infection, regardless of facility (P = 0.001). For dogs at the teaching hospital, hospitalization within the past year was also a risk factor (P = 0.001). MRS infections should be suspected in dogs with pyoderma seen at either primary or tertiary care facilities when a history of antimicrobial drug treatment in the previous year is present. Follow-up cultures are necessary in order to determine susceptibility in dogs with persistent disease after treatment. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 14 Usefulness of cefovecin disk diffusion test in predicting mecA gene carriage in Staphylococcus pseudintermedius and clinical efficacy of cefovecin in dogs with superficial pyoderma K. IYORI*, Y. TOYODA*, K. ISHIHARA , H. FUJITA , K. IDE*, Y. TAMURA , T. IWASAKI* and K. NISHIFUJI* *Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; Department of Veterinary Hygiene & Environment, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan Antibiotic susceptibility testing is widely used in the prediction of the in vivo efficacy of antibiotic therapy in veterinary medicine. Cefovecin (Convenia; Pfizer Inc.) is a third-generation, broad-spectrum cephalosporin antibiotic licensed for treatment of cutaneous infections in dogs. Although the drug has been widely applied to treat pyoderma in dogs, and the in vitro activity against clinical isolates has been analyzed, little is known about the correlation among the in vitro susceptibility of cefovecin, the clinical efficacy in each case, and the presence of the mecA gene in the clinical isolates. The objective of this study was to determine whether an in vitro antimicrobial susceptibility test for cefovecin using a disk diffusion method is useful in predicting mecA gene carriage in Staphylococcus pseudintermedius, as well as to determine the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius. Thirty-six strains of S. pseudintermedius were isolated from cutaneous lesions of 22 dogs with superficial

Abstracts pyoderma. The diagnosis of canine superficial pyoderma was based on compatible clinical signs (papules, pustules, erythema, scales, epidermal collarettes, or combination thereof) along with cytology consistent with staphylococcal infection (infiltration of neutrophils with presence of extra- and intracellular cocci). S. pseudintermedius was identified using multiplex polymerase chain reaction (PCR) to distinguish the thermonuclease gene of the species from that of other staphylococcal species. In vitro antimicrobial susceptibility tests for cefovecin were conducted using the disk diffusion and agar dilution methods recommended by the Clinical and Laboratory Standards Institute. PCR analysis to detect mecA was also performed on all S. pseudintermedius isolates tested. We found a significant linear correlation (r = -0.83; Pearson’s correlation coefficient, two-tailed) between zone diameter of obvious growth inhibition by disk diffusion and minimum inhibitory concentrations for cefovecin. All of the disk diffusion test results were subjected to receiver operating characteristic analysis to estimate a better breakpoint for detection of mecA gene carriage. A zone diameter of 25 mm resulted in an 84.2% sensitivity rate and a 100% specificity rate in detecting mecA gene carriage, whereas a zone diameter of 36 mm resulted in a 100% sensitivity rate and a 43.8% specificity rate. A zone diameter of 26–28 mm resulted in a 94.7% sensitivity rate and a 93.8% specificity rate. Therefore, S. pseudintermedius isolates with a zone diameter of 28 mm or less were regarded as resistant to cefovecin in the present study. We further compared cefovecin disk diffusion results with clinical efficacy of cefovecin in dogs with superficial pyoderma. At 14 days after a single injection of cefovecin (8 mg/kg) subcutaneously, the mean improvement rate of the clinical scores in dogs carrying cefovecin-resistant strains was 40.1%, which was significantly lower than that in dogs carrying cefovecin-susceptible strains (82.7%; Student’s t-test, P < 0.01). These findings indicate that the disk diffusion test for cefovecin will be valuable in predicting mecA gene carriage in S. pseudintermedius, as well as in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius. Funding: Pfizer Japan Inc. Conflict of interest: None declared.

Supporting Original Study 15 Investigations on the dog-demodex relationship: All dogs harbour Demodex mites in the skin, but only dogs with demodicosis have antibodies against Demodex I. RAVERA, L. ALTET, O. FRANCINO, A. SAŃCHEZ, W. ROLDAŃ, S. VILLANUEVA, M. BARDAGI´ and L. FERRER Veterinary School, Universitat Auto`noma de Barcelona, Bellaterra, Barcelona, Spain The prevalence of Demodex mites in the skin of humans is close to 100%, with a mean mite density of 0.7 mites/

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cm2 (facial skin). However, despite the importance of canine demodicosis, the prevalence and density of Demodex parasitism in the dog is not known. Textbooks state that Demodex is a common inhabitant of canine skin, but a formal demonstration of this is lacking. Previous attempts to demonstrate the presence of Demodex in the skin of healthy dogs were unsuccessful, probably due to the low sensitivity of the techniques used. In addition, although it is believed that the immune system controls Demodex populations in dogs, evidence of a specific immune response against Demodex in the dog is also lacking. The objectives of this investigation were to (i) investigate the epidemiology of Demodex in healthy dogs, using a high-sensitivity real time-polymerase chain reaction (PCR), and (ii) detect antibodies against Demodex in the serum of dogs with generalized demodicosis, using dot blot and western blot techniques, before and after treatment. We studied 100 shelter dogs of different breeds and sexes, which were clinically healthy. Fourteen dogs had a diagnosis of generalized demodicosis and were treated with oral ivermectin. For real-time PCR analysis, the samples consisted of groups of 250–300 hairs plugged with roots, taken from different skin points. We did skin surface biopsies as described in the literature. Demodex whole extract was prepared using 200 Demodex canis mites obtained from four dogs with demodicosis. Mites were sonicated and treated with lysis buffer, and protein was quantified using the bicinchoninic acid colorimetric assay (Pierce BCA Protein Assay; Thermo Fisher Scientific, Rockford, IL, USA). Dot blot and western blot were performed using an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA). Real-time PCR results showed the number of positive dogs increasing with the number of cutaneous locations sampled. In the first trial, sampling five locations, 17% of the dogs were positive. In the last trial, sampling 20 locations, all healthy dogs were positive at least at one point. For various reasons, the skin surface biopsies were not useful for detecting and quantifying Demodex mites in the canine skin. These results are the first sound evidence that all or almost all dogs harbour Demodex in the skin, although probably in very low numbers. In the dot blot technique, the serum of dogs with generalized demodicosis reacted with the whole Demodex extract. The reaction was much weaker in the same dogs after therapy and clinical cure, and it was absent in the serum of healthy dogs (which had been positive in the real-time PCR). The western blot detected at least four major Demodex proteins in the serum of dogs with generalized demodicosis. We conclude that most healthy dogs harbour Demodex mites in the skin, although in very low numbers, with the most common locations being the head and abdomen. Despite this parasitism, no specific antibodies against Demodex were detected in the serum of healthy dogs. In contrast, dogs with demodicosis presented antibodies recognizing at least four major Demodex proteins. Funding: Ivan Ravera has obtained a PhD grant from the European Society of Veterinary Dermatology and European College of Veterinary Dermatology. Conflict of interest: None declared.


Plenary Session Abstracts: Saturday Afternoon, 28th July Theme: ONCOLOGY State-of-the-Art Address State of the art: treatment for skin cancer D. J. ARGYLE University of Edinburgh, Midlothian, Edinburgh, UK In this presentation we will explore the current state of the art for skin cancer therapies and how a better understanding of skin biology is opening the door to new therapies. Despite great strides in our understanding of the basic molecular biology of skin cancer, the various forms of the disease remain ones of high morbidity and mortality. Surgery, radiation, and chemotherapy are still the mainstays of therapy for the majority of tumour types, both in human and veterinary oncology. However, dissection of the process of carcinogensis is now allowing for a translation of basic biology into clinical practice. The best example of this is the recent introduction of the first two veterinary tyrosine kinase inhibitors for the treatment of mast cell disease. Dysregulation of the tyrosine kinase pathway in cancer makes this an ideal target, and defects in tyrosine kinase signal transduction have been identified in a number of cancer types, including mast cell tumours, squamous carcinoma, melanoma, and lymphoma. However, in human medicine it is clear that tumour heterogeneity may still allow for the development of drug resistance, and second- and thirdgeneration drugs will be required to overcome this. Initial results with veterinary-specific tyrosine kinase inhibitors are promising, but more work is required to understand the role these drugs may play in combination with other therapies such as radiation and surgery. In addition, these inhibitors may have a stronger role to play in combination drug protocols. Recent evidence suggests that various forms of skin cancers may originate in the skin stem cell pool. This has profound implications for therapy in terms of resistance to conventional therapeutic options. Existing therapies have been developed largely against the bulk population of tumour cells because the therapies are often identified by their ability to shrink tumours. Because most cells in a tumour have limited proliferative potential, a therapy’s ability to shrink a tumour mainly reflects its ability to kill the non-proliferating cells. It seems that stem cells from various tissues tend to be more resistant to chemotherapeutics than are mature cell types from the same tissues. The reasons for this are not clear, but may relate to high levels of expression of anti-apoptotic proteins or ATP-binding cassette (ABC) transporters such as the multi-drug resistance gene. If the same were true of cancer stem cells, then one would predict that these cells would be more resistant to chemotherapeutics than are tumour cells with limited proliferative potential. Even therapies that cause complete regression of tumours might spare enough cancer stem cells to allow re-growth of the tumours. The concept of cancer stem cells is an exciting one, and prospective studies need to be employed in veterinary cancer patients to identify these populations. A greater understanding of carcinogenesis has identified a number of potential targets including signal transduction pathways, angiogenesis, and the immortality of cancer. However, if sub-populations of stem cells exist, then these therapies will have to target both populations. Similarly, new reagents will have to be developed that identify these cells diagnostically. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

Funding: Self funded Conflict of interest: None declared.

Supporting Review The use of tyrosine kinase inhibitors in veterinary medicine C. LONDON The Ohio State University, Columbus, OH, USA Substantial progress in the field of molecular biology has permitted the identification of key abnormalities in cancer cells. These aberrations often involve a class of proteins called tyrosine kinases (TKs), which play critical roles in normal cell signal transduction, acting to modulate critical cellular processes such as growth and differentiation. The TKs work through phosphorylation, binding adenosine triphosphate (ATP) and using it to add phosphate groups to key residues on themselves (a process called autophosphorylation) and on other molecules, thereby initiating a downstream signal inside the cell. This process typically occurs in response to external signals generated by growth factors or other stimuli that initiate the cascade. The TKs can be expressed on the cell surface, in the cytoplasm, and in the nucleus. Dysfunction of TKs is now recognised as a common event in tumours. While this has been best characterised in humans, recent data indicate that dog and cat cancers experience similar dysregulation. Kinases may be dysregulated through a variety of mechanisms, including through mutation, overexpression, fusion proteins, or autocrine loops. The ultimate consequence of this dysfunction is the initiation of signal transduction in the absence of appropriate regulation (i.e. constitutive signaling), which ultimately contributes to uncontrolled cell growth and survival of abnormal cells. Significant efforts have now been directed at developing strategies to inhibit those TKs that participate in tumourigenesis by using direct effects on the cancer cell or modulating the tumour microenvironment (stroma and neovasculature). One of the most successful approaches to date has been through the use of small molecule tyrosine kinase inhibitors (TKIs). Several new TKIs have been approved for the treatment of human cancer, and many more will become available in the near future. In veterinary medicine, the use of TKIs to treat cancers in dogs and cats is relatively recent; however, two are now approved for use in dogs. Toceranib (Palladia) is a multitargeted inhibitor that blocks the function of several TKs including VEGFR, PDGFR, KIT, FLT3, and CSF1R. Toceranib has demonstrated activity against mast cell tumours, sarcomas, and carcinomas. Masitinib (Kinavet) is a small molecule inhibitor of the TKs KIT, PDGFR, and Lyn that has activity against mast cell tumours and possibly some solid tumours. Several clinical trials are ongoing to help guide the use of toceranib and masitinib in veterinary oncology. Funding: Self-funded Conflict of interest: None declared.

Abstracts

Supporting Original Study 16 Contribution of stem cells to epidermal and hair follicle tumours in the dog C. BRACHELENTE*, I. PORCELLATO*, M. SFORNA*, E. LEPRI*, L. MECHELLI* and L. BONGIOVANNI *Department of Biopathological Sciences and Hygiene of Animal and Alimentary Productions, Perugia, Italy; Department of Comparative Biomedical Sciences, Teramo, Italy In recent years, cutaneous stem cells have been implicated not only in epidermal and hair follicle renewal and repair but also in epidermal tumourigenesis. Stem cells have been identified in human, rodent, and, recently, canine hair follicles. However, to our knowledge, no studies have been conducted in the dog to elucidate the role of stem cells in the development of hair follicle tumours. In this study, 10 infundibular keratinizing acanthomas, 10 trichoblastomas, 10 trichoepitheliomas, 10 pilomatricomas, and four tricholemmomas were retrospectively investigated for the expression of keratin 15 (K15) and nestin, two recognized markers of epidermal/follicular and follicular stem cells, respectively. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. Four samples of normal-haired skin from healthy dogs were used as positive controls. Immunohistochemical expression was determined using commercially available antibodies (Keratin 15, clone LHK15, Thermoscientific; Nestin, clone ab7659; Abcam) that had already been tested and described in the veterinary literature. Expression of K15 and nestin in the cytoplasm of neoplastic cells was semi-quantitatively scored as negative (0) (< 25% of positive cells), mild (1) (25–50% of positive cells), moderate (2) (50–75% of positive cells) and intense (3) (> 75% of positive cells). The intensity of the reaction of single cells was further evaluated as faint (+), medium (++) or strong (+++). In normal adult canine skin, K15 was expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region) and multifocally in the basal cell layer of the epidermis. Nestin expression was less pronounced and restricted to the middle portion of the hair follicle. For K15, expression was negative or mild in the majority of infundibular keratinizing acanthomas (9/ 10), pilomatricomas (10/10), and squamous cells carcinomas (27/30). The trichoepitheliomas were the most heterogeneous group regarding the expression of K15, in terms of both percentage of positive cells and intensity of expression. Trichoblastomas were moderately to strongly positive (7/10) and showed the highest intensity of reaction of all the tumours studied. Tricholemmomas were either negative (2/4) or moderately to strongly positive (2/4). In all samples, K15 immunostaining of neoplastic cells and lobuli was generally more prominent in the external or deep portions of the tumour. Although milder and not equivalent, the trend of nestin expression in follicular tumours was similar, with the highest positivity in trichoblastomas and a high variability in trichoepitheliomas. However, the reaction was always very faint, and nestin was therefore judged a non-optimal marker for the study of the role of stem cells in hair follicle tumours of the dog. Our results suggest that hair follicle stem cells in the bulge may play a role in tumourigenesis of tumours originating from this portion, namely trichoepitheliomas and trichoblastomas, where the highest K15 immunostaining was observed. The loss

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of K15 expression in squamous cell carcinomas compared to normal skin suggests that this event could be critical in the malignant transformation of epidermal tumours, although further studies are needed to confirm this. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 17 Epithelial to mesenchymal transition: Immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours L. BONGIOVANNI*, A. D’ANDREA*, M. ROMANUCCI*, D. MALATESTA*, M. CANDOLINI*, L. DELLA SALDA*, L. MECHELLI , M. SFORNA and C. BRACHELENTE *Department of Comparative Biomedical Sciences, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy; Department of Biopathological Sciences and Hygiene of Animal and Alimentary Productions, Faculty of Veterinary Medicine, University of Perugia, Perugia, Italy Epithelial to mesenchymal transition is a multistep process playing an important role in tumour invasion and the development of metastases. Hallmarks of epithelial to mesenchymal transition are loss of several epithelial markers, b-catenin redistribution within the cell, and gain of mesenchymal markers expression, which are regulated by transcription factors, such as Snail, Slug, and Twist. At the site of metastasis, neoplastic cells again change their phenotype, undergoing the mesenchymal-toepithelial transition. Our previous results showed a reduction of membranous b-catenin expression and colocalisation of vimentin and altered subcellular bcatenin distribution in more malignant squamous cell carcinomas Therefore, we performed a comparative immunohistochemical evaluation of the pattern and levels of expression of cytokeratin, vimentin, survivin, and Hsp72 in canine cutaneous epithelial tumours, in order to understand the role of these molecules in cutaneous tumour pathogenesis and progression, including their possible role in the epithelial-to-mesenchymaltransition phenotype. We investigated 10 canine squamous cell carcinomas, one squamous cell carcinoma lymph node metastasis, 30 canine hair follicle tumours (four benign and two malignant pilomatricomas, eight infundibular keratinizing acanthomas, three benign and three malignant trichoepitheliomas, and 10 trichoblastomas), and five normal skin samples by immunohistochemistry using specific antibodies against vimentin, cytokeratin, survivin, and Hsp72. A semi-quantitative method was used to analyse results as follows: 0 to < 5%, ‡ 5 to < 10%, ‡ 10 to < 25%, and ‡ 25% of positive cells. Immunofluorescence was also used to investigate the colocalisation of survivin-vimentin and survivin-Hsp72 expression in selected canine infiltrative squamous cell carcinomas samples and one squamous cell carcinoma lymph node metastasis. In normal epidermis, we found weak Hsp72 cytoplasmic staining and scattered survivin positive nuclei in the basal cell layer. In normal hair follicles, Hsp72 showed a diffuse cytoplasmic staining of the outer root sheath with scattered positive nuclei in the basal cell layer, while survivin-positive nuclei were present in the basal cell layer of the outer root sheath 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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and matrix cells. In benign hair follicle tumours, survivin and Hsp72 showed the highest number of positive cells in trichoblastomas, and survivin exhibited high expression also in pilomatricomas and trichoepitheliomas. In malignant hair follicle tumours, a reduced intensity of cytokeratin expression was observed, associated with an increase of both survivin and Hsp72 expression. In squamous cell carcinomas, cytokeratin staining intensity was decreased in 0 to < 25% of neoplastic cells, while loss of cytokeratin expression and simultaneous vimentin immunolabelling acquisition, suggestive of the epithelialto-mesenchymal-transition phenotype, were evident only in a very low number (< 5%), often single, infiltrative neoplastic cells, detaching from the front of tumour invasion, where high nuclear survivin and cytoplasmic Hsp72 were evident. Survivin-Hsp72 colocalisation was observed in several neoplastic cells. Only a few neoplastic cells in the front of tumour invasion showed vimentinsurvivin colocalisation. These results suggest, in addition to their clear contribution in neoplastic transformation, a possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multi-phase epithelial-tomesenchymal-transition process of cutaneous epithelial tumours in dogs. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 18 Canine inflamed cutaneous T cell lymphoma: A diagnostic conundrum P. F. MOORE, V. K. AFFOLTER and S. M. KELLER University of California, Davis, Davis, CA, USA The objective of this study was to introduce the key features of a relatively recently recognized form of nonepitheliotropic T cell lymphoma in dogs. From 2004 to the present, we have encountered 21 dogs with inflamed T cell lymphoma in the skin. The dogs belonged to 18 breeds and had a median age of 7 years (± 3.2). Thirteen dogs were male, and seven dogs were female. The affected sites included tissues of the head (nine dogs), trunk (nine dogs) and limbs (five dogs); topographic sites were not specified in four dogs. Lesions presented as nodules, plaques, or masses of up to 4 cm in maximal dimension. Lesions were frequently ery-


thematous and crusted. An initial diagnosis of cutaneous reactive histiocytosis (11 dogs) or histiocytic neoplasia (three dogs) was made by primary pathologists. All lesions were assessed by standard histology and immunohistochemical methods to detect canine leukocyte antigens (CD3, CD11d, CD18, CD20, CD45, CD45RA, CD79a). Genomic DNA was extracted from paraffin sections of the lesional tissue, and gene rearrangement analysis of the T cell receptor gamma locus was assessed by polymerase chain reaction (PCR) in 20 of 21 dogs. The cutaneous lesions consisted of densely pleo-cellular infiltration of all levels of the dermis, with variable extension into the subcutis. The lesions often surrounded vessels and adnexae. Epitheliotropism was minimal. Small lymphocytes, plasma cells, and intermediate to large lymphocytes were scattered between prominent histiocytic infiltrates. Eosinophils were prominent in the lesions of only four dogs. The histiocytes did not manifest cytological atypia and radiated from perivascular locations to coalesce in the dermis. The intermediate to large lymphocytes often had large vesicular or hyperchromatic nuclei, a high nuclear:cytoplasmic ratio, and in some regions formed clusters of similar cells. The latter cells expressed CD3 with highly variable intensity ranging from faint or punctate cytoplasmic expression to dense cytoplasmic and surface expression. Molecular clonality analysis of the T cell receptor gamma locus revealed clonal expansion of T cells in a heavy polyclonal background in 16 of 20 dogs. The results in the remaining four dogs were pseudoclonal (two dogs), polyclonal (one dog) or no amplification (one dog).In conclusion, the recognition of inflamed cutaneous T cell lymphoma and its differentiation from reactive cutaneous histiocytosis depends on careful assessment of lymphocyte morphology and immunostaining patterns. Clusters of cytologically atypical lymphocytes with highly variable CD3 expression, especially partial to almost complete loss of expression, are important features. Confirmation of the diagnosis is best accomplished by lymphocyte antigen receptor gene rearrangement analysis. Application of these procedures, which are now more widely available, should enable increased recognition of inflamed T cell lymphoma and ensure that appropriate treatment is selected earlier in the clinical course. Funding: Leukocyte Antigen Biology Laboratory, University of California, Davis. Conflict of interest: None declared.

Equine Dermatology Sessions: Saturday Afternoon, 28th July Supporting Original Study 19 Dermatohistopathological findings in horses with pituitary pars intermedia dysfunction (PPID) before and after treatment with pergolide A. PETERSEN*, M. INNERA*, D. DESJARDINS and H. C. SCHOTT IIà *Michigan State University, College of Veterinary Medicine, Department of Small Animal Clinical Sciences, East Lansing, MI, USA; Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI, USA; àDepartment of Large Animal Clinical Sciences, Veterinary Medical Center, College of Veterinary Medicine, East Lansing, MI, USA Pituitary pars intermedia dysfunction (PPID) in equids develops due to degeneration of hypothalamic dopaminergic neurons extending to melanotropes in the pituitary pars intermedia. A pathognomonic clinical sign in PPIDaffected equids is a long hair coat that fails to shed (hypertrichosis). Pergolide is a dopamine agonist that acts to replace dopaminergic innervation by stimulating dopamine type 2 receptors on pars intermedia melanotropes and has been successfully used to treat PPIDaffected horses for several decades. Previously, we found that hypertrichosis in PPID-affected horses is characterized by persistence of hair follicles in anagen. In this follow-up study we compared hair follicle morphology of eight markedly hypertrichotic horses with PPID before and after 6 months of treatment with pergolide. Dermatohistopathologic findings in PPID-affected horses were also compared to four unaffected (and untreated) aged control horses. Eight-mm-punch skin biopsies were collected from the neck and rump of all 12 horses; pretreatment biopsies were obtained between November 1 and January 31, and biopsies were repeated 6 months later, between May 1 and July 31. PPID status was established on presence or absence of hypertrichosis and overnight dexamethasone suppression test results. Biopsy samples were sectioned at the level of, or slightly below, entry of the sebaceous duct in a plane parallel to the skin surface and were stained with haematoxylin and eosin. Hair cycle growth stage (anagen or telogen) was determined based on morphometric characteristics including degree of tricholemmal keratinization, inner root sheath histology, and volumetric involution of the outer root sheath. The number of hair follicles in anagen or telogen in a 50 mm2 area was counted for each biopsy. Prior to treatment, PPID-affected horses had a higher percentage of anagen follicles (neck – 89%, rump – 75%) and a lower percentage of telogen follicles (neck – 11%, rump – 25%) than control horses (anagen: neck – 15%, rump – 34%; and telogen: neck – 85%, rump – 66%). After treatment with pergolide mesylate, all PPID-affected horses had improved shedding of the hair coat. Further, five of eight PPID-affected horses had normalization of overnight dexamethasone suppression test results after treatment. Dermatohistopathogical findings in PPIDaffected horses after treatment showed that they still had a higher percentage of anagen follicles (neck – 70%, rump – 74%) than telogen follicles (neck – 30%, rump – 26%), and these percentages were not different from the pre-treatment values or to those of control horses:

anagen follicles (neck – 68%, rump – 85%) and telogen follicles (neck – 32%, rump – 15%). Post-treatment biopsies were performed in the late spring through early summer, a time of year when hair follicles are expected to be predominantly in anagen and to shed the telogen hairs. Because PPID-affected horses were observed to have improved shedding with treatment, largely prior to our post treatment sampling, it is likely that their hair follicles proceeded through a telogen phase and had returned to anagen by the time we collected the posttreatment biopsies. More frequent circannual biopsies of both PPID-affected and aged control horses would be required to answer this question. In conclusion, these dermatohistopathological findings provide further support that the clinical observation of severe hypertrichosis in PPID-affected horses is due to prolonged anagen phase of the hair follicles. Funding: Self-funded. Conflict of interest: None declared.

Supporting Original Study 20 Equine sarcoidosis: Clinical signs, diagnosis, treatment, and outcome of 22 cases M. M. SLOET VAN OLDRUITENBORGHOOSTERBAAN* and G. C. M. GRINWIS *Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Equine sarcoidosis may present as a skin condition (generalised or localised) which is clinically characterised by exfoliative dermatitis or as a nodular form which is characterised by granulomatous inflammation of multiple organs. The aim of this study was to evaluate the clinical signs, diagnosis, treatment and outcome of equine patients admitted to Utrecht University Equine Clinic with a histologically confirmed diagnosis of sarcoidosis between 2002 and 2011. Twenty-two cases were diagnosed with sarcoidosis (six geldings, 16 mares; 10 ± 4.6 years old, range 3–17 years; 14 Dutch warmblood horses, two German warmblood horses, two Friesian horses, two Arabian horses, one standardbred horse, and one pony). Diagnosis was based on clinical signs (scaling, crusting, partial alopecia, increased local skin temperature, subcutaneous oedema and/or granuloma) and confirmed by skin biopsy (multifocal nodular to diffuse non-caseating lympho-granulomatous dermatitis with multinucleated giant cells). A standardised treatment was used for all forms of equine sarcoidosis, consisting of an initial high dose of systemic corticosteroids (prednisolone at 1.0–2.0 mg/kg orally q 24 h in the morning, or occasionally dexamethasone at 0.04– 0.08 mg/kg intramuscularly q 24 h, for 7–14 days followed by a lower dose of prednisolone (0.20–1.0 mg/kg orally q 24 h) for several weeks or longer. Products of different manufacturers were used depending on the owner’s private veterinarian. No local treatment was advised since the skin of affected areas was already fragile and sensitive. The three recognised forms and their outcomes were as follows. Generalised sarcoidosis (n = 3): cutaneous signs and/or granulomas, persistent 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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low-grade fever, no pruritus, and sometimes exercise intolerance, painful to touch, mild respiratory distress, weight loss, and/or peripheral lymphadenopathy. One of three cases was euthanized immediately, and the remaining two were euthanized after 2–3 months of unsuccessful treatment. Partially generalised sarcoidosis (n = 4): granulomas present on some parts of the body with or without a localised skin problem on a pigmented or nonpigmented limb. All four cases deteriorated despite treatment and were euthanized after 2–12 months. Localised sarcoidosis (n = 15): localised area(s) on pigmented or non-pigmented lower limb(s) or elsewhere with non-pruritic crusting, scaling and oedema, sometimes painful to the touch, and lameness in severe cases. Fourteen of the 15 cases had signs on one or more lower limbs, and one had a localised area on the body. Two cases showed no or insufficient improvement on treatment and were euthanized. Four cases showed full


recovery after treatment, and one showed partial recovery without treatment. Eight cases improved on prednisolone but required continuous low doses to maintain the improvement. The present outcome is comparable with the follow-up of our previous case series (1999–2001; four geldings, five mares, 5–20 years old) in which two of nine horses with localised sarcoidosis showed a full recovery and seven of nine showed some improvement. Recognition of the different forms of sarcoidosis based on history, clinical appearance, and histopathology assisted owners in making an informed choice between treatment or euthanasia and prevented unnecessary (local) treatments. Localised equine sarcoidosis should always be considered in the differential diagnosis of a localised scaling dermatitis of unknown origin. Funding: Self-funded. Conflict of interest: None declared.

Free Communication Abstracts Session 1: NEOPLASIA FC-01 Sensitivity, specificity and diagnostic accuracy of cytology vis-a`-vis histopathology in the diagnosis of cutaneous and subcutaneous neoplasia in dogs

FC-02 Immunologic and inflammatory changes in canine skin and oral mucosa following radiotherapy as determined by gene expression profiling

N. K. SOOD, B. MEKIBIB and K. GUPTA Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India Neoplasia in dogs is responsible for nearly 16–24% mortality. Among all canine tumours, those of the skin and subcutis are diagnosed more frequently. In veterinary practice, cytology is a quick, easy, inexpensive, and risk free method for the diagnosis, monitoring, and prognosis of these tumours. The present study was conducted to compare the relative efficacy of diagnostic cytology with histopathology in the diagnosis of tumours. One hundred thirty-six clinical lesions of the skin and subcutis from 126 dogs were screened, and 53 neoplasms were identified. From these tumours, fine needle aspiration, tissue impression, scraping, and swab smears were prepared and stained with Romanovsky’s stain. Multiple biopsy samples were also collected from each dog and processed routinely for histopathology for comparison with cytologic findings. Of 53 tumours evaluated by cytology only 47 had a sufficient number of cells for diagnosis. In all, 16 different subtypes of neoplasms were diagnosed, which included epithelial (38%), mesenchymal (28%), round cell (28%), and melanocytic (6%). Malignant neoplasms were less frequent (45.28%) than benign (54.72%). The tumours were either single (81.13%) or multiple (18.87%) and more in male (79.25%) than female dogs (20.75%). The overall diagnostic rate of cytologic specimens was 88.7% with a sensitivity and specificity of 93.2% and 100%, respectively, when compared with histopathology. The positive and negative predictive values of cytology for the diagnosis was 100% and 85.7%, respectively. In summary, cytology proved extremely useful in the diagnosis of cutaneous/subcutaneous neoplasia in dogs. Some cytologic sampling techniques were superior. Funding: Director of Research, GADVASU, Ludhiana, Punjab, India. Conflict of interest: None declared.

A. DIESEL*, S. S. BONDURANT , B. ZHOUà and D. J. DEBOER§ *Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA; Gene Expression Center, University of Wisconsin Biotechnology Center, University of Wisconsin-Madison, Madison, WI, USA; àDepartment of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA; §Department of Medical Sciences, School of Veterinary Medicine, University of WisconsinMadison, Madison, WI, USA Radiation therapy is a standard of care treatment recommendation for nasal tumours in veterinary medicine and for human head and neck cancer. Inflammatory changes in the skin (dry or moist desquamation) and oral mucositis are common adverse acute effects of radiation therapy in this body region. There are few reports regarding the pathogenesis of inflammatory changes in the skin and oral tissue following radiation therapy, and none at all in dogs. A custom canine gene expression microarray (Roche NimbleGen, Madison, WI, USA) was used to evaluate changes on the entire genome level that occur in skin and oral mucosa following exposure to ionizing radiation. Samples were obtained from four client-owned dogs being treated for nasal neoplasia with radiation therapy (helical tomotherapy, 5 Gy per fraction, 10 fractions, total 50 Gy) before and after treatment. Messenger RNA was extracted from tissues and pooled to minimize individual variation (pre and post treatment; skin and oral mucosa) for cDNA synthesis. Gene expression microarray identified 1272 genes in skin and 2141 genes in oral mucosa differentially expressed by at least twofold change in response to treatment. Of those differentially expressed, 327 genes were in common between the two tissues. Functional analysis showed that differentially expressed genes in both tissues were involved in focal adhesion and complement and coagulation cascades. Further study is necessary to determine whether expressed genes correlate with function proteins and whether these may be used for therapeutic trials in the future to minimize side effects of treatment. Funding: American College of Veterinary Dermatology Resident Research Award. Conflict of interest: None declared.


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FC-03 Canine melanoma: epidemiological, histological and genetic aspects and comparison to human melanoma

FC-04 Spectral Doppler ultrasound and its relationship with prognostic factors in canine cutaneous mast cell tumour

E. CADIEU*, , M. GILLARD*, , C. DE BRITO*, , J. ABADIEà, A. S. GUILLORY*, , B. VERGIER§, P. DEVAUCHELLE–, F. DEGORCE**, L. LAGOUTTE*, , B. HEDAN*, and C. ANDRE*, *CNRS, UMR 6061, Institut de Geńe´tique et De´veloppement de Rennes, Rennes, France; Universite´ Rennes 1, UEB, IFR140, Faculte´ de Me´decine, Rennes, France; àLaboratoire d’Histopathologie Animale, Oniris, Ecole Nationale Ve´te´rinaire, Nantes, France; §Service de Pathologie, CHU Bordeaux et Universite´ Bordeaux Segalen, Bordeaux, France; –Centre de Cance´rologie Ve´te´rinaire, ENVA, Maisons Alfort, France; **Laboratoire d’Anatomie Pathologique Ve´te´rinaire du Sud-Ouest LAPVSO, Toulouse, France Spontaneously occurring melanomas are frequent in dogs and are found in the same locations as in humans: skin, mucosal sites, and eye. Interestingly, several canine melanoma types are breed specific, with very clearly predisposed breeds, and black-coated dogs are overrepresented. In addition, the oral melanoma is more frequent and aggressive than the cutaneous form. Histopathological examinations of 2350 affected dogs showed that poodles are at high risk to develop oral melanoma, while schnauzers, Rottweilers and Beaucerons are most often affected by cutaneous melanoma. An in-depth epidemiological, clinical and morphological analysis of 147 canine melanoma cases helped advance knowledge of the different types of melanoma and showed interesting correlations between anatomic sites and malignancy, severity, coat colours, clinical outcomes, and histopathologic data. Several lines of evidence prompt us to propose that canine melanoma presents homologies with human intradermal, non ultraviolet (UV) dependent melanoma. Over 300 blood samples from melanoma affected dogs and 150 melanoma tissues were collected to perform genetic analyses. Comparative sequencing of seven genes relevant for human melanoma classification, because of the presence of specific somatic alterations, was performed in 35 dogs affected by different melanoma types. This work highlighted somatic mutations in two genes, as well as constitutional mutations in melanocortin 1 receptor (MC1R), that correlated with canine coat colours. These findings advance the understanding of canine melanoma, and also support canine melanoma as a potential model for the pathogenesis of human non-UV dependent melanoma. Funding: CNRS, the European Commission (FP7-LUPA project-GA-201370) LIGUE NATIONALE CONTRE LE CANCER. Conflict of interest: None declared.

S. S. COSTA*, E. M. TERRA , R. T. NETOà, M. T. COSTA and R. L. AMORIMà *College of Veterinary Medicine, Pioneer Union of Social Integration, Brası´lia/Distrito Federal, Brazil; College of Agricultural and Veterinarian Sciences, Saõ Paulo State University, Jaboticabal/Saõ Paulo, Brazil; àCollege of Veterinary Medicine and Animal Science, Saõ Paulo State University, Botucatu/Saõ Paulo, Brazil Cutaneous mast cell tumour (MCT) is a common malignant neoplasm in dogs. Our goal was to correlate Doppler ultrasound (US) vascular features with clinical and immunohistochemical findings in canine MCT. We included 20 dogs with single (12/20) and multiple (8/20) cutaneous MCT, from distinct breeds, sexes and ages. We investigated the usefulness of spectral Doppler US in determining the potential of tumour recurrence and metastasis. Also, we correlated Doppler US with KIT protein expression (CD 117) and intratumoural microvessel density [(IMD) anti-factor VIII], number and size of tumours, ulceration, survival time, and histological grade using systems proposed by Patnaik et al. (1984) and Kiupel et al. (2011). By using Doppler US, we have identified vessels in 54% of tumours. There was no association among intratumoural vessel identification and IMD, KIT protein, histological grades, recurrence, metastasis or survival time (P > 0.05). Doppler US findings have correlated with size and tumour ulceration (P < 0.05). Pulsatility and resistivity indices obtained on Doppler US may be promising tools in the differentiation of high and low grade MCT in the preoperative period, rather than only the detection of blood vessels, since the average of both indices in high grade tumours were higher than in low grade tumours. We have observed a tendency of tumours with blood vessels detected by Doppler US to show shorter survival rates and regional lymph node metastasis. Further studies should be conducted so that this technique can be used as a noninvasive method to characterize vascularization and blood flow in canine cutaneous MCTs. Funding: Saõ Paulo State Research Foundation (Fapesp). Conflict of interest: None declared.


FC-05 Complete canine papillomavirus life cycle in pigmented plaques C. E. LANGE, K. TOBLER, E. M. SCHRANER, E. VETSCH, N. FISCHER and C. FAVROT Vetsuisse Faculty, University of Zurich, Zurich, Switzerland Canine papillomaviruses (CPVs) have been identified in various benign and malignant neoplastic skin disorders. The most frequent manifestations of CPV infections are classical warts and pigmented plaques. Although the aetiology of canine oral papillomatosis is well established, knowledge about the role of CPVs in the development of pigmented plaques remains vague. Indeed, as CPV DNA may frequently be found on clinically healthy canine skin, its detection in lesions

Abstracts cannot be regarded as a sufficient indicator of causality. Whether CPVs are actually active in pigmented plaques and involved in their development is consequently an open question. To answer this, two distinct clinical cases of canine pigmented plaques were evaluated in greater detail. We report: (i) the histological findings support the clinical diagnosis of pigmented viral plaques; (ii) sequencing of amplified CPV specific DNA isolated from infected tissue reveals two genomes of novel CPV types, both putatively belonging to the genus Chi; (iii) viral gene transcription can be shown by RT-PCR assay of mRNA from the identified CPVs in the respective lesions;

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(iv) viral particles are detected by electron microscopy in homogenate as well as in nuclei of keratinocytes in pigmented plaques. In conclusion, we linked the clinical lesions of pigmented plaques to histological changes, the presence of CPV specific DNA, viral gene transcription, and the presence of viral particles in and from the lesions. Thus, the findings outline the replicative cycle of CPVs in pigmented plaques and support the causal relationship between these viruses and the described disorders. Funding: This study was supported by the Krebsliga Schweiz. Conflict of interest: None declared.


Free Communication Abstracts Session 2: MISCELLANEOUS DISEASES FC-06 A case of pili torti in a domestic short-haired cat E. MAINA*, S. COLOMBO*, F. ABRAMO and G. PASQUINELLIà *Servizi Dermatologici Veterinari, Cuneo e Legnano, Italy; Dipartimento di Patologia Animale, Universita` degli Studi di Pisa, Pisa, Italy; àServizio di Citopatologia, Dipartimento di Patologia Sperimentale, Universita` di Bologna, Policlinico S. Orsola-Malpighi, Bologna, Italy Pili torti is a hereditary human disease, characterised by distortions of the hair follicle, which leads to flattening and twisting of the hair shaft by 90–360. A similar condition was reported in a litter of kittens with generalized hair loss, associated with systemic signs, followed by death at a young age. A 1 year-old castrated male tricoloured domestic short-haired cat was presented for noninflammatory and nonpruritic symmetrical multifocal alopecia involving the head, pinnae, a front limb, tail and chest of 1 month duration. The cat was otherwise healthy. Differential diagnoses included congenital alopecia, demodicosis, dermatophytosis, telogen effluvium, alopecia areata, pseudopelade, and paraneoplastic alopecia. Microscopic examination of hair shafts revealed pili torti and a normal mixture of anagen and telogen roots, occasionally spiral in shape, without evidence of mites or dermatophytes. Histopathological examination showed the same hair shaft abnormality with normal epidermis, dermis, hair follicles and other adnexa. A blood sample was taken for genetic testing as the cat was a tricoloured male, but the karyogram was normal. Scanning electron microscopy confirmed a hair shaft dysplasia characterized by pili torti, similar to that observed in humans. To the authors’ knowledge, this is the first case report of pili torti in a healthy young adult cat. Funding: Self funded. Conflict of interest: None declared.

FC-07 Canine mucocutaneous lupus erythematosus: a nondiscoid variant of chronic cutaneous lupus erythematosus T. OLIVRY and K. E. LINDER Center of Comparative Medicine and Translational Research, NC State University, Raleigh, NC, USA Lesions of human chronic cutaneous lupus erythematosus (LE) are uncommonly seen on mucosae, while they are typically absent in dogs with generalized or localized cutaneous discoid LE. Our objective is to report the clinical, histological and immunological characteristics of canine chronic mucocutaneous LE (MCLE). Four adult dogs, including three German shepherd dogs, were affected with chronic, recurrent mucosal erosions and ulcers. Oral, genital and anal mucosae and/or mucocutaneous junctions were affected in two, three and three dogs, respectively. There were no systemic signs other than pain upon lesion palpation and dyschezia, which was noted whenever anal lesions were present. Lesions failed to respond to oral and/or topical antibiotics. In three dogs for which follow up was available, all lesions resolved rapidly after administering a niacinamide-tetra 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

cycline combination (500 mg each, orally, three times daily). In all dogs, biopsies from perilesional skin or mucosae revealed lymphocytic interface dermatitis with basal cell apoptosis and a thickened basement membrane. Direct immunofluorescence confirmed the presence of a thick, irregular band of IgG (4/4 dogs), IgM (4/ 4), IgA (2/4), and C3 (4/4) at the basement membrane zone. Circulating antibasement membrane autoantibodies were not detected in any dog by indirect immunofluorescence using normal canine salt-split buccal mucosa as a substrate. A low antinuclear antibodies serum titer was found in only one dog. Altogether, these dogs exhibited clinical, histological and immunological lesions compatible with those of chronic mucosal LE in humans. These observations argue for the addition of MCLE to the spectrum of canine cutaneous lupus-specific clinical variants. Funding: Self funded. Conflict of interest: None declared.

FC-08 Malassezia pachydermatis overgrowth in six cats with generalized dermatitis O. CROSAZ*, A. LEGRAS*, F. VILAPLANAGROSSO , J. DEBEAUPUITS , B. HUBERT*, G. MARIGNAC* and J. GUILLOT* *Parasitology, Mycology, Dermatology, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France; Medicine, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France Malassezia yeasts are not considered common pathogens in cats. However, the isolation of high populations of Malassezia yeasts has been associated with allergic skin diseases, immunosuppressive viral infections, and serious internal diseases. In 2010 and 2011, we observed six cats with generalized dermatitis associated with Malassezia overgrowth. The cats were presented to the dermatology consultants of the Veterinary School of Alfort, France. Elevated numbers of yeasts were identified in lesional skin by cytology and culture. Malassezia pachydermatis was the species isolated. Skin lesions commonly occurred on the face (5/6 cats), ventral neck (5/6), abdomen (4/6) and ear canals, and were characterized by some degree of alopecia, erythema and crusting. In most cases, pruritus was intense. Underlying diseases included cutaneous adverse food reaction (cats 1 and 2, one cat had concurrent hypereosinophilic syndrome), feline infectious peritonitis virus infection (cats 3 and 4), post-herpetic erythema (cat 5), and superficial necrolytic dermatitis (cat 6). Cats 3–6 were in poor health and subsequently died. Cats 1 and 2 were treated with an antiseptic and antifungal shampoo. After 3–4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed, but relapses occurred as long as the origin of the adverse food reaction was not elucidated. These observations confirm that Malassezia overgrowth should be considered as a marker of serious underlying disease or allergic conditions. Funding: Self funded. Conflict of interest: None declared.

Abstracts

FC-09 A mouse model of dermatophilosis: comparison of different mouse strains M. DE REYNAL, M. EL JACK, B. CATCHPOLE and D. H. LLOYD Royal Veterinary College, London, UK Dermatophilosis is a skin disease affecting a wide range of animals worldwide and caused by Dermatophilus congolensis. Previous experimental studies have focused on primary infection, and little is known about the pathogenesis of the disease and the nature of the immune response to multiple exposures to D. congolensis. We have developed a mouse model of dermatophilosis, using a prime-boost-challenge inoculation protocol to compare the inflammatory and immune responses between two mouse strains, BALB/c and C57BL/6. Mice were topically inoculated with D. congolensis on three occasions, and development of clinical disease was evaluated. Skin biopsies were obtained for histological study of local inflammatory responses. Cytokine responses were evaluated following in vitro T-cell stimulation, and serum antibodies were assessed by ELISA. Evaluation of innate and adaptive immune parameters during primary and challenge infections demonstrated clear differences between the two mouse strains. In BALB/c mice, an initial transient inflammatory influx of neutrophils and macrophages at the infected sites was followed by a T helper (Th)2-type immune response characterised by a strong immunoglobulin (Ig) G1 antibody response and a weak cellular response, preventing spread of the infection. C57BL/6 mice displayed a weak initial innate response followed by a potent Th1 D. congolensis-specific response, resulting in the development of an immune mediated chronic disease. For the first time, differing immune responses have been demonstrated between mouse strains displaying distinct phenotypes. This model provides a platform to further study the pathogenesis of dermatophilosis addressing the specific role of innate and immune mechanisms deployed against infection. Funding: European Union 4th Framework Programme and Council for Assisting Refugee Academics. Conflict of interest: None declared.

FC-10 A case of Ehlers-Danlos syndrome in a Japanese monkey (Macaca fuscata) A. SHIMIZU*, ,à, S. IKOMA§, M. NAGATA–, N. MURAYAMA–, Y. SHIMURA§ and A. ISHIKO* *The First Department of Dermatology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan; Department of Dermatology, Keio University of School of Medicine, Tokyo, Japan; àShimizu Animal Hospital, Chiba, Japan; §Kushiro Municipal Zoo, Hokkaido, Japan; –ASC Dermatology Service, Tokyo, Japan Ehlers–Danlos syndrome (EDS) is a hereditary connective tissue disorder characterized by hyperextensibility of the skin, hypermobility of the joints and generalized connective tissue fragility. To date, EDS has been reported in several animal species, but has not been reported in monkeys. In this study, with the use of precise electron microscopic (EM) observation, we report a suspected case of EDS in a Japanese monkey (Macaca fuscata). The monkey was a 5 year-old female that since birth had

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hyperextensibility of the skin, repeated traumatic injury, and atrophic scar formation with hair loss. Her mother and siblings had no symptoms of EDS, although her father could not be identified. Clinically, we suspected EDS, and skin biopsy was taken from the affected monkey and as controls her mother and a healthy nonsibling monkey. Histopathologically, no abnormality was found in the epidermis, dermis or hair follicles of the affected monkey. On the other hand, EM findings demonstrated irregular diameter and shapes of the cross-section of collagen fibrils. Moreover, many ‘flower-like’ cross-sections, which may be characteristic for EDS, were observed. Then we statistically analysed the diameter of collagen fibrils. The affected monkey showed significantly decreased diameter (64.0 ± 15.1 nm) compared to the non sibling normal monkey (95.9 ± 13.2 nm, P < 0.01). Interestingly, the mother also demonstrated slightly smaller diameter of collagen fibrils (82.0 ± 16.8 nm) compared to the non sibling normal monkey (P < 0.01), which might indicate the mother was a heterozygous carrier. Funding: This study was self funded. Conflict of interest: None declared.

FC-11 Microbiological characteristics of ulcerative dermatitis in koi (Cyprinus carpio) B. PALMEIRO* and S. RANKIN *Lehigh Valley Veterinary Dermatology & Fish Hospital, Allentown, PA, USA; School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA Ulcerative dermatitis (UD) is one of the most common dermatologic conditions affecting ornamental fish and an extremely common presenting complaint to the aquatic veterinary practitioner. The purpose of this study was to describe the microbiological characteristics of UD in koi including: comparison of bacterial culture results from superficial swab and sterile tissue cultures, antimicrobial susceptibility of bacterial isolates and comparison of biochemical identification to 16S rRNA sequencing identification. Eighteen adult koi with UD were sampled under anaesthesia. Superficial swab cultures and sterile tissue biopsy cultures were performed from the same lesion. Bacterial isolates were identified biochemically (Trek Sensititre: Trek, Cleveland, OH, USA) and via 16S rRNA sequencing (ABI 310/MicroSeq: Applied Biosystems, Carlsbad, CA, USA). Susceptibility was determined via Kirby-Bauer disk diffusion. A total of 75 bacterial isolates were obtained from 18 fish (mean isolates/swab 2.5; mean isolates/tissue culture 1.7). The same isolate was identified on swab and tissue cultures in 8/18 (44%) fish; no fish had completely identical isolates on swab and tissue cultures. Sixty-six isolates were available for 16S rRNA sequencing identification (ID); biochemical ID agreed completely with DNA sequencing ID in 11/66 (17%) isolates. Resistance rates were high and no single antibiotic was effective for all isolates. Superficial swab cultures in koi with UD have poor correlation with sterile tissue culture results. Culture with sensitivity is recommended in clinical cases due to high rates of bacterial resistance. Biochemical ID poorly correlated with DNA sequencing results, likely secondary to the limited database and unique biochemical/metabolic properties of aquatic bacteria. Funding: University of Pennsylvania. Conflict of interest: None declared. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

Free Communication Abstracts Session 3: PARASITIC FC-12 Histopathological findings and correlation with clinical data for 95 cases of canine demodicosis E. FLORANT*, M. MIALOT , J. GUILLOTà and G. MARIGNACà *Clinique Ve´te´rinaire Les Sablons, Plaisir, France; Alfort-Idexx Laboratory, Alfortville, France; àUnite´ de Parasitologie-Mycologie-Dermatologie, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons Alfort, France The aim of this retrospective study was to compare in a representative sample, the histopathological features of canine demodicosis with clinical data. All cases of demodicosis (n = 109) diagnosed in 2009 at Alfort-Idexx Vetlab laboratory were re-examined applying a scoring system for the main lesions and patterns. Demodex mites were counted (mean and maximal number per follicle, and percentage of follicles parasitized). Four tumour associated cases were excluded. For statistical analysis, 95 cases with complete data, were included: 61 young dogs (including 10 French bulldogs) and 34 adults (including seven shih tzus). There were three pododemodicosis, six localized, and 86 generalized forms (including 55 with coexisting pyoderma). Disease duration ranged from 15 days to over 3 months. Demodex mite number was significantly higher in dogs with pyoderma. In 14 cases, not correlated with disease duration, neither mural folliculitis nor interface dermatitis was detected. Mural folliculitis was absent or moderate in cases with higher number of parasites, except in localized forms. Interface dermatitis was not observed in early cases, and appeared progressively with an eventual appearance of melanophages and pigmentary incontinence, particularly in adults. Perifolliculitis was frequently (88/95) and rapidly observed with no correlation with parasite burden. Parafollicular granulomas were more frequent with coexisting pyoderma in young animals (41/61) than in adults (9/34) without correlation of the number of mites. This study demonstrates differences in the patterns between young and adult dogs, depending on the clinical form and the duration of the disease. It also suggests a lack of immune response in severe cases. Funding: Self funded. Conflict of interest: None declared.

FC-13 Treatment of canine generalized demodicosis secondary to hyperadrenocorticism with topical moxidectin and imidacloprid H. P. HUANG* and Y. H. LIEN *Institute of Veterinary Clinical Science, Veterinary School, National Taiwan University, Taipei, Taiwan; Azu Clinic for Animals, Taipei, Taiwan Canine generalized demodicosis secondary to hyperadrenocorticism is usually intractable. Eleven dogs affected with hyperadrenocorticism and generalized demodicosis characterized by no response to ivermectin (0.5 mg/kg, 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

orally, once daily for > 4 weeks) and refractory to milbemycin oxime (0.5 mg/kg, orally, once daily for > 4 weeks) were included in this study. The diagnosis of generalized demodicosis was based on finding more than three live adult demodectic mites from > 3/4 deep scrapings. The diagnosis of hyperadrenocorticism was based on clinical signs and laboratory profiles consistent with hyperadrenocorticism, affirmative results from ACTH stimulation tests, and ultrasonographic findings of the adrenal glands. Spot-on 10% imidacloprid and 2.5% moxidectin (Advocate: Bayer HealthCare AG, Leverkusen, Germany) at the dose of 0.1 mL/kg was applied once weekly for 12 weeks. All dogs were reevaluated every 2 weeks. Deep scrapings of four sites and routine blood tests were performed every visit. Trilostane, the concomitant treatment for hyperadrenocorticism, remained at the same dosage throughout the period of this study. The mean total live adult mite counts before, 4- and 8-weeks after weekly spot-on moxidectin/ imidacloprid were 20.1 ± 6.3 (range, 13–33), 0.5 ± 0.7 (range, 0–2; 6/11 were negative), and 0.2 ± 0.4 (range, 0–1; 9/11 were negative), respectively, which reached statistical significance (P < 0.001). Ten of 11 dogs (90.1%) achieved clinical remission by demonstrating no demodectic mites at any life stage for consecutive scrapings over 8 weeks. No side effects in terms of clinical aspects or on routine blood tests were found during the period of the study. Funding: Self funded. Conflict of interest: None declared.

FC-14 Perception of owners concerning the presence of flea infestation in dogs and cats and flea control N. KIMURA*, M. JOBEZ , L. DESQUILBETà, B. HUBERT*, R. CHERMETTE*, S. PERROTà, F. BEUGNET§, J. GUILLOT* and G. MARIGNAC* *Unite´ de Parasitologie-Mycologie-Dermatologie, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; Veto-A-Dom, Montrouge, France; àInstitut de Recherche Clinique de l’Animal, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maison-Alfort, France; §Merial, Lyon, France Achieving good owner compliance during flea control remains an ongoing challenge for the veterinary profession. To improve treatment and better understand the perception of owners regarding fleas and flea control, a questionnaire (12 questions) was given to pet owners during dermatology or vaccination consultations from March to July 2011. A total of 299 questionnaires were collected and 298 were analysed. Just 25% of owners considered flea infestation to be rare in dogs and cats. Whereas 42% of owners thought the main reason a pet had fleas was the lack of regular flea treatment, 25% thought it was free access to the outside, and 13% thought it was contact with stray animals. Also, 58% of owners believed fleas were mainly found on animals, and 50% believed that fleas found on pets were different from those present at home. Only 30% of owners considered it

Abstracts important to eliminate fleas in the surrounding environment, and 32% considered it sufficient to treat animals on which fleas were seen. Although 53% of owners knew that fleas could transmit pathogens to animals only 27% were aware pathogens could be transmitted to humans. Spot-on formulations were used most frequently (72%). More owners (57%) bought anti-flea products at veterinary clinics and 21% at pharmacies. The price was not a limiting factor. Despite television advertising, articles in newspapers, and veterinary advice, the knowledge of flea control by pet owners, including those whose animal suffers from skin disease, is inaccurate, which is probably responsible for the lack of compliance during flea control programs. Funding: The present study was self funded. This work is part of the veterinary thesis of M. Jobez. Conflict of interest: N Kimura has received a 1 year grant from the French Embassy in Japan, financed for 50% by Merial Japan and 50% by the French Government. F Beugnet is employee of Merial SAS.

FC-15 Knockdown effect of some insecticides on isolates of the cat flea Ctenocephalides felis collected from dogs and domestic cats in Japan S. HAYASHIYA*, , Y. NAKAMURA*, , M. HAYASHIYA* and T. FUKASE *Hayashiya Animal Hospitals, Uji-shi, Kyoto, Japan; Hayashiya Institute of Life Sciences, Uji-shi, Kyoto, Japan Many compounds have been developed as effective insecticides against fleas on dogs and domestic cats. However, refractory cases have been occasionally identified with insecticide products, so it is assumed that different flea isolates may show different sensitivity to each insecticide. In the present research, sensitivity of flea isolates was examined to some insecticides by in vitro knockdown tests. Thirty isolates of the cat flea Ctenocephalides felis were respectively collected from dogs and cats in Japan. Each isolate of the fleas was examined for its sensitivity to permethrin of the pyrethroid class, fipronil of the phenylpyrazole class and imidacloprid of the nicotinoid class by bringing the fleas in contact with various amounts of these chemicals in a petri dish. The number of fleas ‘knocked down’ was counted 24 h after exposure to the insecticides. Many of the flea isolates showed high sensitivity to all three chemicals tested, but some isolates showed low sensitivity to one or two of the chemicals. The number of isolates with low sensitivity to permethrin, fipronil and imidacloprid was five, four, and four isolates from dogs, and four, four, and seven isolates

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from cats, respectively. From these results, it is considered that some isolates of fleas show low sensitivity to a certain kind of insecticides. Accordingly, when fleas on dogs or domestic cats are not satisfactorily eliminated by an insecticide remedy, low sensitivity of fleas to that drug should be considered, and the use of another compound of a different insecticide class is recommended. Funding: Hayashiya Animal Hospitals. Conflict of interest: None declared.

FC-16 Effectiveness of phytogenic fly repellent and ectoparasiticide product against dipteran flies B. NARALADKER*, M. SAXENA , B. S. KHILLARE*, S. T. KALWAGHE*, G. M. CHIGURE*, K. RAVIKANTH and S. MAINI *College of Veterinary and Animal Sciences, MAFSU, Parbhani Maharashtra, India; Ayurvet Limited, Baddi, India Flies and midges are a nuisance and cause irritation to animals and transmit several diseases. Extreme discomfort caused by constant fly and midge attacks can result in reduced weight gain, lower milk yield in cattle and poor milk quality. Repellents have been suggested as a means to alleviate fly nuisance. Development of pesticide resistance in house flies has prompted development of physical and biological control measures. A study was conducted at a livestock dairy farm that had a history of irritation caused by Culicoides peregrinus and C. schultzei. During the summer season, the farm was treated with the herbal fly repellent product ‘Keetgaurd liquid’ (M/S Ayurvet Limited, Baddi, India) recommended as one part with 20 parts of water for application directly on the animal, and one part with 40 parts of water for application on drainage channels and animal premises. The herbal fly repellent product was assessed for oviposition deterrent, ovicidal and larvicidal effect, and for its efficacy to minimize the count of larval and adult Culicoides spp. in the drainage channels around cattle sheds after application of product. Keetgaurd liquid was found to be quite efficacious as a fly repellent for livestock dwellings. It had good larvicidal potential in addition to ovicidal and oviposition deterrent activity. It has not been found to cause deleterious or adverse effects such as irritation, loss of production, or mortality in the experimental animals, rather it is safe for animal usage and for application in animal premises. Funding: Ayurvet Limited. Conflict of interest: None declared.


Free Communication Abstracts Session 4: THERAPY FC-17 A low dose azathioprine/glucocorticoid protocol for the treatment of canine pemphigus foliaceus S. BORIO and C. NOLI Servzi di Dermatologia Veterinaria, Turin and Peveragno, Italy Treatment of canine pemphigus foliaceus (CPF) is based on orally administered prednisolone (2–6 mg/kg) or dexamethasone (0.2–0.6 mg/kg), often with azathioprine (1.5–2.5 mg/kg). These may cause severe side effects, leading to euthanasia. The aim of this study was the retrospective evaluation of a low dose immunosuppressive protocol for CPF. Cases with sufficient follow-up were retrieved from a dermatological caseload. The protocol included daily azathioprine (1–2 mg/kg) and glucocorticoids (1–2 mg/kg prednisone or 0.1–0.2 mg/kg dexamethasone). After 14–28 days, glucocorticoids were given every 48 h, then tapered by one quarter dose every 2 weeks and finally discontinued. One month after glucocorticoid withdrawal, azathioprine was tapered and eventually stopped. Data on starting drug doses, time to resolution, adverse effects, and maintenance protocols were recorded. Three mixed-breed and eight pure-breed dogs with CPF were administered 0.8– 1.8 mg/kg azathioprine and 0.8–1.5 mg/kg prednisone (nine cases) or 0.025–0.1 mg/kg dexamethasone (two cases). Eight dogs were given concomitant antibiotics for 1 month. In five dogs lymphocyte counts decreased below 800 · 103/lL: azathioprine was withdrawn in three dogs and the dose was reduced by half in two dogs. In all cases, clinical remission was achieved in 1– 3 months. In five dogs, therapy was discontinued without relapse, while five dogs were maintained in remission with azathioprine (0.6–1.8 mg/kg), in two of these also with low dose glucocorticoids every 48 h. One dog was euthanized due to hepatic failure 1 month after initiation of azathioprine. This study suggests lower induction dosages of azathioprine and glucocorticoids may be effective for treating CPF. Funding: Self funded. Conflict of interest: None declared.

FC-18 Treatment of solar dermatitis with firocoxib in five dogs: clinical outcome and cyclooxygenase isoform-2 expression. F. ALBANESE*, C. CAPORALI*, F. ABRAMO , G. VICHIà and F. MILLANTA *Clinica San Clemente, Arezzo, Italy; Department of Animal Pathology, Pisa, Italy; àPrivate practitioner, Ancona, Italy Chronically sun-damaged skin in dogs is referred to as solar dermatitis. Sun avoidance, solar suits and sunscreens are the first treatments in dogs. The aim of this study was to determine cyclooxygenase 2 (COX-2) expression in the skin of four Dogo Argentino and one pitbull-mix dogs (age 3.5–9.0 years), two females and three males, chronically affected by solar dermatitis, and to investigate the therapeutic use of firocoxib, a COX-2 selective inhibitor (Previcox; Merial Italia SpA, Assago, 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

MI, Italy). Dermatological lesions were alopecia, erythema, shiny, firm skin and comedones mainly in the glabrous ventral abdomen. Histopathology revealed sublaminar fibrosis, elastosis, actinic comedones, epidermal hyperplasia, dyskeratosis and plaques of actinic keratosis. Immunohistochemistry revealed granular to diffuse cytoplasmic COX-2 (UCS Diagnostic, Morlupo, Rome, Italy) expression in keratinocytes of the hyperplastic and dyskeratotic areas. Oral treatment with firocoxib at a dosage of 5 mg/kg/day was initiated in all dogs. Clinical follow up at day 50 and 180 of treatment in four dogs and at day 60 of treatment in one dog showed overall improvement. By owner consent biopsies were taken from the same areas collected at baseline, and histology and COX-2 expression revealed: dermal changes were still present, hyperplasia and dyskeratosis were decreased in all but one dog, and COX-2 expression was reduced and less intense. In conclusion clinical follow up showed a general improvement, which histologically correlated with the regularization of the epidermal proliferation rather than recovery of dermal changes. A role for COX-2 might thus be hypothesised in the pathogenesis of canine solar dermatitis. Funding: Merial Italia SpA. Conflict of interest: None declared.

FC-19 Changes in ear pinnae dermal blood flow caused by various haemodynamic drugs in dogs: detection by laser Doppler flow meter K. IDE, S. HIRATSUKA, K. SHIMADA, T. IWASAKI and K. NISHIFUJI Tokyo University of Agriculture and Technology, Tokyo, Japan Laser Doppler flow meter (LDF) is a tool to measure dermal blood flow used in human medicine. We have previously evaluated dermal blood flow measured by LDF in canine ear pinnae, in both healthy controls and dogs with auricular ischemic dermatopathy. The objective of this study was to determine whether LDF could measure the changes in the dermal blood flow following administration of various hemodynamic drugs. Dibutyryl cyclic AMP (DBcAMP) (40 lg/kg/min i.v.) or atropine (0.04 mg/kg s.c.) or medetomidine (25 lg/kg i.m.) were administered to healthy beagle dogs (5 dogs/drug). Dermal blood flow of both sides of ear pinnae was measured before and after the drug administration (post 15, 30 and 60 min). In the DBcAMP group, dermal blood flow was significantly higher after 30 and 60 min (35.2 ± 3.0 and 36.1 ± 4.6 mL/min/100 g, respectively), compared to before administration (25.4 ± 4.8 mL/min/100 g; P < 0.05). In the atropine group, dermal blood flow was significantly higher after 30 and 60 min (35.2 ± 7.6 and 34.8 ± 6.7 mL/min/100 g, respectively), compared to before administration (25.8 ± 7.4 mL/min/100 g; P < 0.05). In the medetomidine group, dermal blood flow at 15 min after the drug administration (17.3 ± 4.4 mL/ min/100 g) was significantly lower than before administration (30.2 ± 7.0 mL/min/100 g; P < 0.05). In conclusion, LDF data efficiently reflects alterations of dermal

Abstracts blood flow caused by the hemodynamic drugs. This method may be useful to monitor changes of ear pinnae blood flow in dogs with auricular ischemic dermatopathy. Funding: Self funded. Conflict of interest: None declared.

FC-20 TrisEDTA significantly potentiates the bactericidal activity of silver sulfadiazine against multi-drug resistant Pseudomonas aeruginosa L. M. BUCKLEY*, N. A. MCEWAN*, P. GRAHAM and T. NUTTALL* *The University of Liverpool School of Veterinary Science, Neston, Cheshire, UK; Nationwide Laboratories, Poulton-le-Flyde, Lancashire, UK Pseudomonas aeruginosa commonly complicates chronic otitis in dogs. Topical therapeutic options for these cases can be limited due to damage to the tympanic membrane and/or the presence of multidrug resistance. The aim of this in vitro study was to determine the minimum bactericidal concentrations (MBCs) of silver sulfadiazine, with and without the addition of ethylenediaminetetraacetic acid-tromethamine (trisEDTA), against multidrug resistant- (i.e. resistant to at least three classes of antibiotic) P. aeruginosa (n = 12) isolated from cases of canine otitis. Isolates were incubated for 90 min with silver sulfadiazine (at concentrations of 4–500 lg/mL) alone and in combination with trisEDTA (tris 4.5 mg/mL; EDTA 1.2 mg/mL). Positive and negative controls were included, and all wells were in triplicate. Ten microlitre aliquots from each well were transferred to sheep blood agar and incubated for 18 h at 37C. Bacterial growth was recorded as the number of colony forming units or as confluent growth. The MBCs were recorded as the lowest concentration of silver sulfadiazine ± trisEDTA that completely prevented bacterial growth. The trisEDTA had no antimicrobial activity when used alone. The addition of trisEDTA significantly (P < 0.001) reduced the MBCs of silver sulfadiazine against these multidrugresistant P. aeruginosa isolates [median MBCs: silver sulfadiazine alone 23.4 lg/mL (range 4–500); silver sulfadiazine/trisEDTA 4 lg/mL (range < 4–7.8)]. In 11/12 isolates, moreover, the silver sulfadiazine/trisEDTA combination was bactericidal at all concentrations tested (i.e. MBC < 4 lL). These findings may be of use to clinicians when managing canine otitis complicated by multidrugresistant P. aeruginosa. Funding: The PA isolates were provided by Nationwide Laboratories, Poulton-le-Flyde, UK. The study was otherwise self-funded. Conflict of interest: PG is an employee of Nationwide Laboratories.

FC-21 The residual antibacterial activity of canine hairs after treatment with antibacterial shampoos I. KLOOS, R. K. STRAUBINGER, C. HORSTMANN and R. S. MUELLER Ludwig Maximilian University, Munich, Germany The aim of the study was the comparison of the residual antibacterial activity of hair shafts against Staphylococ-

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cus pseudintermedius after shampooing with seven different shampoos and a combination of shampoo and conditioner. The shampoos used in this study contained the active ingredients chlorhexidine in different concentrations (0.8%, 3% and 4%), the combination of chlorhexidine 2% and miconazole 2%, benzoyl peroxide 2.5% and, ethyl lactate 10%. One shampoo vehicle was the control. Healthy dogs (n = 42) were shampooed on day 1, day 4, day 7 and day 10 with their assigned shampoo. Hair samples were taken on day 0, day 10, day 12 and day 14 and day 17. They were weighed and placed onto a Mueller–Hinton agar streaked with Staphylococcus pseudintermedius. After the incubation time of 24 h at 37 the agar plates were photographed and the zone inhibition was measured. The biggest zone of inhibition on all sampling days was seen with the shampoos containing chlorhexidine 2% and 3% and the combination of shampoo and conditioner. Shampoos containing 4% chlorhexidine and 0.8% chlorhexidine inhibited bacterial growth to a lesser extent. No bacterial inhibition was seen with the control and the active ingredients benzoyl peroxide and ethyl lactate. Despite the concentration of the active ingredients, the composition of the shampoo plays an important part in its antibacterial activity. Based on this study the antibacterial activity of hair shafts was most pronounced after use of shampoos containing 2% and 3% chlorhexidine and the combination of shampoo and conditioner. Funding: Dermcare-Vet, Springwood, Queensland, Australia. Conflict of interest: During her dissertation, I Kloos was financially supported by Dermcare-Vet, Springwood, Queensland, Australia. In the last 5 years, R Mueller has obtained funding, lectured or consulted for Bayer Animal Health, Boehringer Ingelheim, Dechra Veterinary Products, Intervet, Merial, Novartis Animal Health, Pfizer Animal Health, Procter & Gamble, Royal Canin, Selectavet, and Virbac.

FC-22 Evaluation of masitinib (MasivetÒ) efficacy on feline interface lymphocytic dermatitis and mural folliculitis with apoptosis and hyperkeratosis: a case report X. LANGON Clinique Ve´te´rinaire du Bournet, Seyssins, France Feline thymoma-associated exfoliative dermatitis is a rare syndrome characterized by progressive erythema, scaling and alopecia that begins on the head and pinnae and then generalises. Histological features include interface lymphocytic mural folliculitis and dermatitis with hyperkeratosis, variable apoptosis, and often with loss of sebaceous glands. A 9 year-old male-neutered domestic short-haired cat was presented for an acute onset of exfoliative dermatitis. The clinical observations and the histopathological pattern revealing interface dermatitis and mural folliculitis, hyperkeratosis and apoptosis were suggestive of thymoma-associated exfoliative dermatitis. No thymoma was found by imagery examinations and no other abnormality was identified. Oral prednisolone (2 mg/kg/day) was given without clinical improvement. A protocol comprising masitinib (Masivet: AB Science, Paris, France) 10 mg/kg/day in combination with low 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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dose methylprednisolone (0.4 mg/kg/day) was given with successful outcome. Five months later, chemotherapy was stopped without lesion recurrence. Feline thymomaassociated exfoliative dermatitis is an immunologic reaction pattern. It is not associated with one single cause, but has been associated with thymoma. Masitinib is an oral tyrosine kinase inhibitor, specifically targeting the tyrosine kinase receptors c-Kit and platelet-derived growth factor receptors (PDGFR), as well as the intracellular kinases Lyn, Fyn, and Lck. In vitro studies


have shown that T-cell activation and proliferation is substantially reduced by the anti-proliferative action of masitinib due to PDGFR and Lck inhibition. This reported case demonstrates the efficacy of masitinib on this exfoliative dermatitis syndrome, either by direct influence on T cells, or on an unknown underlying causal factor. Funding: Self funded. Conflict of interest: None declared.

Free Communication Abstracts Session 5: OTITIS FC-23 Developing an objective clinical score for canine otitis externa E. BENSIGNOR*, C. SPERANZA-GASTAL , W. SEEWALD and T. NUTTALLà *Centre Hospitalier Veterinaire, Paris, France; Novartis Animal Health, Paris, France; àThe University of Liverpool School of Veterinary Science, Neston, Cheshire, UK The lack of an accepted clinical scoring system in canine otitis externa makes it difficult to compare clinical trials. The aim of this study was to develop a clinical score using 0–2, 0–3 or 0–5 assessments of erythema, oedema/ swelling, erosion/ulceration, exudates, and pain of the ear canals. Additional data included odour, owner pruritus scores, tympanic membrane condition, treatment outcome, and neutrophil, bacterial, and Malassezia counts. Twenty healthy dogs (40 ears) and 50 dogs with otitis (90 ears) were recruited. There were no significant differences between scores for the vertical and horizontal canals, with correlation coefficients > 0.9 for all parameters. Correlation coefficients for the 0–3 and 0–5 scales were also > 0.9 for all parameters. There were no differences between the 0–5 and 0–3 scales, but the 0–2 scale was more variable for most parameters. Pain and pruritus did not correlate well with clinical scores, and were associated with suppurative and erythroceruminous otitis, respectively. Neutrophil and microbial counts were highly variable and could not be used to generate satisfactory cut-off values to differentiate healthy and affected ears or determine the response to therapy. This study shows that a 0–3 scale for erythema, oedema/ swelling, erosion/ulceration, and exudate can be reliably used to assess the response to treatment in canine otitis externa, but other parameters are not useful. The total score for this scale was from 0 to 12; a score of ‡ 4 differentiated affected from healthy ears with 70% sensitivity and 100% specificity, and a score £ 2 was 99% sensitive for clinical success. Funding: This study was funded by Novartis Animal Health. Conflict of interest: E. Bensignor and T. J. Nutall have received other research, lecture and consultancy fees from Novartis Animal Health. C. Speranza-Gastal and W. Seewald are employees of Novartis Animal Health.

FC-24 Study to compare owner perception of their dog’s hearing and brainstem auditory-evoked response findings in 45 dogs C. L. BALL and S. PATERSON Dermatology Department, Rutland House Referrals, St. Helens, UK The study objective was to assess owner ability to determine the degree of hearing impairment in their pet dogs with otitis. Owners of 45 dogs with otitis completed a questionnaire designed to investigate their opinion of their pet’s hearing and the ability of the dog

to respond to common household noises. Brainstem auditory-evoked response (BAER) measurements were taken from the dogs under general anaesthesia using a standard technique (Nihon Kohden Neuropack l machine). BAER results were assessed for minimal hearing threshold (MHT) in decibels (dB) and categorised into five grades of impairment severity; grade zero (no impairment, MHT 25 dB or less), grade one (slight impairment, MHT 26–40 dB), grade two (moderate impairment, MHT 41–60 dB), grade three (severe impairment, MHT 60–80 dB), and grade four (profound impairment, MHT 81 dB or more). All owners correctly determined absence of hearing impairment in grade zero cases (13 of 13). Owners were unable to detect slight (11 of 11), moderate (two of two), or severe (one of one) unilateral hearing deficits. In nine cases of bilateral hearing loss where one ear was graded at slight impairment, only 33% of owners noticed hearing reduction. Owners were consistently able to detect bilateral hearing deficits of grade two and above in their dog (10 of 10). Dogs with mild or unilateral hearing deficits appear to adapt well within the home environment meaning that owners are frequently unaware of any hearing impairment. Owners were not able to accurately predict hearing deficits in their dogs until moderate bilateral hearing deficits were present. Funding: Self funded. Conflict of interest: None declared.

FC-25 In vitro antimicrobial activity of Pseudomonas aeruginosa isolated from dogs with otitis externa A. CORONA, A. VERCELLI and L. CORNEGLIANI Ambulatorio Veterinario Associato, Torino, Italy Pseudomonas aeruginosa is an aerobic, gram-negative bacillus and it is the most common organism associated with chronic otitis externa in dogs. These bacteria often display resistance to a great variety of antimicrobial drugs and induce difficult therapeutic management of otitis. The purpose of this study was to compare the in vitro activities of nine antibiotics against 100 strains of P. aeruginosa isolated from dogs with recurrent otitis externa, from 2009 to 2010, in a veterinary laboratory analysis in Italy. Swabs were obtained by insertion into external ear canals with bacterial infection and inoculated onto blood agar and Pseudomonas selective agar. One hundred P. aeruginosa strains grown in purity were used for this study. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method on Muller Hinton agar for amoxicillin-clavulanate, aztreonam, cefadroxil, cefalexin, enrofloxacin, gentamicin, marbofloxacin, piperacillin, and tobramycin. Pseudomonas aeruginosa was susceptible to piperacillin (82%), tobramycin and gentamicin (69% and 65% respectively), aztreonam (62%), and marbofloxacin (57%). It was resistant to cefalexin (97%), cefadroxil (95%), amoxicillin-clavulanate (93%), and enrofloxacin (51%). The data confirmed a high resistance of P. aeruginosa to antibiotics 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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registered for use in veterinary medicine and used very frequently in Italy. For this reason the veterinarians should always perform bacterial culture and antimicrobial susceptibility testing before starting therapy. Piperacillin is of great interest and should be considered as a new veterinary drug for antimicrobial treatment of Pseudomonas otitis. Funding: Self funded. Conflict of interest: None declared.

FC-26 Treatment of primary secretory otitis media (PSOM) with myringotomy in dogs with Chiari-like malformation: 21 dogs (28 bullae) with 24 month follow-up D. J. MARINO and C. A. LOUGHIN Long Island Veterinary Specialists, Plainview, NY, USA Primary secretory otitis media (PSOM) is a disease that commonly affects Cavalier King Charles spaniels (CKCS). Affected dogs have a mucus plug in the tympanic cavity that causes the tympanic membrane to bulge. Clinical signs include severe head or cervical pain and facial/ cervical pruritus resembling those of Chiari-like malformation (CLM). Traditionally, a diagnosis of PSOM is made by otoscopic examination or computed tomography (CT) scan; however, magnetic resonance imaging (MRI) has been found to be superior. The purpose of this study was to describe the effectiveness of myringotomy and flush /aspiration (MFA) in the treatment of PSOM in dogs with CLM. Magnetic resonance (MR) imaging was performed in all dogs at the time of diagnosis and every 6 months for 2 years. Of the 21 dogs, 12 (57.1%) had right bullae, two (9.6%) had left bullae, and seven (33.3%) had bilateral bullae with PSOM initially. Three (10.7%) bullae were found normal on MR 6 months after the initial MFA, four (14.3%) 12 months after initial treatment and second MFA, nine (32.1%) 18 months after initial treatment and third MFA, 8 (28.5%) 24 months after initial treatment and fourth MFA. Based on these findings, MFA is an effective treatment for PSOM in dogs with CLM; however, multiple MFA treatments are necessary in most cases. Funding: Self funded. Conflict of interest: None declared.

FC-27 Aural haematoma in dogs: evaluation of a simplified medical treatment using in situ methylprednisolone acetate V. BRUET, L. IMPARATO, A. ROUSSEL, A. JOURDAN and P. BOURDEAU Dermatology, Parasitology and Mycology Unit, Veterinary School, ONIRIS, Nantes, France Although aural haematoma is a common disorder, little information is available. The purpose of this prospective study was to evaluate the effectiveness of a simplified treatment for aural haematomas. Dogs included (19) were presented to the veterinary hospital in Oniris. On day 0 (D0), aural haematomas were drained, flushed and the site locally injected with methylprednisolone acetate (2 mg/kg), on sedated animals. On day 7 (D7), the haematoma was drained and flushed, if present. On day 15, in cases of relapse, surgery was performed. Statistical 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

analyses included Mann–Whitney and Fischer tests (P < 0.05) [reference population (RP) used = population from Oniris]. Mean age was 9.3 years; significantly older than RP. The three most represented breeds were golden retriever* (four), Labrador (four), and the French bulldog* (three) (*overrepresented compared to RP). Aural haematomas were principally unilateral (17 dogs) and localised to the right pinna (14 dogs). Identified underlying or associated conditions were: bacterial otitis (three), fungal otitis (eight), parasitic otitis (two), canine atopic dermatitis (four), trauma (three), flea bite hypersensitivity (three), sarcoptic mange (one), flea infestation (one), vasculopathy (one). Successful treatment was obtained in 18 cases [11 (D0), seven (D7)] but recurrence was observed for six dogs [15 days (one), 1 month (three), 2 months (one), 3 months (one)]. This study highlights the multifactorial origin of aural hematomas. This medical treatment seems as effective as surgical treatment (data from Oniris) with a 68% success rate. This non-traumatic approach could be proposed as a first treatment for aural haematomas. Funding: Self funded. Conflict of interest: None declared.

FC-28 Otitis externa prevention with OticArmor, a veterinary otic liquid bandage: a 14 day study in client-owned dogs A. E. SCHICK* and K. M. HOLD *Dermatology for Animals, Avondale, AZ, USA; Kayster Consulting, Belmont, CA, USA This study was conducted to determine the efficacy of OticArmor liquid bandage (AllAccem, Inc., San Carlos, CA, USA) to control the signs of canine otitis externa (OE). Six client-owned dogs with mild bilateral OE were included in this study. Inclusion criteria were as follows: clinical signs of OE including pruritus, erythema, or exudate, presence of intact tympanic membranes, and cytologic evidence of increased numbers of Malassezia and/or bacteria. After otoscopic examination, clinical signs (pruritus, erythema, and exudates) were scored from 0 to 3, with 0 = no signs, 1 = mild, 2 = moderate, and 3 = marked. Roll swabs were then taken from both ears and stained with Wright’s stain. Malassezia and bacterial counts were averaged for 10 high power fields (HPFs). Both ears were first cleaned with a standard flushing solution, then thoroughly rinsed with saline and dried. Subsequently, OticArmor was applied to the right ear, while the left ear was left as a control. Fourteen days following application, clinical scores and cytology were evaluated. Clinical scores and Malassezia/ bacterial counts were compared between the control ear and OticArmor treated ear. A significant reduction (P < 0.05 paired two-tailed t-test) in overall clinical scores of 37% was observed in the OticArmor treated ear compared to the control ear at day 14. Individual scores were reduced by 67% for pruritus, 22% for erythema, 43% for exudate, 24% for Malassezia, 56% for cocci, and 41% for rods. This study supports the use of OticArmor for controlling the signs of mild canine OE. Funding: AllAccem, Inc. Conflict of interest:A Schick and K Hold are AllAccem Inc. consultants.

Free Communication Abstracts Session 6: ALLERGY – PATHOGENESIS AND DIAGNOSIS FC-29 Immunohistochemical characterization of gelatinases and their inhibitor in feline eosinophilic dermatoses I. PORCELLATO, L. MECHELLI and C. BRACHELENTE Department of Biopathological Sciences and Hygiene of Animal and Alimentary Productions, Faculty of Veterinary Medicine, University of Perugia, Perugia, Italy Feline eosinophilic dermatoses include skin diseases characterized by eosinophil infiltration, flame figures, and/or foci of coagulative necrosis. The pathogenesis still remains unknown, though a hypersensitivity reaction is suspected. Gelatinase A [matrix metalloproteinase (MMP-2)] and gelatinase B (MMP-9) and their inhibitor TIMP-2 play an important role in collagen fibre degradation, but their involvement in physiological and pathophysiological events is still under debate. The aim of this study was to document the expression and the possible role of gelatinases and their inhibitor in feline eosinophilic dermatoses. Specimens from cats diagnosed with eosinophilic dermatoses were immunohistochemically tested for MMP-2, MMP-9, and TIMP-2. In 90% of cases, MMP-2 showed cytoplasmic reaction in plump spindle cells interpreted as fibroblasts and in variable numbers of inflammatory cells. In all cases, endothelial cells of small intralesional vessels showed a strong positivity. Unlike MMP-2, the expression of MMP-9 and TIMP-2 was seldom observed. The highest positivity for MMP-2 was detected in specimens with areas of coagulative necrosis, while in samples with numerous flame figures the positivity was lower. An imbalance between MMP-2 and TIMP-2 expression could be an indirect evidence for massive collagenolysis and explain the necrotic foci. Moreover, inflammatory cells showed a critical role in the production of MMP-2, therefore, contributing to the histopathological findings. MMP-9 showed positive reaction only in neutrophils, leading to the hypothesis of a minor involvement of this gelatinase in these lesions. To our knowledge, this is the first study investigating the presence and the role of gelatinases in the pathogenesis of feline eosinophilic dermatoses. Funding: Self funded. Conflict of interest: None declared.

FC-30 Development of a model of IL-31 induced pruritus in beagle dogs W. HUMPHREY, T. FLECK, M. ALEO, E. COSCARELLI, B. GALVAN, M. MULLINS, B. HUMMEL, J. SHELLY, J. MESSAMORE, S. MAHABIR, A. GONZALES and R. MCCALL Pfizer Animal Health, Kalamazoo, MI, USA Skin biopsies from pruritic and non-pruritic lesions in cases of human and mouse atopic dermatitis (AD) have revealed an up-regulation in interleukin-31 mRNA levels implicating a contributory role of this cytokine in the development of pruritus. Using recombinant canine IL31 (cIL-31 or IL-31), we have developed an anti-pruritic

screening model in dogs using exogenous IL-31 to induce episodes of pruritus in the presence/absence of test article treatments. In this model, dogs were acclimated to single housing runs equipped with ceiling cameras. Pruritic behaviors displayed by the dogs such as licking, scratching, head shaking, and body rubbing were observed by video surveillance (in real-time) over preset time intervals (usually £ 2 h) before and/or after intravenous injection of IL-31. A categorical scoring system (yes/no determination of displayed pruritus made over consecutive discrete 1 min intervals) was used to provide a measure of pruritic reactivity of each animal during these preset time intervals. IL-31 produced significant pruritus compared to mock protein or saline injections. The model was validated by demonstrating that administered prednisolone significantly decreased IL-31 induced pruritus. Additionally, the janus kinase inhibitor, oclacitinib, reduced IL-31 induced pruritus in the dog. These data indicate that IL-31 produces pruritus in the dog and that this can be used as a basis for a model to identify antipruritic compounds. Funding: Pfizer Animal Health. Conflict of interest: Oclacitinib is a Pfizer Animal Health investigational drug.

FC-31 Efficacy of an anti-IgE monoclonal antibody in an allergen induced type I hypersensitivity model S. DUNHAM, D. WHEELER, M. ALEO, M. DUPUIS, J. BEAM, R. WALTERS, W. DUNKLE, Y. ZHU, C. RUGG, L. BERGERON, C. STRIETZEL, B. LEONE, D. DUNYAK, W. HUMPHREY, J. MESSAMORE, J. SHELLY, P. AEED, P. CLARE, G. BAINBRIDGE, K. GREENWOOD, J. HARDHAM, R. MCCALL, A. GONZALES and O. MARTINON Pfizer Animal Health, Kalamazoo, MI, USA Atopic dermatitis (AD) is one of the most common pruritic skin diseases in dogs presenting to veterinarians. The disease appears to have a genetic component and is often correlated with exposure to environmental allergens followed by an increase in allergen-specific IgE. To inform the development of new therapeutic alternatives we have characterized fully caninized anti-IgE monoclonal antibodies (mAb) in a disease model of IgE-mediated type I hypersensitivity. Antibodies evaluated in these studies retain the necessary characteristics of an efficacious therapeutic anti-IgE mAb including: (i) the ability of the mAb to inhibit IgE from binding its high affinity receptor, FCR1, on mast cells and (ii) lack of ability to cross link IgE already bound to the surface of mast cells. To evaluate the in vivo efficacy of lead candidates we have sensitized beagles to house dust mite (HDM) allergens and assessed how subcutaneous administration of the mAb affects circulating free IgE levels and allergen-specific wheal and flare. The results of these studies demonstrated a dose dependant reduction in sensitivity to HDM extract in sensitized dogs for up to 5 weeks post mAb administration and that this wheal and flare reduction correlated with a reduction of free IgE. Taken together these findings 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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suggest that these mAbs have great promise to simultaneously evaluate the role of IgE in the pathogenesis of canine atopic dermatitis and to define the utility of these molecules in the treatment of atopic disease. Funding: Privately funded. Conflict of interest: All authors are current or past employees of Pfizer animal health.

FC-32 Positive and negative predictive values of a sarcoptes specific IgG ELISA in a tested population of pruritic dogs L. M. BUCKLEY, V. M. SCHMIDT, N. A. MCEWAN and T. NUTTALL The University of Liverpool School of Veterinary Science, Neston, Cheshire, UK Infestation with Sarcoptes scabiei var. canis is an important differential for canine atopic dermatitis. Sarcoptes specific IgG assays are widely used in the investigation of pruritus in dogs. However, sarcoptes mite and Dermatophagoides sp. house dust mite antigens can cross-react, and false positive reactions could be seen in atopic dogs. This study analysed the performance of a sarcoptes specific IgG ELISA in 470 pruritic dogs. The diagnosis of sarcoptic mange was based on the demonstration of mites, eggs or faecal pellets, and/or a complete response to selamectin (Stronghold; Pfizer Animal Health, Walton Oaks, UK) or moxidectin/imidacloprid (Advocate; Bayer Animal Health, Newbury, UK) given every 2 weeks for three applications. The diagnosis of atopic dermatitis was based on accepted criteria, and no response to 6–8 week parasite control (as above) and elimination diet trials. There were 16 dogs with sarcoptic mange, with mites, eggs, or faecal pellets detected in 11 of 16 (69%), and 15 of 16 (94%) of these dogs were ELISA positive. There were 454 atopic dogs, of which 31 of 454 (7%) were ELISA positive. The specificity of the test was 93% and the sensitivity was 94%. The negative predictive value was 99.8% and the positive predictive value was 33%. All the atopic dogs with a positive sarcoptes ELISA were sensitised to Dermatophagoides sp. In conclusion, a negative ELISA can be used to reliably eliminate sarcoptes infestation in pruritic dogs. However, positive ELISAs are of little diagnostic value where atopic dermatitis is the most common diagnosis in the tested population. Funding: Self funded. Conflict of interest: None declared.

FC-33 Development and validation of a scale for the rapid scoring of lesion severity in canine atopic dermatitis J. D. PLANT*, K. GORTEL , M. KOVALIKà, N. L. POLISSAR§ and M. B. NERADILEK§ *SkinVet Clinic, Lake Oswego, OR, USA; Lake Country Veterinary Specialist Hospital, Lake Country, BC, Canada; àDivision of Veterinary Clinical Sciences, Roslin, Midlothian, UK; §The Mountain-Whisper-Light Statistics, Seattle, WA, USA The Canine Atopic Dermatitis Extent and Severity Index (CADESI-03) requires 248 individual evaluations per 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

dog, limiting its widespread use. The objective of this study was to develop and validate a canine atopic dermatitis lesion scale that is practical for use in both clinical and research settings. Veterinary dermatology specialists were invited to participate through the vetderm listserve. Content validity was achieved by incorporating feedback from veterinary dermatologists on the first iteration of the Canine Atopic Dermatitis Lesion Index (CADLI). The final construct included a score (0– 5) of the extent and severity of two feature clusters in five body regions. Multiple assessments of atopic dogs by two observers were analyzed to determine construct validity, criterion validity, reliability, bias, and precision. Fiftyseven dogs were evaluated by 11 veterinary dermatologists in six countries. CADLI scores correlated with overall assessment (r = 0.50–0.71; P < 0.001) and pruritus severity (r = 0.45–0.53; P < 0.001), establishing construct validity. CADLI strongly correlated with CADESI-03 (r = 0.84–0.88; P < 0.001), establishing criterion validity. Intra-observer CADLI correlation (r = 0.98; P < 0.001) and inter-observer CADLI correlation (r = 0.89–0.91; P < 0.001) were excellent. BlandAltman precision ranged from 1.68 (intra-observer) to 4.69 units (inter-observer) on the 0–50 scale. The estimated thresholds for CADLI to predict overall assessment values were 5, 7, and 23. The mean time required to complete CADLI (115 s) and CADESI-03 (754 s) were significantly different (P < 0.001). The CADLI demonstrated suitable validity and offers a significant time-saving advantage over CADESI-03 for the lesion scoring of dogs with atopic dermatitis. Funding: Self funded. Conflict of interest: None declared.

FC-34 Responsiveness and validity of the SCORFAD, an extent and severity scale for feline hypersensitivity dermatitis J. STEFFAN*, T. OLIVRY , S. KINGà and W. SEEWALD* *Novartis Animal Health, Basel, Switzerland; Department of Clinical Sciences and Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA; àNovartis Animal Health US, Inc., Greensboro, NC, USA Hypersensitivity dermatitides (HD) are common in cats, causing pruritus and various patterns of skin lesions including head-and-neck excoriations, self-induced alopecia, eosinophilic plaques, and miliary dermatitis. The design of a novel scale (SCORFAD) to assess the value of different criteria used as outcome measures for treatment of feline HD was validated retrospectively with data generated in a multicentre clinical trial enrolling 100 cats with HD treated with placebo or two dosages of ciclosporin (Atopica for Cats; Novartis Animal Health, Basel, Switzerland). Construct of the scale was evaluated with the correlation between the scale and overall assessment of clinical response by investigators at study end. The final SCORFAD, as well as absolute or percentage reductions in SCORFAD from baseline values, were significantly correlated with investigator’s global assessment of improvement. Sensitivity to change was assessed by examining baseline and final SCORFAD following placebo or ciclosporin treatment. Threshold

Abstracts values of various outcome measures used to discriminate success from failure were also calculated: while 86% of cats with a 40–60% SCORFAD reduction from baseline values were considered to have had a successful response to treatment, 89% of cats with a SCORFAD reduction from baseline lower than 40% were considered to have a disease that had failed to respond to treatment. As the SCORFAD was found to exhibit satisfactory content,

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construct, criterion, and sensitivity to change, it is therefore proposed for use as a validated tool for the assessment of disease severity and response to therapeutic interventions in clinical trials for feline HD. Funding: The study was funded by Novartis Animal Health. Conflict of interest: Authors except for T. Olivry are employees of Novartis Animal Health.


Free Communication Abstracts Session 7: ALLERGY THERAPY – PART 1 FC-35 A multicentre clinical trial to evaluate the efficacy and field safety of oclacitinib S. COSGROVE, J. WREN, V. KING, D. WHEELER and M. STEGEMANN Pfizer Incorporated, New York, NY, USA The objective of this study was to demonstrate the efficacy and safety of a novel janus kinase inhibitor, oclacitinib, for control of atopic dermatitis (AD) in client-owned dogs. Twenty-three veterinary dermatologists enrolled 341 dogs, randomly allocated in a 1:1 ratio, to either oclacitinib (0.4 mg/kg twice daily orally for 14 days) or a matched placebo. Dogs had a documented history of chronic, nonseasonal AD. Minimum enrolment criteria included: an owner’s survey description of ‘moderate itching/dermatitis’ and a dermatologist’s score of 25 using the Canine Atopic Dermatitis Extent and Severity Index (CADESI02). Owner visual analog scale (VAS) pruritus measurements (100 mm scale) and dermatologist CADESI-02 scores were analyzed with linear mixed models for repeated measures with a covariate. Baseline VAS means were 74 (oclacitinib) and 75 (placebo). VAS least squares mean (lsmean) measurements for oclacitinib were significantly reduced compared with placebo (P < 0.0001) for all observation days; D1: 55 vs. 66; D2: 43 vs. 65; D7: 30 vs. 68 and D14: 24 vs. 70. Oclacitinib treatment produced a significant (P < 0.0001) decrease in the CADESI-02 lsmean scores relative to placebo. On D0, CADESI-02 mean scores for oclacitinib and placebo were 56 and 59. By D14, lsmean scores were 26 and 57 respectively. Diarrhoea and/or emesis were the most frequently reported abnormal health events. In this study, oclacitinib used at 0.4 mg/kg twice daily for 14 days was effective and safe for control of canine AD. Owners observed significant improvement in VAS compared to placebo from D1 to D14. The dermatologists D14 CADESI-02 scores mirrored these findings. Funding: Pfizer Incorporated. Conflict of interest: All authors are employees and shareholders of Pfizer Incorporated.

FC-36 Comparison of the janus kinase (JAK) inhibitor, oclacitinib, and prednisolone in canine models of pruritus T. FLECK, W. HUMPHREY, E. COSCARELLI, B. GALVAN, M. ALEO, A. GONZALES, J. SHELLY, S. MAHABIR and R. MCCALL Pfizer Animal Health, Kalamazoo, MI, USA Using recombinant canine IL-31 we have developed a model of pruritus in the dog. The objective of this study was to compare the efficacy and onset of action of the janus kinase (JAK) inhibitor oclacitinib and prednisolone in our IL-31 model and a flea allergic dog model. Oclacitinib pretreatment significantly reduced pruritus induced by IL-31 in a dose-related manner. Significant reductions in pruritus were noted at single oral doses ranging between 0.05 and 0.4 mg/kg. The maximum reduction in pruritus was 80% compared to placebo controls. The onset and duration of effect of an oral dose 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

of 0.4 mg/kg oclacitinib was determined. Oclacitinib significantly reduced pruritus when given 1, 6, 10, and 16 h prior to IL-31 injection but not 22 h prior to injection. Oclacitinib reduced pruritus within 1 h in a flea allergic dog model. Prednisolone (0.25 and 0.5 mg/kg) failed to significantly reduce pruritus when given 1 h prior to IL-31 injection, but when a 0.5 mg/kg dose was given 10 h prior to IL-31 challenge, a significant 37% reduction in pruritic response was observed. In a separate study 1 mg/kg oral prednisolone failed to significantly reduce pruritus when administered 2 and 6 h prior to IL31, reinforcing the time-dependency of the anti-pruritic response. Prednisolone (1 mg/kg) had an onset of antipruritic activity of 8–12 h in flea allergic dogs. These data indicate that the JAK inhibitor oclacitinib produced a greater suppression of pruritus and had a faster onset of action than prednisolone in canine models of pruritus. Funding: Pfizer Animal Health. Conflict of interest: Oclacitinib is a Pfizer Animal Health investigational drug.

FC-37 Oclacitinib for the treatment of pruritus and lesions associated with canine flea-allergic dermatitis. D. W. WHEELER*, J. CIVIL , M. PAYNEJOHNSON , M. R. STEGEMANN and S. B. COSGROVE* *Veterinary Medicine Research and Development, Pfizer Animal Health, Kalamazoo, MI, USA; Veterinary Medicine Research and Development, Pfizer Animal Health, Zaventem, Belgium A novel drug, oclacitinib, was tested for its ability to reduce clinical signs associated with canine flea-allergic dermatitis. Thirty-six flea allergic dogs were repeatedly infested with unfed fleas over a 29 day period. Dogs were treated orally twice daily with oclacitinib during the last 2 weeks of the study. Treatments included: T01: placebo, T02: 0.4 mg/kg oclacitinib, and T03: 0.8 mg/kg oclacitinib. Nocturnal pruritic behavior (licking, chewing, scratching, etc.) was videotaped twice before and three times during drug administration. Erythema and skin lesions (other than erythema) were evaluated using a 10 cm visual analog scale (VAS) prior to flea infestation, prior to oclacitinib administration, and following oclacitinib administration. Within 24 h dogs in T02 and T03 had > 58% decrease in pruritus compared to T01. After 2 weeks oclacitinib-treated dogs had > 76% decrease in pruritus. Dogs in T02 and T03 had significantly less pruritus than dogs in T01 (P £ 0.0120) for all 3 days post-treatment. Mean VAS scores for erythema and lesions prior to and after flea infestation were similar for all treatments. After treatment with oclacitinib, dogs in T02 and T03 had VAS scores that were 31.6% to 55.6% of T01 scores. VAS scores were significantly reduced for both erythema (P < 0.0001) and lesions (P £ 0.0025) compared to T01, whereas there were no significant differences between T02 and T03. Under the conditions of this study, oclacitinib administered at 0.4 mg/kg twice daily for 14 days, rapidly, safely, and significantly

Abstracts reduced pruritus, erythema, and lesions in flea-allergic dogs. Funding: Veterinary Medicine Research and Development, Pfizer Animal Health, Kalamazoo, Michigan, USA. Conflict of interest: All authors are current or former employees of Pfizer Animal Health. Oclacitinib is a Pfizer Animal Health investigational drug.

FC-38 Recombinant feline interferon-x as oral or subcutaneous treatment of canine atopic dermatitis P. LITZLBAUER, K. WEBER and R. S. MUELLER Centre for Clinical Veterinary Medicine, Ludwig Maximilian University, Munich, Germany Recently, recombinant feline interferon-x has been reported effective for short term treatment of canine atopic dermatitis. The aim of this study was to determine whether 26 atopic dogs develop antibodies against Virbagen omega (Virbac, Carros, France) during long-term therapy and to compare the efficacy of oral and subcutaneous (SC) administration. One group (n = 15) was treated with SC injections of Virbagen omega on days 0, 3, 7, 14, 21, 35, 56, and 90. The other group (n = 11) was treated orally once daily. Dosages (depending on weight) ranged from 1 to 4 million international units (IU) in the SC group and from 500 to 2000 IU in the oral group. On days 0 and 120 blood samples were collected, clinicians evaluated the canine atopic dermatitis extent and severity index (CADESI) and medication scores, and owners evaluated a pruritus score. For antibody detection an indirect ELISA, using Virbagen omega as antigen, was performed. There was no significant change in the mass extinction coefficient between samples from day 0 and 120 in both groups (SC group mean values 0.3605 and 0.3231, P = 0.33 and oral group 0.3933 and 0.4152, P = 0.68 respectively), indicating that antibody development could not be demonstrated. Comparison of pruritus scores, CADESI, and total scores between days 0 and 120 showed improvement in both groups; however, significant improvement could only be detected in the oral group with CADESI and total scores (61%, P = 0.0414 and 36%, P = 0.019, respectively). The results obtained with oral treatment should be further verified in larger, randomised, controlled clinical trials. Funding: This study was supported financially in part by a grant from the German Society of Veterinary Dermatology, Virbac provided part of the recombinant feline interferon-x (Virbagen omega). Conflict of interest: Virbac provided part of the recombinant feline interferon-x for this study. In the last 5 years, R. Mueller has obtained funding, lectured or consulted, among others, for Virbac.

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FC-39 Long-term maintenance therapy of canine atopic dermatitis with a 0.0584% hydrocortisone aceponate spray (Cortavance) used on two consecutive days each week A. M. LOURENÇO-MARTINS*, B. SAÕ-BRAZ*, V. M. SCHMIDT , C. A. REMEà and T. NUTTALL *Technical University of Lisbon Faculty of Veterinary Science, Lisbon, Portugal; The University of Liverpool School of Veterinary Science, Neston, Cheshire, UK; à Virbac Animal Health, Carros, France A 0.0584% hydrocortisone aceponate (HCA) spray (Cortavance; Virbac Animal Health, Carros, France) is efficacious and well tolerated in canine atopic dermatitis (cAD). The aim of this randomised, double-blinded pilot study was to evaluate the efficacy of a long term maintenance treatment regimen with HCA. Dogs with AD (n = 41; diagnosed according to accepted criteria) were treated once daily to remission (two sprays from 10 cm away to each 10 · 10 cm area of affected skin). Dogs were then randomly assigned to receive either the HCA (n = 21) spray or a placebo (n = 20) spray on two consecutive days each week (weekend therapy). All dogs were on appropriate flea control. Essential fatty acids, allergen specific immunotherapy, and emollient shampoos were permitted if in use before the trial, dogs were stable, and the regimen did not change. No topical or systemic anti-inflammatory or antimicrobial agents were permitted. Four dogs were lost to follow-up and four were withdrawn after receiving prohibited medication. Intention to treat analysis was used. At D0, all the dogs were in remission or had mild AD based on their CADESI-03 scores. The time to relapse requiring topical or systemic anti-inflammatory or antimicrobial therapy was significantly higher in the HCA group (median 115 days; SD 64; range 31–260) compared to the placebo group (median 33 days; SD 13.7; range 15–61) (P < 0.0001). No adverse events attributable to the HCA spray were seen. These results indicate long term therapy of cAD with an HCA spray administered on two consecutive days each week is effective and well-tolerated. Funding: This study was funded by Virbac Animal Health. Conflict of interest: C Reme is an employee of Virbac Animal Health. T Nuttall has received other research, lecture and consultancy fees from Virbac Animal Health.

FC-40 Efficacy of an anti-pruritic shampoo for dogs with mild to moderate allergic pruritus: a randomised, double-blind, placebo-controlled study J. SCHILLING and R. S. MUELLER Ludwig Maximilian University, Munich, Germany Shampoo therapy is frequently used in pruritic dogs; however, there are few double-blinded, placebo-controlled studies of this form of therapy. This randomised, double-blinded, placebo-controlled study evaluated the efficacy of a commercial medicated shampoo (DermaTopic: Almapharm, Kempten, Germany) containing chlorhexidine, lactoferrin, piroctone olamine, chitosan, and essential fatty acids in 27 dogs with mild to moderate 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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allergic pruritus without secondary skin infections. All dogs received shampoo therapy with either DermaTopic or a shampoo vehicle as placebo twice weekly for 4 weeks. The extent of pruritus was evaluated before the study and then on a daily basis by the owners using a visual analogue scale. Before beginning the treatment and after 4 weeks the skin lesions were evaluated by an experienced clinician using a validated lesion score (Canine Atopic Dermatitis Extent and Severity Index – CADESI). The pruritus was reduced significantly by both DermaTopic and placebo; however, there was no significant difference between both groups. There was no statistically significant difference in the CADESI scores


pre- and post-treatment in either group or between the two types of treatment. This study provides further evidence of the benefit of shampoo therapy for pruritic dogs. Funding: This study was financially supported by Almapharm. Conflict of interest: In the last 5 years, R Mueller has obtained funding, lectured or consulted for Bayer Animal Health, Boehringer Ingelheim, Dechra Veterinary Products, Intervet, Merial, Novartis Animal Health, Pfizer Animal Health, Procter & Gamble, Royal Canin, Selectavet, and Virbac.

Free Communication Abstracts Session 8: EQUINE 1 FC-41 Seasonal differences in cytokine expression in skin of Shetland ponies suffering from insect bite hypersensitivity

FC-42 Improved diagnosis of insect bite hypersensitivity in horses with allergens from indigenous Culicoides obsoletus

C. MEULENBROEKS*, D. M. W. ZAISS*, N. VAN DER MEIDE , M. M. SLOET VAN OLDRUITENBORGH-OOSTERBAANà, V. P. M. G. RUTTEN*,§ and T. WILLEMSE*,– *Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands; Department of Cell Biology & Immunology, Wageningen University, Wageningen, The Netherlands; àDepartment of Equine Sciences, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands; §Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa; –Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands Equine insect bite hypersensitivity (IBH) is an allergic skin disease of horses with close to worldwide distribution. IBH is an IgE-mediated, pruritic skin disorder caused primarily by Culicoides spp. Differences in skin pathology and immune responsiveness between IBH ‘on and off seasons’ may be indicative for processes underlying the disease. Skin biopsies and blood samples were taken from 17 ponies with IBH (age 3–20 years) and 14 ponies with no history of IBH (age 2–19 years), both in winter and in summer (no exposure vs. extensive exposure to C. obsoletus). Sampling in summer included biopsies of chronic lesional skin. Culicoides obsoletus extract-specific serum IgE levels were determined using ELISA. Skin mRNA expression of IL4, IL10, IL13, CD3f, FoxP3, IFNc and 18s was measured by qRTPCR. IgE serum levels in summer were significantly higher in ponies with IBH than in healthy controls. Expression of CD3f, IFNc, IL4, and IL13 was overall upregulated in healthy, non-lesional, and chronic lesional skin in summer. Only IL13 expression was significantly lower in summer if chronic lesional skin and healthy controls were compared. IL10 and FoxP3 could not be detected. Results indicate that in summer, skin immune responsiveness is activated in ponies with and without IBH as compared to winter, potentially due to exposure to Culicoides and other insects. The lower expression of IL13 in chronic lesional skin suggests a downregulation of Th2 immune responsiveness in the chronic phase of IBH. Funding: Dutch Technology foundation (STW-NWO); Dutch Federation of horse breeding (Vereniging Koepel Fokkerij); ALK-Abello´/Artu Biologicals. Conflict of interest: None declared.

N. M. A. VAN DER MEIDE*, C. VAN ALTENA*, A. SCHURINK , B. WAGNERà, W. LEIBOLD§, J. ROHWER§, C. MEULENBROEKS–, M. M. SLOET VAN OLDRUITENBORGH-OOSTERBAAN**, H. F. J. SAVELKOUL* and E. TIJHAAR* *Cell Biology and Immunology Group, Wageningen University, Wageningen, The Netherlands; Animal Breeding and Genomics Centre, Wageningen University, Wageningen, The Netherlands; àPopulation Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY, USA; §Immunology Unit, University of Veterinary Medicine Hannover, Hannover, Germany; –Department of Infectious diseases and Immunology, Faculty of Veterinary Medicine Utrecht, Utrecht, The Netherlands; **Department of Equine Sciences, Faculty of Veterinary Medicine Utrecht, Utrecht, The Netherlands Insect Bite Hypersensitivity (IBH) is a common type I skin allergy in horses, often caused by the bites of Culicoides species. Currently diagnostic tests are not very reliable and there is no treatment available. Culicoides obsoletus was identified as the major species responsible for IBH in The Netherlands. In the literature, C. sonorensis (North America) and C. nubeculosus (Europe) are often chosen for the identification of allergens, because these species are readily available from laboratory bred colonies. Culicoides obsoletus insects still need to be collected from the wild. The objective of this study was to evaluate if the use of C. obsoletus would improve the diagnosis of IBH for horses in The Netherlands. Whole body extracts of C. obsoletus, C. sonorensis, and C. nubeculosus were compared in different in vivo and in vitro diagnostic tests using clinically well-defined IBH affected (n = 10) and healthy control horses (n = 10). Culicoides obsoletus whole body extracts induced larger skin wheals when injected intradermally in IBH affected horses compared to C. sonorensis and C. nubeculosus extracts. Compared to the other extracts, C. obsoletus extract also showed better binding to IgE (ELISA) in serum and induced higher histamine release (functional test) in whole blood from IBH affected horses. Only with C. obsoletus extract could IBH affected horses be well discriminated from healthy control horses in all tests. In conclusion, C. obsoletus considerably improves diagnosis of IBH. At the moment a number of candidate C. obsoletus allergens are being produced as recombinant proteins and evaluated in diagnostic tests. Funding: Dutch Technology Foundation STW (STWNWO); Dutch Federation of horse breeding (Koepel Fokkerij); ALK-Abello´/Artu Biologicals. Conflict of interest: None declared.


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FC-43 A retrospective study into the causes and incidence of recurrent urticaria in the horse S. PATERSON Rutland House Veterinary Hospital, St. Helens, UK The case records and intradermal allergen test results were assessed from 20 horses presenting between January 2008 and August 2011. In all cases the signs of urticaria were recurrent, having occurred in previous years at a similar time. A diagnosis of seasonal urticaria through aeroallergens exposure was made by exclusion of other causes including insect reactions, seasonal food substances, or physical urticaria. Intradermal allergy testing was undertaken using 46 individual allergens. The time period for active disease was recorded for each horse. Seventy-five percent of horses were thoroughbreds or thoroughbred crosses. The mean age of onset of the urticaria was 10.1 years with a range of 4–15 years. There was marked clustering of results, with the majority of horses showing signs of urticaria in the autumn and early winter. Seventy-five percent of all horses had active disease in November followed by December (60%), October (55%), August (50%), and September (45%). The months with the largest numbers of reactions were November (46), January (45), December and October (39). Within each month the percentage of reactions to each group of allergens was recorded. In November 32.6% of reactions were to mite extracts, 26.1% weeds, 23.9% moulds. These results tend to suggest that recurrent urticaria in the horse is more common in the autumn and early winter in the UK than in the summer. Aeroallergens of importance as a cause for recurrent urticaria in the horse may include moulds, mite extracts, and weed pollens. Funding: Self funded. Conflict of interest: None declared.

FC-44 A prospective evaluation of clinical and immunological responses to allergen-specific immunotherapy in horses with atopic dermatitis N. E. RADWANSKI*, D. O. MORRIS , R. C. BOSTON , R. CERUNDOLOà and K. W. LEE§ *Mid-Atlantic Equine Medical Center, Ringoes, NJ, USA; School of Veterinary Medicine University of Pennsylvania, Philadelphia, PA, USA; àDick White Referrals Veterinary Specialist Centre, Suffolk, UK; § Greer Laboratories, Inc, Lenoir, NC, USA The purpose of this study was to determine the effects of allergen-specific immunotherapy (ASIT) over time in atopic horses. Nineteen horses with a clinical diagnosis of atopic dermatitis (AD) were enrolled, and received ASIT for up to 24 months. Horses were randomised to one of three treatment groups: ASIT based upon intradermal test (IDT) results (n = 7); allergen-specific IgE results by ELISA [Greer Laboratories, Lenoir, NC, USA (n = 6)]; or a composite of IDT and ELISA results (n = 6).


Serum concentrations of allergen-specific IgE and IgG were measured for 60 allergens at initial evaluation (time 0) and every 4 months thereafter. Horse owners recorded visual analog scale (VAS) scores at times 0, 12 months, and 24 months. Eleven horses completed the 24-month evaluation. Logistical regression analysis showed the mean VAS score to decrease by 1.2 units [95% CI: 1.28, 1.14; (P < 0.0001)] per each 12-month period of ASIT therapy. There were no significant differences in VAS scores according to seasonality of symptoms. There was excellent agreement between allergen-specific IgE concentrations (time 0) and both immediate and delayed IDT results (P < 0.00001), and between immediate IDT and IgG results (P = 0.003). Specific concentration of serum IgE and IgG decreased significantly (P < 0.05) for 13% and 38% of allergens, respectively, that were included in ASIT. Results suggest that ASIT provides significant clinical benefit, and supports roles for both allergen-specific IgE and IgG in the pathogenesis of equine AD. These data also suggest that clinical benefit from ASIT may result from reduction of allergen-specific IgE and IgG concentrations in serum. Funding: Study funded by Greer Laboratories, Inc. Conflict of interest: K. Lee is currently employed by Greer Laboratories, Inc.

FC-45 Skin thinning effects of topically administered glucocorticoids in dogs and horses M. KIETZMANN and S. KEMMET Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany Skin thinning effects of four different glucocorticoids were studied in dogs and horses. In dogs, hydrocortisone, triamcinolone acetonide, betamethasone valerate, and clobetasol propionate were administered once daily onto the outer ear skin over 12 consecutive days. Horses were treated topically (thoracic skin) with hydrocortisone, diflorasone diacetate, mometasone furoate, and clobetasol propionate once daily over 12 days. The skin thickness was measured by means of the CT (compression, thickness) method which allows the simultaneous determination of the skin compressibility and the skin fold thickness. In dogs, the skin thinning effect was most prominent for clobetasol propionate, followed by betamethasone valerate and triamcinolone acetonide. In horses, the skin thinning effect of diflorasone diacetate, mometasone furoate, and clobetasol propionate was quite similar. Hydrocortisone showed only a weak skin thinning effect in dogs and horses. The study confirms that skin thinning effects have to be considered as clinically relevant side effects of topically administered glucocorticoids in dogs and horses. Species differences as well as the potency ranking of glucocorticoids were demonstrated. Funding: Self funded. Conflict of interest: None declared.

Abstracts

FC-46 Results of 7300 thyroid hormone profiles in horses: 2002–2008 H. SCHOTT II, P. SCHENCK, K. REFSAL and R. NACHREINER Michigan State University, East Lansing, MI, USA Hypothyroidism remains a poorly characterized disorder in horses. Due to lack of assays for equine thyrotropin (TSH), hypothyroidism in horses is supported by low values for circulating thyroid hormone concentrations. This study examined results of 7300 equine thyroid hormone profiles submitted from 2002 to 2008. Total thyroxine (TT4), free thyroxine (FT4), total triodothyronine (TT3), and free triodothyronine (FT3) were measured by validated radioimmunoassays. TT4 was within reference range (RR) values in 62%, low in 18%, and elevated in 20% of samples. FT4 was within RR values in

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83%, low in 16%, and elevated in 1% of samples. TT3 was within RR values in 58%, low in 41%, and elevated in 1% of samples. FT3 was within RR values in 62%, low in 29%, and elevated in 9% of samples. There were no effects of age or sex on the percentage of either increased or decreased thyroid hormone concentrations. Low values for one or more thyroid hormones in 16–41% of samples provided support for a diagnosis of hypothyroidism. However, other factors may have contributed to low values of circulating thyroid hormone concentrations. In conclusion, accurate interpretation of circulating thyroid hormone concentrations in horses requires complete knowledge of the patient’s condition. Further, limitations of circulating thyroid hormone concentrations to assess thyroid gland function argue for the need of an assay to measure endogenous TSH in horses. Funding: Self funded. Conflict of interest: None declared.


Free Communication Abstracts Session 9: ALLERGY THERAPY – PART 2 FC-47 Pharmacokinetics of ciclosporin after intravenous and subcutaneous administration in cats S. DIAZ*, H. P. LEFEBVRE , B. MELDRUM* and D. PANCIERA* *VA-MD Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA; Clinical Research Unit & Physiology, National Veterinary School of Toulouse, Toulouse, France Ciclosporin A (CsA) has been classically used as an immunosuppressive agent in kidney transplantation in cats. More recently, CsA has been reported to be effective for the management of several feline dermatoses. Oral CsA absorption, however, is relatively limited and varies substantially between individuals. Subcutaneous (SQ) administration of CsA could be an alternative route for administration. The objectives of this study were to characterise the pharmacokinetic profile of CsA after intravenous (IV) and SQ administration of Sandimmune 50 mg/mL injectable solution (Novartis Pharmaceuticals, NJ, USA) in cats. Five healthy adult cats received a single IV bolus (2.5 mg/kg) of CsA. After 2 weeks, they received CsA (2.5 mg/kg) SQ every 48 h for 14 days. Repeated blood samples were obtained after IV bolus and the last SQ dose. Blood CsA was measured by high-performance liquid chromatography-tandem mass spectrometry. Non compartmental pharmacokinetic (PK) analysis was performed. No adverse effects were observed in any cat. Following IV bolus, clearance, steady state, volume of distribution, mean residence time, and elimination half-life were 0.35 ± 0.10 (mean ± SD) L/h/kg, 3.51 ± 0.62 L/kg, 10.6 ± 2.9 and 10.5 ± 2.5 h, respectively. Following SQ administration, the maximal concentration (461 ± 116 ng/mL) was observed at 1.9 ± 1.7 h (range: 0.25–4 h). The area under the curve from time 0 to infinity, an indicator of exposure, showed a between-individual coefficient of variation of 25%. The estimated SQ bioavailability was 95 ± 7.8%. The IV PK parameters are similar to those previously reported. Subcutaneous CsA bioavailability is very good. The SQ route, therefore, could represent a promising route of administration in cats. Funding: Winn Feline Foundation and the VA-MD Regional College of Veterinary Medicine. Conflict of interest: None declared.

FC-48 A randomized, double-blinded, placebo-controlled, multi-centre, confirmatory efficacy field trial for the evaluation of ciclosporin at a target dose of 7 mg/kg administered orally for 6 weeks in the control of feline hypersensitivity dermatitis in cats S. KING, E. S. ROBERTS and L. M. ROYCROFT Novartis Animal Health United States, Inc., Greensboro, NC, USA The field efficacy and safety of ciclosporin (ATOPICA for Cats; Novartis Animal Health US, Inc., Greensboro, 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

NC, USA) was evaluated for the control of feline hypersensitivity dermatitis (FHD) characterised by head and neck excoriations, self-induced alopecia, eosinophilic plaques and miliary dermatitis. Cats ‡ 6 months old, at least 1.4 kg body weight, identified with FHD along with non seasonal pruritus were eligible for inclusion. Twohundred and seventeen cats received ciclosporin (7.0 mg/ kg) or placebo once daily for 6 weeks. Lesion severity and extent, pruritus and overall clinical improvement were evaluated. Clinical safety was assessed by reviewing adverse event (AE) reports, clinical pathology, and body weight. Treatment with ciclosporin significantly reduced mean total lesion scores (65.1% vs. 9.2%), resulted in a greater percent of cats classified as a success by the owner (78.6% vs. 26.2%) and showed superior investigator overall clinical improvement scores, a reduction in extent of lesions and a mean improvement in owner assessment of pruritus. The mean value for investigator assessment of pruritus was numerically lower in the ciclosporin treated group. Mild, transient digestive tract disorders (vomiting, diarrhoea, hypersalivation) were the most frequently reported AEs. Ten ciclosporin treated cats were positive for Toxoplasma in the absence of clinical signs of disease. Cats treated with ciclosporin tended to lose weight. Evaluation of clinical pathology indices showed no clinically relevant abnormalities associated with ciclosporin treatment. Administration of ciclosporin at a target dose of 7 mg/kg once daily for 6 weeks was effective and appeared to be well tolerated in the control of FHD. Funding: The study was funded by Novartis Animal Health. Conflict of interest: Authors are employees of Novartis Animal Health.

FC-49 Dose tapering regimen in cats with hypersensitivity dermatitis administered ciclosporin at a target dose of 7 mg/kg orally S. KING*, J. STEFFAN , W. SEEWALD and L. M. ROYCROFT* *Novartis Animal Health United States, Inc., Greensboro, NC, USA; Novartis Animal Health, Basel, Switzerland The ability to taper the frequency of ciclosporin (Atopica for Cats; Novartis Animal Health US, Inc Greensboro, NC, USA) administration in cats identified with hypersensitivity dermatitis was evaluated in two open field studies. Upon inclusion, all cats received ciclosporin at 7 mg/kg once daily for 4 weeks which could be subsequently tapered at 4-week intervals to every other day (EOD) then twice weekly (TW) according to the investigator assessed overall clinical response. If the response was excellent/good, the dose frequency was tapered, if moderate, the dose frequency was maintained and, if poor/unchanged, the cat could be removed from study. In Study 1, the dose frequency was tapered to EOD on day 28 in 70% of the 71 evaluable cats. On day 56, 57% of 65 evaluable cats had the frequency of administration reduced to TW while 15% of

Abstracts cats remained on EOD and 22% required daily administration. In Study 2, the dose frequency was tapered to EOD on day 28 in 80.1% of the 136 evaluable cats, while on day 56, 63% of the 100 evaluable cats were dosed TW, 22% EOD and 15% once daily. A small proportion of cats in both studies had the dose frequency reduced but subsequently increased due to poor response. Eight (8/ 88) and 7.8 (15/191) percent of enrolled cats in Studies 1 and 2, respectively, were withdrawn for lack of efficacy. Tapering the dose frequency was achieved without a clinically relevant loss of response in the majority of cats administered ciclosporin at 7 mg/kg. Funding: The study was funded by Novartis Animal Health. Conflict of interest: Authors are employees of Novartis Animal Health.

FC-50 A prospective, blinded, controlled pilot study to evaluate the effects of ciclosporin (AtopicaÒ) on skin barrier function in canine atopic dermatitis by measurement of transepidermal water loss R. MARSELLA, D. FELD and K. AHRENS College of Veterinary Medicine, University of Florida, Gainesville, FL, USA Skin barrier dysfunction plays an important role in canine and human atopic dermatitis (AD). In dogs skin barrier impairment may be secondary to inflammation and it would be expected to improve once inflammation is decreased. Since glucocorticoids induce skin atrophy, ciclosporin may represent a better alternative to improve skin barrier in AD. The purpose of this study was to investigate the effects of glucocorticoids and ciclosporin on skin barrier function by measuring transepidermal water loss (TEWL) before and after 4 weeks of therapy. Twenty-seven privately owned dogs diagnosed with AD were randomly allocated in a double-blinded study to receive either prednisolone (0.5 mg/kg daily for the first week, then every other day for the next 3 weeks) or ciclosporin (5 mg/kg once daily for 4 weeks). Severity of clinical signs was scored using the Canine Atopic Dermatitis and Extent Severity Index (CADESI). Skin barrier was assessed by measuring TEWL from pinna, axilla, and inguinal area. Measurements were taken in triplicates using a closed chamber device. For CADESI, analysis of variance (ANOVA) only showed a significant effect of time (P = 0.03; end < beginning). No significant effect of time or group was found for TEWL. The only significance for TEWL was found for body region (P < 0.0001; axilla > inguinal > pinna). Larger studies may be needed to conclusively address the role played by inflammation on skin barrier dysfunction in dogs. Funding: Self funded. Conflict of interest: None declared.

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FC-51 Treatment with ciclosporin A has minimal impact on calcium metabolism in dogs with atopic dermatitis M. KOVALIK*, R. J. MELLANBY*, , H. EVANSà, J. BERRY§, A. H. VAN DEN BROEK*, and K. L. THODAY*, *The University of Edinburgh, Dermatology Unit, Division of Veterinary Clinical Sciences, The Hospital for Small Animals, Edinburgh, UK; The Roslin Institute, Edinburgh, UK; àCambridge Specialist Laboratory Services, Cambridge, UK; §Vitamin D Research Group, Medicine, Manchester Royal Infirmary, Manchester, UK Ciclosporin A (CsA) is widely used in the management of a range of canine dermatological conditions including atopic dermatitis (AD). In humans, CsA therapy has been linked to disturbances in calcium metabolism and skeletal disorders yet, the impact of CsA on calcium homeostasis in dogs is poorly understood. This study reports the concentrations of 12 h fasting serum calcium, phosphate, albumin, creatinine, 25 hydroxyvitamin D, 1,25 dihydroxyvitamin D and plasma concentrations of ionised calcium and parathyroid hormone (PTH), together with the urinary fractional excretion of calcium and phosphate. These parameters were evaluated in 16 dogs with spontaneous AD before and after a 6-week course of once daily CsA at the licensed dose of 5 mg/kg. The degree of skin lesions and pruritus were respectively assessed using the canine AD extent and severity index (CADESI)-03 and the Edinburgh pruritus scale (EPS) prior to and at the end of the study. The CADESI-03 and the EPS scale scores had decreased in all dogs at the end of the study. Plasma PTH concentrations were significantly increased (P = 0.02) following CsA treatment whereas all other biochemical parameters were not significantly different from the initial values. There was no correlation between post-treatment PTH and CsA trough concentrations (r = )0.08, P = 0.78). The increase in PTH was generally mild and the proportion of dogs with PTH concentrations above the reference range was not significantly different following treatment. This study indicates that CsA has minimal impact on calcium metabolism in dogs with AD when used at therapeutically effective doses. Funding: The Petplan Charitable Trust. Conflict of interest: None declared.

FC-52 Pharmacokinetic and toxicity of methotrexate in the dog and its efficacy in canine atopic dermatitis D. PIN, I. FOUREL, C. LUSSIEZ, C. GUINET, P. BERNY and E. VIDEMONT Vetagro Sup Campus Ve´te´rinaire de Lyon, Marcy l’Etoile, France The study aims were to determine the pharmacokinetic and toxicity of methotrexate (MTX) after oral administration in healthy dogs and, to evaluate its efficacy in canine atopic dermatitis (AD) in an open prospective study. For the pharmacokinetic study, blood samples were collected from three healthy beagles at 0.5, 1, 2, 3, 4, 5, 7.5, 10, 13, 17, and 24 h after administering 5 mg of 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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MTX. Six healthy beagle dogs were given MTX once weekly at 2.5, 5, 7.5, 10, and 12.5 mg during 1, 4, 3, 6, and 7 weeks, respectively, to evaluate toxicity. The maximum serum concentration (Cmax) varied among dogs (93.1–184.1 ng/mL) and the time Cmax was achieved (Tmax) varied between 0.5 and 2 h. A rapid decrease in serum concentration was observed until 7.5 h after administration, followed by a very slow decrease until 24 h. Cmax and elimination rate increased as the MTX dose increased. No clinically relevant abnormalities of the tested laboratory parameters were noticed. Ten client-owned dogs with perennial AD were given 2.5 mg of MTX once weekly during 5 months and 5,


7.5, or 10 mg once weekly where inadequate response to therapy was noted. Skin lesions and pruritus were assessed using the CADESI-02 and a 10-point visual analogue scale (PVAS). Nausea and/or vomiting, 48 h after MTX administration, was observed in five dogs. CADESI scores (P < 0.024) and pruritus values (P < 0.009) were significantly lower after 5 months of therapy compared to pre-treatment. The study results suggest that MTX is safe and effective for the treatment of canine AD. Funding: Self funded. Conflict of interest: None declared.

Free Communication Abstracts Session 10: ACVD RESIDENTS FC-53 Pain due to otitis externa in 36 dogs. Part 1: Owner perception and clinician’s assessment of pain R. MOUNT, A. SCHICK and T. LEWIS Dermatology for Animals, Phoenix, AZ, USA Canine otitis externa is a common sequela to numerous primary dermatoses including atopic dermatitis and adverse food reactions. Ear pain is commonly reported in people with otitis. To the author’s knowledge, the assessment of otic pain has not been reported in the veterinary literature. The objective of this pilot study was to determine if owners subjectively perceive pain in their dogs with active otitis externa. A secondary objective was to compare the investigator’s objective measurement of pain to the owner’s assessment. Thirty six dogs that presented to a private practice dermatology clinic with active otitis externa were evaluated by a single investigator. Prior to clinical assessment, the owners were asked to rank their dog’s pain from 0 to 10 using a Visual Analog Scale. The investigator was blinded to the owner’s assessment. The investigator used a modified version of the Colorado State University acute pain scale to objectively quantify the pain level in each dog. Multiple parameters, including reaction to palpation of canals and guarding of ears, were used to evaluate pain. Owners subjectively perceived pain in 28 of 36 (77.8%) cases. The investigator objectively scored pain in 31 of 36 (86.1%) dogs. A pooled two-sample t-test showed no significant difference (P = 0.78) between owners’ subjective perception of pain and the investigator’s pain score. Our results provide evidence that pain is present in most cases of canine otitis and pain management should be considered as part of the management of canine otitis externa. Funding: Self funded. Conflict of interest: None declared.

FC-54 Pain due to otitis externa in 36 dogs. Part 2: Clinician’s assessment of pain and assessment of clinical severity of otitis externa R. MOUNT, A. SCHICK and T. LEWIS Dermatology for Animals, Phoenix, AZ, USA In part one of this study a correlation between owner’s assessment and investigator’s pain scores was established in 36 dogs. The objective of this pilot study was to correlate the severity of canine otitis externa (COE) with the level of pain in these 36 dogs. In order to objectively determine severity of disease a clinical scale was developed. The scale was modeled after the Canine Atopic Dermatitis Extent and Severity Index (CADESI). To develop the scale, 40 diplomates of the American College of Veterinary Dermatology were surveyed and asked to rank eight clinical signs from most to least important in their assessment of severity of COE. The five clinical signs with the highest average rank, including stenosis of canal, firmness of canal, erythema of canal and pinna, presence of purulent exudate and presence of ulcerations/

erosions, were used for the scale. Each clinical sign was ranked from 0 (absent) to 3 (severe) with a maximum total score of 15. Based on the total score the severity of disease was classified as mild (1–5), moderate (6–10) or severe (11–15). Using this scale the original 36 dogs were given a clinical severity score. The modified Colorado State University acute pain scale scores from study one were used for comparison. Pearson’s correlation test did not show a strong linear relationship (R = 0.652; P < 0.001) between level of pain and level of severity of clinical disease. The lack of a strong correlation possibly resulted from the small sample size and the relatively small number of dogs with severe disease. Funding: Self funded. Conflict of interest: None declared.

FC-55 Comparison of once daily vs. twice weekly terbinafine administration for the treatment of canine Malassezia dermatitis – a pilot study D. BERGER*, T. LEWIS*, A. SCHICK* and R. STONE *Dermatology for Animals, Gilbert, AZ, USA; Department of Industrial and Manufacturing Systems Engineering, Iowa State University, Ames, IA, USA This randomized, single-blinded clinical trial was conducted to evaluate the clinical efficacy of twice weekly vs. once daily oral administration of terbinafine for canine Malassezia dermatitis. Twenty client-owned dogs with Malassezia dermatitis were included. Dogs were randomly assigned to receive terbinafine at 30 mg/kg either once daily for 21 days (n = 10) or once daily on two consecutive days per week for six doses (n = 10). At day 0 (visit 1) and day 21 (visit 2), a mean yeast count (MYC) was calculated from eight sites diagnosed with Malassezia colonization via adhesive tape-strip cytology. Clinical lesion scores (CLS) were assigned by one of the investigators to the same eight sites on days 0 and 21. Additionally, owners assessed pruritus using a visual analog scale (VAS) at both visits. Treatment groups were compared using repeated measures analyses of variance and paired t-test. Both treatment groups showed a significant (P < 0.01) reduction in MYC, CLS, and VAS, although VAS did not reach clinical significance. There was no significant difference between treatment groups regarding reduction in MYC (P = 0.34) and CLS (P = 0.89). Pruritus was significantly decreased in the twice-weekly treatment group compared to the daily group (P = 0.047). Seven of 20 dogs had either an abnormal laboratory or owner reported adverse event during treatment. There was no difference between treatment groups regarding owner reported adverse events. One dog in the daily treatment group had elevated alanine aminotransferase level. These results suggest that twice-weekly terbinafine administration could be an alternate treatment regimen for Malassezia dermatitis in dogs. Funding: American College of Veterinary Dermatology resident research grant. Conflict of interest: None declared. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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FC-56 The presence of intraepithelial lymphocytes in healthy, haired skin of dogs and alpacas M. CLARK, J. PETERS-KENNEDY, D. SCOTT, L. DONG and S. MCDONOUGH Cornell University, Ithaca, NY, USA A small population of resident T-lymphocytes is present in the healthy epidermis of the skin from humans, mice, cattle, and sheep. Resident lymphocytes were not found in the epidermis or adnexal epithelia of healthy skin from cats and horses. One 6 mm biopsy specimen from normal skin of the dorsal-lateral thorax from 29 dogs was examined histologically and immunohistochemically for the presence of CD3+ cells (T- lymphocytes) and Pax5+ cells (B-lymphocytes), in the epidermis and adnexal epithelia. All examinations were negative. It appears that lymphocytes rarely occur in the epidermis and adnexal epithelia of normal dog skin. Hence, the presence of lymphocytes in these structures in skin biopsy specimens from dogs should be considered abnormal. In a separate study, one 6 mm biopsy specimen from normal skin of the dorsal-lateral thorax from 32 alpacas was examined histologically and immunohistochemically for the presence of CD3+ cells, and CD79a+ cells (B-lymphocytes) in the epidermis and adnexal epithelia. CD79a lymphocytes were not found in samples from normal alpaca skin. CD3+ lymphocytes were found in 32/32 of the samples examined. Total numbers of CD3+ lymphocytes in each sample ranged from one to 53 with varying numbers present in the epidermis, follicular epithelium, sebaceous gland epithelium and epithelium of epitrichial sweat glands. Hence, the presence of lymphocytes in these structures in skin biopsy specimens from alpacas should not be considered abnormal. Funding: Self funded. Conflict of interest: None declared.


FC-57 Case-control study of Staphylococcus lugdunensis infection isolates from small companion animals K. A. ROOK*, D. C. BROWN*, S. C. RANKIN and D. O. MORRIS* *Department of Clinical Studies, School of Veterinary Medicine University of Pennsylvania, Philadelphia, PA, USA; Department of Pathobiology, School of Veterinary Medicine University of Pennsylvania, Philadelphia, PA, USA Coagulase-negative Staphylococcus lugdunesis has recently been shown to cause invasive infections of people, which are similar in pathogenic effect to those caused by S. aureus. Because little is known about the pathogenicity of S. lugdunensis in companion animals, a retrospective evaluation was performed. Thirty-three cases of S. lugdunensis (25 dogs, six cats, two small mammals) were identified between January, 2003 and August, 2011. Two S. pseudintermedius controls, which were identified by the microbiology laboratory immediately before and after each S. lugdunensis, were host-species matched to each case. A case-control analysis was then used to compare potential risk factors for infection, body sites affected, and whether cases/controls had been treated with antimicrobial drugs based upon susceptibility test results. In univariate analysis, S. pseudintermedius isolation was significantly associated with skin infections (P < 0.0001), while S. lugdunensis isolation was associated with the respiratory tract (P = 0.03) and other deep tissues (P = 0.005). Cases were less likely than controls to have been treated based upon antimicrobial susceptibility test results (P = 0.02). A conditional logistic regression analysis showed isolation of S. lugdunensis to be associated with intact male status [Odds Ratio (OR) = 7.39 (95% Confidence Interval (CI): 1.28, 42.48), P = 0.025]; recent (within 30 days prior to bacterial culture) corticosteroid administration [OR = 9.27 (95% CI: 1.76, 48.74), P = 0.009]; and in-patient status [OR = 7.37 (95% CI: 1.86, 29.15), P = 0.004]. These results suggest that S. lugdunesis may cause invasive infections in companion animals, which should be treated with antimicrobials based upon susceptibility tests when available. Funding: University of Pennsylvania. Conflict of interest: None declared.

Free Communication Abstracts Session 11: ACVD RESIDENTS FC-58 Evaluation of canine adverse food reactions by patch testing C. JOHANSEN and R. S. MUELLER Centre for Clinical Veterinary Medicine, Ludwig Maximilian University Munich, Munich, Germany It is challenging, emotionally and financially, for many pet owners to perform an effective diet trial required for the diagnose of adverse food reaction. Patch testing with food allergens was recently reported to be helpful in choosing ingredients for an elimination diet. The aim of this study was to evaluate patch testing with the currently fed dog food in dogs suspected of adverse food reaction. In 10 dogs with adverse food reaction, a patch test was performed on the lateral chest, 48 h after clipping, with the original commercial dry food in the form of a kibble mixed with water to pasty consistency. Vaseline was used as the negative control. After 48 h reactions were evaluated and, nine of 10 dogs with adverse food reaction showed erythema on the tested sites. Three dogs with adverse food reaction were tested with their elimination diet (both with the carbohydrate and protein sources) instead of the original food and no skin lesion at the patch test site was visible. Patch testing with the original food resulted in a negative reaction in nine of 10 animals without adverse food reaction. Based on these data, the sensitivity and specificity of patch testing for adverse food reaction are 90%. In conclusion, patch testing with the original food seems useful in the evaluation of adverse food reaction in dogs. Funding: Royal Canin, Aimargues, France. Conflict of interest: Cornelia Johansen’s residency is supported by Royal Canin, Aimargues, France. In the last 5 years, Ralf Mueller has obtained funding, lectured or consulted for Bayer Animal Health, Boehringer Ingelheim, Dechra Veterinary Products, Intervet, Merial, Novartis Animal Health, Pfizer Animal Health, Procter & Gamble, Royal Canin, Selectavet, and Virbac.

FC-59 Accuracy and precision of compounded ciclosporin capsules and solution M. UMSTEAD*, D. BOOTHE , C. CRUZESPINDOLA , J. MACDONALD*, R. KENNIS* and D. ANGARANO* *Department of Clinical Science, Auburn University, Auburn, AL, USA; Department of Anatomy, Physiology, and Pharmacology, Auburn University, Auburn, AL, USA Compounded products do not undergo Federal Drug Administration (FDA) mandated premarket assessment. Accordingly, compounded products should be prescribed only if no approved product is available. This study describes the accuracy and precision of ciclosporin (CsA) compounded capsules (10 and 300 mg) or solutions (50 and 150 mg/mL). We hypothesize that > 25% of compounded CsA products will fail to be between 90% and 110% of the labelled strength. Each compounded CsA was acquired by prescription from five pharmacies at

three different times, 14–45 days apart. FDA approved Atopica (Novartis Animal Health, Greensboro, NC, USA) (10 and 100 mg) and human USP generics (50 and 100 mg capsules and 100 mg/mL solution) served as positive controls. Accuracy was based on CsA strength as measured by high performance liquid chromatography (HPLC), and precision on replications (n = 3) from each pharmacy (n = 5). Accuracy of positive controls ranged from 90% to 102.65%. For compounded preparations, accuracy (actual mean + SD (mg, % of predicted) among all pharmacies was 10.13 ± 0.98 (10 mg, 101.26%), 290.06 ± 9.58 (300 mg, 96.69%), 44.79 ± 9.92 (50 mg/mL, 89.57%), and 127.11 ± 18.37 (150 mg/mL, 84.74%); the least accurate product deviated 33.8% from labelled strength. Precision among all pharmacies for capsules ranged from 0.58% to 8.72% and for solutions from 0.67% to 14.34%. The greatest imprecision for any single pharmacy was 29.41%. We conclude that compounded CsA capsules, but not solutions, tend to be both accurate and precise. This study supports the need for standardization of CsA compounding recipes. Funding: This study was supported by a grant from the American College of Veterinary Dermatology and by the Department of Clinical Science, Auburn University, Auburn, Alabama. Conflict of interest: None declared.

FC-60 Effect of antibiotics on the survival of a canine keratinocyte cell line, not a predictor of cutaneous drug hypersensitivity reactions K. L. VOIE, R. LUNDEEN, K. L. CAMPBELL and S. N. LAVERGNE University of Illinois, Urbana, IL, USA The pathogenesis of drug hypersensitivity is poorly understood, but ‘danger’ signals, such as cell necrosis or oxidative stress, are thought to play a role. Our central hypothesis was that keratinocytes exposed to antibiotics that are commonly associated with drug hypersensitivity would induce greater cell toxicity than antibiotics not commonly associated with such reactions. Four antibiotics commonly associated with cutaneous drug hypersensitivity (amoxicillin, cefalexin, sulfadimethoxine, and sulfamethoxazole) were investigated for their potential to kill a proliferative canine epidermal keratinocyte cell line (CPEK, CellnTec, Bern, Switzerland), in comparison to two antibiotics rarely associated with drug hypersensitivity reactions (amikacin and enrofloxacin). Five concentrations were tested: doses covering the therapeutic plasma concentration range; one dose below and one dose significantly above this range. Cell viability was evaluated using the Cell Titer Glo kit that measures ATP levels (Promega, Madison, WI, USA). A time curve was performed using the two therapeutic concentrations at 0.5, 1, 2, 4, 8, and 24 h. The media was used as a negative control; DMSO 50% and triton X0.1% were used as positive controls. At therapeutic concentrations, over a period of 30 min–24 h, none of the drugs significantly 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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affected the proliferation and survival of the CPEK cells compared to the media controls. At the highest tested concentrations for 24 h, only enrofloxacin (500 lmol/L) induced significant cell death compared to media. Direct drug-induced cell death does not appear to explain why b-lactams and sulfonamides are more often associated with skin drug hypersensitivity than fluoroquinolones and aminoglycosides. Funding:American College of Veterinary Dermatology Resident Research Award. Conflict of interest: None declared.

FC-61 Minimum effective dosage and safety of methylprednisolone and triamcinolone for the induction and maintenance treatment of pruritus in allergic cats. A double-blinded, randomized, prospective study E. C. GANZ*, C. E. GRIFFIN , D. A. KEYSà and T. A. FLATGARD* *Animal Dermatology Clinic, Marina del Rey, CA, USA; Animal Dermatology Clinic, San Diego, CA, USA; à Independent Statistical Consultant, Athens, GA, USA Triamcinolone (T) and methylprednisolone (M) have been considered almost equipotent, and varying doses are used for control of pruritus associated with feline allergic


dermatitis. The first objective was to determine effective doses of methylprednisolone (Pfizer, New York, NY, USA) and triamcinolone (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA) required to induce remission from pruritus associated with feline allergic dermatitis. The second objective was to compare the efficacy of several different alternate day (EOD) maintenance doses. The third objective was to determine whether laboratory abnormalities occurred at the effective doses. Thirty two client-owned allergic cats were randomly assigned to the M or T groups. Owners reported on pruritus score and behavioural changes weekly. Remission was defined as a pruritus score of equal to or < 2 of 10, with zero defined as the least and 10 as the most pruritic. Serum chemistry, complete blood count, fructosamine, and urinalysis were assessed on day 0, at the end of the 7–14 day induction phase, and at study completion. Mean once daily doses required for induction were 1.41 mg/kg for M and 0.18 mg/kg for T. Mean EOD maintenance doses were 0.54 mg/kg for M and 0.05 mg/kg for T. There was a statistically significant decrease in eosinophils and increase in fructosamine for both groups from baseline to study completion. In no case did fructosamine exceed the upper reference limit. These results suggest that triamcinolone is 10 times more potent than methylprednisolone, and that these doses are efficacious and safe for controlling pruritus in allergic cats. Funding: Novartis ACVD Resident Research Award. Conflict of interest: None declared.

Free Communication Abstracts Session 12: EQUINE 2 FC-62 Novel localized, lympho-histiocytic, granulomatous single limb dermatitis in fourteen horses K. BERGVALL* and A. SHOKRAI *Department of Clinical Sciences, University of Agriculture, Uppsala, Sweden; Department of Pathology, Swedish Veterinary Institute, Uppsala, Sweden This report describes the clinical and histopathological characteristics of a localized, lympho-histiocytic, granulomatous dermatitis in horses. Eight mares, five geldings, and one stallion, 11–22 years old at onset of clinical signs, were presented with a 1–3 years history of welldemarcated alopecic, erythematous, dry and lichenified skin with sparse crusting and swelling, confined to one leg. The condition was slowly progressive and non responsive to topical or systemic antibiotics, non steroidal anti-inflammatory drugs (NSAID), oral prednisolone (0.4 mg/kg every 48 h), and various ‘mud fever creams’. Systemic dexamethasone temporarily improved the condition in one horse. Histopathology revealed dermal lympho-histiocytic, granulomatous inflammation with giant cells. Special stains were negative for microorganisms and tissue culture revealed no growth. All but two horses responded to topical tacrolimus (Protopic 0.1%; Astellas Pharma AB, Malmo¨, Sweden) treatment and one horse to oral prednisolone (Prednisolon; Pfizer AB, Sollentuna, Sweden) at 1 mg/kg once daily and topical betamethasone (Betnovat 0.1%; GlaxoSmithKline AB, Solna, Sweden). Treatment was discontinued without relapse in one horse (1 year follow-up). The remaining horses needed continued treatment on a weekend basis or at relapse after discontinuing treatment. Two horses were re-biopsied at clinical resolution and had similar histopathology, however, milder. The condition remained confined to one leg in all horses during the follow-up period (9 months–6 years) and was not associated with concurrent disease. This dermatitis does not seem to spontaneously resolve but was responsive to immune modulation. The pathogenesis of this condition remains to be elucidated. Funding: Self funded. Conflict of interest: None declared.

FC-63 Clinical and histological response to topical application of Xxterra and Aldara on healthy horse skin K. BERGVALL*, H. BROSTRO¨M* and R. GRANDON *Department of Clinical Sciences, University of Agriculture, Uppsala, Sweden; Department of Pathology, University of Agriculture, Uppsala, Sweden The objective of this study was to evaluate the clinical and histological effects of topical applications of sanguinarine and zinc sulphate (Xxterra; N-Vet AB, Uppsala, Sweden) and imiquimod (Aldara; Meda AB, Solna, Sweden) to healthy horse skin. Xxterra and Aldara, have been used to treat equine sarcoids; however, there is no report of their effect on healthy horse skin. Two healthy standardbred mares, 13 and 16 years old, were clipped on each side of the neck and biopsied. One week later, 0.5 mL of Aldara and Xxterra were applied to a 3 cm diameter area, on each side of the neck, respectively, for 3 weeks. Aldara was applied three times weekly according to the protocol used in a previous sarcoid treatment study and Xxterra once daily for 5 days, then twice weekly according to the manufacturer’s recommendations. The lesions were photographed and evaluated daily. One week after discontinuing treatment the areas were biopsied and sent blinded to a pathologist. Both horses developed similar degree of eroded, crusted lesions with moderate swelling and pain when touched. Histopathology revealed orthokeratotic hyperkeratosis and epidermal hyperplasia and a diffuse lymphocytic infiltration in the superficial dermis in both treatments. Fibrosis of the deep dermis was seen in Xxterra treated skin and oedema of the tunica adventitia of subcutaneous vessels was noticed in Aldara treated skin. In conclusion, Xxterra and Aldara applied to healthy horse skin initiated an inflammatory response characterised clinically by crusting, swelling and pain and histologically by a lymphocytic reaction, fibrosis (Xxterra) and vessel oedema (Aldara). Funding: Self funded. Conflict of interest: None declared.


Free Communication Abstracts Session 13: SKIN BIOLOGY AND ECOLOGY FC-64 Production of IL-31 by canine Th2 cells and identification of inflammatory and neuronal target cells E. E. MCCANDLESS, J. MESSAMORE, C. RUGG, G. J. FICI and A. J. GONZALES Pfizer Animal Health, Kalamazoo, MI, USA Recent reports have suggested that the cytokine IL-31 may have a role in atopic dermatitis. Although IL-31 has been implicated in inflammatory responses and pruritus, very little is still understood regarding its interactions with canine cells. The purpose of the following work was to investigate the mechanism by which IL-31 may be involved in the propagation of disease by identifying a cellular source and a responder. Differential stimulation of canine peripheral blood mononuclear cells (PBMCs) and immunoassay analysis suggested T cells as a source of IL-31. Further evaluation showed that co-stimulation of house dust mite specific T helper type 2 (Th2) polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced relatively high levels of IL-31 compared to either stimulant alone. Using real-time polymerase chain reaction (PCR), the canine monocytic line DH82 cells were shown to express the IL-31 receptor alpha chain. These cells were confirmed to be IL-31-responsive via induction of the MAP kinase signal cascade upon IL-31 treatment. Dorsal root ganglia were also examined by PCR and were found to express the IL-31 receptor alpha chain. These results indicate that canine Th2 cells are a source of IL-31 and suggest that both a monocytic cell line and neuronal cells express the IL-31 receptor. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus via induction of signalling in macrophages and direct neuronal interaction. Funding: Pfizer Animal Health, Kalamazoo, MI, USA. Conflict of interest: All authors are employees and shareholders of Pfizer Incorporated.

FC-65 Detection of toll-like receptors (TLRs) on canine skin M. BARDAGI, V. L. MARTIŃEZ-DIÁZ, L. ALTET, I. RAVERA, D. FONDEVILA, O. FRANCINO and L. FERRER Universitat Auto`noma de Barcelona, Bellaterra, Spain Among the different components of the innate immune response, Toll-like receptors (TLRs) have attracted most interest as key recognition molecules. Despite the importance of TLRs in skin biology and disease in human beings, there is no information available about the expression of these receptors in canine skin. The aims of the present study were to investigate the expression of TLRs in canine keratinocytes by means of real-time quantitative polymerase chain reaction (RT-qPCR), confocal immunofluorescence (CIF) and immunohisto 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

chemistry. Confocal immunofluorescence and RT-qPCR were performed on cultured keratinocytes (immortalized cell line CPEK and primary cultures). Immunohistochemistry was performed on canine skin biopsies. Immunohistochemistry and CIF were performed using antibodies (Abs) raised against human TLR2 and TLR4. The RT-qPCR detected transcription of TLR1, TLR2, TLR4, TLR5 and TLR9 but not TLR7. Using CIF, expression of both TLR2 and TLR4 could be detected in cultured keratinocytes. Toll-like receptor 2 was expressed in a granular pattern on the cytoplasm, and TLR4 also in a granular pattern but, mainly in the perinuclear cytoplasm. Using the same antibodies, TLR2 could be detected on paraffin sections but not TLR4. In general, this pattern of expression of TLRs is similar to the pattern described in human beings. These results open a new line of research to investigate the role played by TLRs in the pathogenesis of various canine dermatologic diseases and the eventual modulation of these receptors by different agents. Funding: Affinity Petcare. Conflict of interest: None declared.

FC-66 Mucosal bacteria community profiles in the absence of antibiotics – a pilot study in healthy dogs V. M. SCHMIDT*, N. J. WILLIAMS*, S. DAWSON*, S. SHAW , N. A. MCEWAN* and T. NUTTALL* *The University of Liverpool School of Veterinary Science, Neston, Cheshire, UK; Derm4pets, Chalfont St Giles, Buckinghamshire, UK Antibiotics may exert a selection pressure on the normal microbial flora of the gastrointestinal tract and mucosal surfaces, increasing the prevalence of antibiotic-resistant bacteria. This study characterised the mucosal staphylococcal and faecal Escherichia coli populations of healthy Labrador retriever dogs that had not received antibiotics for at least 12 months. Bacteria were isolated from mucosal swabs (nasal and perineal; n = 73 dogs) and faeces (n = 68 dogs). Staphylococcal spp. and E. coli were identified by phenotypic, cytological, biochemical and genetic characteristics, and tested for antibiotic susceptibility by disc diffusion and minimum inhibitory concentration. Isolates with antibiotic resistance were further investigated for the carriage of resistance genes (mecA; blaCMY2; blaCTX-M; blaQNR) and typed (spa; SCCmec) by polymerase chain reaction (PCR). Speciation of the staphylococcal isolates was performed using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Staphylococci was isolated from 98.6% (72/73) of the dogs; 36% (27/73) carried at least one meticillin-resistant mecA positive isolate and 47% (35/73) carried at least one multi-drug resistant isolate (MDR; resistant to three or more antibiotic classes). The majority of dogs carried coagulase-negative (95%; 70/73) rather than coagulase-positive staphylococci (47%; 35/73), and meticillin-resistance and MDR were associated with coagulase-negative isolates.

Abstracts Escherichia coli were isolated from 95.5% (65/68) of the dogs; 30% (20/68) carried at least one MDR isolate but only 2.9% (2/68) carried an ESBL (extended-spectrum beta-lactamase) producing E. coli and only 4.4% (3/68) an E. coli with AmpC beta-lactamase (blaCMY2). This pilot study provides a baseline to examine changes in the canine bacterial flora following antibiotic therapy. Funding: This study was funded by Pfizer Animal Health. Conflict of interest: The authors have received other research, lecture and consultancy fees from Pfizer Animal Health.

FC-67 Prospective cohort pilot study of the faecal Escherichia coli community profiles in a group of healthy dogs in the absence of antibiotics V. M. SCHMIDT, T. NUTTALL, S. DAWSON, N. A. MCEWAN and N. J. WILLIAMS The University of Liverpool School of Veterinary Science, Neston, Cheshire, UK This study investigated variation of the intestinal microflora over time in a group of healthy dogs that had not received antibiotics for at least 3 months. Faecal samples were tested daily for 7 days, weekly for 4 weeks and monthly for 2 months from 23 dogs (299 samples). Four additional dogs were sampled for at least the first week (50 samples), but were lost to follow up. Escherichia coli were identified phenotypically and by polymerase chain reaction (PCR) assay. Isolates with antibiotic resistance were investigated for resistance gene carriage (blaCMY; blaCTX-M; blaQNR) by PCR. Macro-restriction pulsedfield gel electrophoresis (PFGE) was used to determine the genetic relatedness of non-resistant and resistant E. coli. Consistent E. coli phenotypes and genotypes were detected in 52% (14/27) of dogs during the first 7 days, but at every sample point of the study in only 4% (2/23) of dogs. Fifty-two percent (12/23) of dogs, however, had some reoccurring isolates throughout the full study period. Fifty-nine percent (16/23) of dogs had a multidrug resistant isolate (MDR; resistance to > 3 antibiotic classes) and 30% an ESBL (extended-spectrum betalactamase) producing E. coli in at least one sample. The majority of these isolates were from four dogs in one household fed an organic raw meat diet. There was no association of isolates with previous antibiotic therapy. This pilot study provides a baseline to examine changes in the faecal flora following antibiotic therapy, but shows that variations with time and external influences, such as diet, need to be taken into account. Funding: This study was funded by Pfizer Animal Health. Conflict of interest: The authors have received other research, lecture and consultancy fees from Pfizer Animal Health.

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FC-68 The aerobic and anaerobic flora of the tympanic cavity of clinically normal cats K. WILDERMUTH and R. A. ROSYCHUK Department of Clinical Sciences, Colorado State University College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO, USA This study investigated the aerobic and anaerobic bacterial flora of the tympanic cavities (TCs) of clinically and otoscopically normal cats. Samples were collected via ventral bulla osteotomy from 24 cats (48 TCs). The TCs appeared grossly normal in all cats. A portion of the bulla septum with associated epithelium of both the ventromedial and dorsolateral chambers was harvested aseptically. These chambers were also swabbed with a sterile cotton tipped swab. The two samples from each TC were combined for aerobic and anaerobic bacterial culture and antimicrobial sensitivity testing. Both chambers of each TC were swabbed for cytologic examination. Bacteria were cultured from 9/48 TCs (7/24 cats). One single organism was cultured from one TC of four cats (Clostridium sp., Bacteroides sp, Escherichia coli and Corynebacterium sp.); Moraxella sp. and Avibacterium volantium from one TC of one cat; Staphylococcus xylosus from both TCs of one cat; Clostridium sp, Porphyromonas sp., Pasteurella multocida, Stenotrophomonas maltophila and non hemolytic Streptococcus from one TC and Bacterioides sp., Pasteurella multocida, Stenotrophomonas maltophila and non-hemolytic Streptococcus from the other TC of one cat. Bacteria were not noted on cytologic examination. Antimicrobial sensitivity was available for 7/12 aerobic isolates. Resistance was noted to clindamycin and amikacin (Escherichia coli, Stenotrophomonas maltophila, Pasteurella multocida), ampicillin (Pasteurella multocida) and erythromycin (Pasteurella multocida, Escherichia coli); intermediate sensitivity to amikacin and erythromycin (Pasteurella multocida) and ceftazidime and polymyxin B (Staphylococcus xylosus). The tympanic cavity appears to be sterile in most cats, but may also harbour a variable, apparently ‘normal’ bacterial flora. Funding: Self funded. Conflict of interest: None declared.

FC-69 Cutaneous carriage of Malassezia species in healthy and diseased cats in the Paris area A. LEGRAS*, O. CROSAZ*, E. PETIT , B. HUBERT*, L. DESQUILBETà, R. CHERMETTE* and J. GUILLOT* *Parasitology, Mycology, Dermatology, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France; INRA, USC Bipar, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France; àAnimal Production and Public Health, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France Little is known about the presence and diversity of Malassezia yeasts on the skin of cats. The aim of this study was to evaluate the prevalence of Malassezia species on the skin of different feline breeds with or without cutaneous lesions. A total of 112 cats were evaluated by cytological examination and fungal culture 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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using modified Dixon’s agar. Samples were obtained from the left external ear canal by swabbing and from the claws on the right front paw, the anus and the back using contact plates. In 54 cats, erythema, alopecia and/or crusts were noted. In 58 cats, no cutaneous lesions were observed. Cytological examination was rarely positive for Malassezia yeasts and the number of microorganisms (yeasts or bacteria) was systematically low. Malassezia growth was noted, from at least one sampled site, in 83% of 17 Devon rex, sphynx, Peterbald and Donskoy cats. Some of these cats had greasy seborrhoea. Conversely, only 15% of 41 healthy cats from other breeds yielded positive cultures. In cats with cutaneous lesions, the


prevalence of positive cultures was 30%. Polymerase chain reaction and direct sequencing of a large subunit of rRNA gene revealed the presence of M. pachydermatis, M. nana, M. sympodialis and M. slooffiae. The non-lipid dependent species, M. pachydermatis, was predominant whereas M. nana was primarily isolated from the ear canal. These data support recent reports of high Malassezia species colonisation in Devon rex and sphynx cats. Peterbald and Donskoy cats may also be predisposed to colonisation by Malassezia species. Funding: Self funded. Conflict of interest: None declared.

Poster Abstracts Topic 1: ADVERSE REACTIONS TO FOOD P-001 The occurrence and the features of food allergy in Hungarian dogs N. TARPATAKI* and T. NAGY *Department and Clinic of Internal Medicine, Faculty of Veterinary Science, Szent Istvań University, Budapest, Hungary; PrimAVet Praxis, Budapest, Hungary The aim of this study was to investigate the occurrence and features of canine food allergy in Hungary. In this study, 39 of 84 dogs (46%) proved to be food allergic based on the results of elimination diet trials, followed by recurrence of symptoms during challenge with the original diet. These 39 dogs did not have symptoms for 6 months or 1 year after being maintained on a monodiet. In 51% of the cases the onset of clinical signs started before 1 year of age. Ninety-seven percent of the dogs suffered from chronic skin disease with pruritus. In 31% of the cases, the animals had gastrointestinal signs and otitis externa with the same frequency as either a permanent or relapsing symptom. Secondary skin infection was caused by Staphylococcus pseudintermedius in 36% of cases and by Malassezia pachydermatis in 3%. The food allergens were identified in 74% of the cases using a provocation test. The most frequent allergen was chicken (75%) followed by beef (32%). Twenty-five percent of the dogs were allergic to one type of protein, and 75% to several types. West Highland white terriers and Hungarian Vizslas were relatively more frequent among the food allergic dogs. We performed correlation analyses and obtained novel information about the association of breed, age, housing, seasonality, symptoms, and allergens. This survey will give small animal dermatologists insight regarding food allergy in Hungary. Funding: Self funded. Conflict of Interest: None declared.

P-002 Retrospective study of homemade elimination diets prescribed during one year in a university consultation: (1) observance of 155 canine and feline cases A. MARIN*, O. CROSAZ*, B. HUBERT*, J. F. JAMET*, L. YAGUIYAN-COLLIARD and G. MARIGNAC* *Unite´ de Parasitologie-Mycologie-Dermatologie, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; UMES, Service de Nutrition Clinique, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; Clinique Ve´te´rinaire Rouget de l’Isle, Choisy le Roi, France Dog and cat owners’ observance and diligence in strictly feeding an elimination diet is considered low. In this study we assessed an elimination diet prescription and owners’ reactions. Based on data from the dermatology files at the Ecole Nationale Ve´te´rinaire d’Alfort, a questionnaire was sent to owners regarding the progress of the homemade elimination diet prescribed to their pets and the current dermatological status of the animals (1 year or more after the prescription of an elimination

diet). During 2008–2009, 110/696 dogs and 45/233 cats (n = 155) seen were prescribed a restricted homemade diet. Seventy-seven of 155 owners did not keep a followup appointment. Reasons cited by the 37/77 owners who responded to the questionnaire were: improvement of clinical signs (14), travel distance (9), length of waiting (6), follow-up by another veterinarian (4), and disappointment about the initial consultation (4) (e.g. misdiagnosis, lack of advice, treatment not suitable). Fiftynine of 110 dogs (53.6%) and 19/45 cats (42.2%) strictly received the elimination diet, and 11/110 dogs and 5/45 cats received a modified diet (addition of other ingredients, change of the protein source). Fifteen of 110 dogs and 8/45 cats were not fed the elimination diet. It was more difficult to maintain cats (48.3%) than dogs (22.5%) on the elimination diet. Cost was the most commonly reported problem by both cat and dog owners. Refusal of the new diet was common in cats, and difficulty/reluctance to get a novel ingredient (e.g. horse) was common in dogs. Funding: Self funded. Conflict of Interest: None declared.

P-003 Retrospective study of homemade elimination diets prescribed during 1 year in a university consultation: (2) clinical signs of 28 canine and feline cases A. MARIN*, O. CROSAZ*, B. HUBERT*, J. F. JAMET, C. HADJAJE-DARMON*, S. RENNA, L. YAGUIYAN-COLLIARD§ and G. MARIGNAC* *Unite´ de Parasitologie-Mycologie-Dermatologie, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; Clinique Rouget de l’Isle, Choisy le Roi, France; Clinique Ve´te´rinaire, Catanzaro, Italy; §UMES, Service de Nutrition Clinique, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France Food allergy is frequently suspected in dogs and cats suffering from a pruritic condition. We assessed how commonly this diagnosis was made in our dermatology service and evaluated the clinical features encountered. Data were collected from the 929 dermatology consultations performed over 1 year and through a questionnaire sent to 155 owners (of 110 dogs and 45 cats) 1 year or more after the prescription of a homemade elimination diet. A provocation test with the original diet was positive in 10/51 animals, and clinical signs recurred in one to 7 days, except for one animal that required 25 days. Eighteen owners declined provocation test because of sustained improvement of their animal on elimination diet, 23 animals did not follow the diet, and information was not obtained for 63 animals. Twentyeight of 145 (18%) animals were considered food-allergic (CFA): 10 confirmed (seven dogs, three cats) and 18 highly suspected (six dogs, 12 cats). No breed or sex predisposition was found. Average age of onset was 4 years in both species, while four dogs and three cats (25%) had symptoms before the age of one. Symptoms and skin lesions varied greatly. Pruritus was present in


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26/28 animals. In dogs, the most common lesions were erythema (76.9%), alopecia (69.2%), otitis externa (69.2%), and crusts (23.1%). In cats, the head and neck pruritus was the most common presentation: 73.3% of cats in the CFA group and 100% of the 12 cats in the confirmed group. Crusts (80%), alopecia, and ulceration (40% each) were common. Funding: Self funded. This work was part of the veterinary thesis of Alix Marin. Conflict of Interest: None declared.

P-004 Survey of IgE and lymphocyte reactivity to environmental and food allergens in 319 dogs suspected to have allergic skin diseases A. SUTO*, Y. SUTO*, N. ONOHARA*, T. OKAYAMA and K. MASUDA *Suto Animal Hospital, Urayasu, Chiba, Japan; Animal Allergy Clinical Laboratories, Inc., Sagamihara, Kanagawa, Japan This study investigated the proportion of responsive allergens in a large number of dogs suspected to have allergic skin diseases using a quantitative allergenspecific IgE test and lymphocyte test. After we ruled out other pruritic diseases, 319 dogs suspected to have allergies were included in this study. A quantitative allergen-specific IgE test was performed against both environmental and food allergens, and lymphocyte responsiveness was assessed against food allergens. In the IgE test, 97 (30.4%), 135 (42.3%), and 33 (10.3%) of 319 cases had positive reactions to environmental allergens alone, environmental and food allergens, and food allergens alone, respectively, and 54 (16.9%) cases had negative results. The lymphocyte test revealed that 244 of 319 dogs (76.5%) had positive lymphocyte reactions to food allergens, and 75 dogs (23.5%) did not. Among the 151 dogs that did not have IgE against food allergens, 125 dogs (82.8%) had positive lymphocyte responses to food allergens. Likewise, of the 168 dogs that had IgE antibodies to food, 119 (70.8%) had positive lymphocyte responsiveness to food allergens. Therefore, the linkage between IgE and lymphocyte reactions to food allergens was not evident, and we considered this lack of association to reflect independent immune reactions in dogs reactive to food allergens. These results show the distribution of responsive allergens in the allergic dogs and, suggest that the lymphocyte test against food allergens has the potential to


become a new method for diagnosing food allergy in the veterinary field. Funding: Self funded. Conflict of Interest: This study was funded by Animal Allergy Clinical Laboratories.

P-005 Possible involvement of lymphocyte responses to food allergens in dogs with atopic-like dermatitis K. KAWANO, T. OKAYAMA, K. MASUDA and T. MIZUNO Primo Animal Hospital Sagamihara Chuo, Kanagawa, Japan Atopic-like dermatitis (ALD) in dogs is defined as a disease that shows similar clinical features to canine atopic dermatitis but lacks detectable allergen-specific IgE. Since the pathogenesis of ALD remains unclear, we investigated lymphocyte responsiveness to food allergens in dogs with ALD by the combination of cell culture and flow cytometry. Using Favrot’s criteria, we diagnosed 26 dogs with no detectable antigen-specific IgE as ALD and further examined them. The peripheral blood mononuclear cells of each dog were cultured with or without 18 different food allergens. The percentages of CD4+CD25+ cells were examined by flow-cytometry. According to our previous study, lymphocyte responsiveness to the tested allergens was determined to be positive when the value exceeded 1.2%. The dogs were then fed an elimination diet that did not contain food allergens positive on the lymphocyte test. Pruritus was assessed before and after the elimination diet using a 10point scale. Each dog reacted from one to 14 allergens on the lymphocyte response test. The values of the positive test results ranged from 1.2% to 6.6% with an average and standard deviation of 2.8 and 1.3, respectively. The average pruritus score of the dogs decreased from 6.3 to 1.8 after the elimination diet trial. Lymphocyte reactions against food allergens were demonstrated in all the ALD dogs, and the elimination of reactive food allergens was effective in relieving clinical symptoms of ALD. Lymphocyte-mediated allergic reaction to food allergens was considered to play at least a partial role in the pathogenesis of canine ALD. Funding: Primo Animal Hospital Sagamihara Chuo. Conflict of Interest: K Masuda is CEO and stockholder of Animal Allergy Clinical Laboratories, Inc.

Topic 2: PATHOGENESIS OF ALLERGIC DISEASES P-006 Metabolic fingerprinting: a new tool for evaluation of canine atopic dermatitis V. BRUET*, H. DUMON, P. BOURDEAU*, J. C. DESFONTIS and L. MARTIN *Dermatology, Parasitology and Mycology Unit, Veterinary School, ONIRIS, Nantes, France; Nutrition and Endocrinology Unit, Veterinary School, ONIRIS, Nantes, France; Animal Pathophysiology and Functional Pharmacology Unit, Veterinary School, ONIRIS, Nantes, France In human medicine, holistic approaches are increasingly used to improve diagnosis. Among these, Fourier transform infrared spectroscopy (FT-IR) presents considerable interest due to its rapid screening ability combined with its low cost. FT-IR gives an absorption spectrum of all the biochemical substances present in a biological sample, resulting in a characteristic ‘fingerprint’. We evaluated the ability of FT-IR to distinguish dogs with atopic dermatitis (CAD; n = 21) from healthy control dogs (n = 10) and from dogs presenting other skin diseases (OD, n = 15). For FT-IR analysis, aliquots of plasma diluted in water were deposited on a ZnSe window and dried under vacuum. Four spectra were acquired for each window and each plasma was tested in triplicate. Discriminant analysis was performed on the spectra to assess the differences among groups. Absorption bands differing between CAD and OD dogs were determined by a t-test at each spectral frequency (P < 0.05). Spectra of the three groups of dogs differed significantly (P < 0.0001). Cross-validation procedures were carried out for 27 dogs (random selection of 13 CAD, 10 OD and four controls). Twelve of 13 dogs with CAD and all the OD and controls were correctly segregated, yielding 96.3% accuracy. The t-test results showed an increased absorption for CAD over OD for six absorption bands (P < 0.001). According to the literature, three of these bands are characteristic of specific proteins: IgG4, IgG2, IgG3, and apolipoprotein C (which is overexpressed in mice with atopic dermatitis). In conclusion, this new method appears suitable for a rapid screening of dogs suspected of atopic dermatitis. Funding: Self funded. Conflict of Interest: None declared.

P-007 Comparative analysis of dogs with atopic dermatitis identifies a clade of susceptible breeds H. MAZRIER, L. J. VOGELNEST, R. M. TAYLOR and P. WILLIAMSON Faculty of Veterinary Science, University of Sydney, Sydney, NSW, Australia The breed structure of dogs provides a powerful model for analysis of complex phenotypes. Canine atopic dermatitis (AD) has environmental and genetic components, and shares many features with human AD. Atopic dermatitis occurs in at least 100 dog breeds. While many studies report highly represented breeds, most have not analyzed relative risk (RR). Recently, genomic and candidate gene studies on canine AD have been reported,

but the molecular basis remains largely unresolved. Increased relative risk for AD identified in multiple, genetically isolated, pure-bred populations around the world, such as Australian dog breeds, may enable the identification of contributing genes. We reviewed records from dogs with confirmed AD, seen by dermatology specialists/residents, from an Australian cohort of over 23 000 dogs attending two Sydney University veterinary teaching hospitals between 2001 and 2009, in Sydney (365) and Camden (487) centres. Breed prevalence was calculated and breeds with RR ‡ 1.5 identified. We compared our results to 11 controlled canine AD prevalence studies (1971–2010), which reported highly represented dog breeds relative to a general population. Breeds were evaluated against the established canine breed haplotype structure. We identified 15 dog breeds with increased relative risk, of which seven had a prevalence above 10%. Haplotype analysis suggests a common genetic origin for many of the susceptible dog breeds. One specific clade contains a concentration of highly represented AD dog breeds including those with increased RR. Follow-up research will include functional genomics and immunological studies in this clade, aiming to improve understanding of the pathogenesis of canine AD. Funding: Australian National Kennel Council Canine Research Foundation, Australian Companion Animal Health Foundation, Margot Roslyn Flood Bequest 2010 & J. M. and J. R. Stewart Legacy 2010, Endeavour International Postgraduate Research Scholarships, The University of Sydney International Postgraduate Award, Goldie and Susie Lesue Scholarship 2009 & Neil and Allie Lesue Scholarship 2010. Conflict of Interest: None declared.

P-008 Dermatophagoides farinae-sensitized beagles are Th2-polarized and have antigen-specific IgE consistent with dogs with atopic dermatitis M. ALEO, C. RUGG, E. MCCANDLESS, L. SLY, B. GALVAN, T. FLECK, W. HUMPHREY, E. COSCARELLI, S. MAHABIR, R. MCCALL and A. GONZALES Pfizer Animal Health, Kalamazoo, MI, USA Canine atopic dermatitis (AD) has been reported to be associated with increased levels of allergen-specific IgE, as well as a Th1/Th2 cytokine imbalance (Th2 dominant) characterized by a low IFN-c/IL-4 ratio. A relevant model of this condition should demonstrate immunological changes similar to those seen in clinical AD cases. To establish such a model, normal healthy beagle dogs were sensitized to house dust mites (HDM, Dermatophagoides farinae) over a 6-week period with a series of three subcutaneous injections of HDM. Sensitization to HDM was confirmed with intradermal testing. The intradermal testing included six serial dilutions of D. farinae to determine the degree of sensitivity, as well as positive (histamine, Sigma-Aldrich; St Louis, MO, USA) and negative (phosphate buffered saline) controls. Sensitized dogs had at least a two-fold increase in circulating free and HDM-specific IgE for at least 7 weeks post-sensitization. IFN-c and IL-4 protein levels were also measured 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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pre- and post-sensitization in isolated peripheral blood mononuclear cells by ELISpot. The HDM-sensitized dogs had increased antigen-specific IL-4 levels and reduced IFN-c/IL-4 ratios post-sensitization, thus confirming a cytokine profile polarized to Th2. Therefore, this model possesses the immunological imbalances frequently seen in AD dogs and may be a useful tool to investigate potential restorative therapies. Funding:Pfizer Animal Health. Conflict of Interest: All authors are employees of Pfizer Animal Health.

P-009 Changes in the CD4/CD8-positive T lymphocyte ratio in the blood of atopic and non-atopic dogs N. TARPATAKI*, M. TERENYI and S. Z. NAGY *Department and Clinic of Internal Medicine, Faculty of Veterinary Science, Szent Istvań University, Budapest, Hungary; Central Agricultural Office, Budapest, Hungary; NEO-VET Veterinary Praxis, Gy} or, Hungary The aim of this study was to evaluate the association between changes in the ratio of CD4+/CD8+- T lymphocytes in peripheral blood of dogs suffering from various skin diseases, and the clinical examination findings. We analysed blood samples from 26 dogs suffering from skin diseases and from seven healthy dogs (controls) by flow cytometry. In the blood of the seven control dogs, the average CD4+/CD8+ T lymphocytes was 1.527. In the 23 atopic dogs the average ratio was 2.104. In the 16 atopic dogs without secondary pyoderma the average ratio was 2.972. In the two dogs with generalized demodicosis the ratios were 1.682 and 2026, and in the dog with systemic lupus erythematosus the ratio was 3.342. The ratio of CD4+/CD8+ T lymphocytes was significantly lower in atopic dogs with secondary pyoderma (1.022, P < 0.01) and in dogs suffering from pododermatitis (1.054, P < 0.05) compared to the healthy controls. The CD4+/CD8+ ratio was significantly higher than in healthy controls in dogs under 3 years of age (2.830, P < 0.05), with gastrointestinal signs (3.019, P < 0.05), and in dogs without lichenification (2.701, P < 0.05). In atopic dogs without secondary bacterial overgrowth, the CD4+/CD8+ ratio of T lymphocytes was significantly higher (2.972, P < 0.01) than in the healthy controls. The CD4+/CD8+ ratio of T lymphocytes in the peripheral blood in atopic dogs was significantly higher than in the healthy controls, and it may be decreased by secondary pyoderma treatment. Funding: Self funded. Conflict of Interest: None declared.

P-010 Prospective study of the association between passive smoking and allergic dermatitides in 161 dogs D. KA*, G. MARIGNAC*, L. DESQUILBET, L. FREYBURGER, B. HUBERT*, D. GARELIK§ and S. PERROT *Unite´ de Parasitologie-Mycologie-Dermatologie, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; Institut de Recherche Clinique Animal, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; Universite´ de Lyon, VetAgro Sup, Sepsis, Inflammation et He´mostase, Marcy l’Etoile, France; §Unite´ Tabacologie Service de Pneumologie, AP-HP.G.H.U. Pitie´-Salpe´trie`re, Paris, France The onset of allergic dermatitides and the appearance of related skin lesions are influenced by various environmental factors in dogs and in humans. Several studies have showed an association between secondhand smoke and the development of allergic dermatitides in children. However, there are no published studies investigating the same association in the dog. We prospectively enrolled 161 dogs during a 6-month period in both dermatology and vaccination consultations. Passive smoke exposure was assessed through a questionnaire administered to the dog owners. Allergic status of the dog was assessed based on Favrot et al.’s criteria for the diagnosis of chronic atopic dermatitis. Logistic regression was used, and association analyses were adjusted for potential confounders such as sex, age, and breed (predisposed vs. not predisposed). Tobacco consumption alone (cigarette consumption) was not significantly associated with the presence of allergic dermatitides. However, an exponential-shaped dose–response association was found between level of exposure to passive smoking (cigarette consumption divided by home surface) and the presence of allergic dermatitides. When compared to unexposed dogs, dogs with the highest exposure (in the last quartile of level of passive exposure) were more likely to present allergic dermatitides (adjusted odds ratio, 4.38; 95% confidence interval, 1.10–17.44; P = 0.03). Dogs with a high level of exposure to tobacco smoke may be at a higher risk for developing allergic dermatitides than unexposed dogs. Funding: Self funded. Conflict of Interest: None declared.

P-011 Mite-allergen challenges reduce the ceramide content of lesional and non-lesional stratum corneum in an experimental dog model of atopic dermatitis J. STAHL*, J. PAPS*, W. BAEUMER* and T. OLIVRY *Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Foundation Hannover, Hannover, Germany; Department of Clinical Sciences, College of Veterinary Medicine, and Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA Previous studies established that dogs with atopic dermatitis exhibit deficiencies in stratum corneum cera-


Abstracts mides, which represent an important stratum corneum lipid subclass. The aim of the present study was to determine the effect of mite-allergen challenge on the stratum corneum ceramide profile of five atopic Maltesebeagle dogs experimentally sensitized to house dust mites. Pre-challenge stratum corneum samples were obtained from the right axilla by removing it by cyanoacrylate stripping. One week later, the dogs were challenged with topically applied mite allergens to the right axilla, which resulted in mild to moderate inflammatory reactions 24 and 48 h later. Two weeks after challenge, we obtained stratum corneum samples of lesional and non-lesional skin areas (the latter 10 cm away from application sites). The different stratum corneum lipid classes were assessed blindly by highperformance thin-layer chromatography. Significantly

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lower amounts of ceramides [AH], [AP], [AS], [NP], [EOP], [NS], and [EOS] were observed in lesional stratum corneum compared to prechallenge samples, while no significant effect was found in the amount of other lipid classes such as cholesterol or free fatty acids. The ceramide profile of non-lesional skin showed generally the same trend of reduction. These findings suggest that the allergic reactions caused by mite-allergens lead to selected reduction of stratum corneum ceramides at the site of inflammation and also at sites different from those of allergen application. These observations argue in favour of the ceramides deficiency observed in canine atopic skin being, at least in part, secondary to inflammation. Funding: Self funded. Conflict of Interest: None declared.


Topic 3: ALLERGY TESTING P-012 Differences in allergy skin testing among dermatologists within the same geographical region in the USA P. HENSEL University of Georgia, Athens, GA, USA Intradermal testing is commonly used by dermatologists. Appropriate selection of allergens varies by geographical region and vegetation, and allergens are often selected through recommendations in the literature or by the allergen-providing companies. This study determined how much intradermal testing differs among dermatologists in the same geographical region. We contacted veterinary dermatologists from 15 locations in the same geographical region and requested their list of allergens, injection volume, and intradermal test concentrations (ITCs). Detailed information was received from 10 dermatology clinics. All sites were using the same source (Greer; Lenoir, NC) for their allergens. Overall, a total of 115 allergens have been used, including grasses (15), weeds (23), trees (30), moulds (18), epidermals (13), dust mites (6), and insects (10), but only an average of 59 allergens (range: 47–84) were used by each location. Of 115 allergens, 19 were used by all clinics, and another 15 were used by 9/10 clinics. All other allergens were used less frequently. Injection volume varied from 0.05 to 0.1 mL. All locations used histamine as a positive control at a concentration of 1:100 000 w/v (0.01 mg/mL) in 9/ 10, and 1:10 000 w/v (0.1 mg/mL) in 2/10. Intradermal test concentrations were 1000–8000 PNU for plant allergens, 1000–1750 PNU for moulds, 100–2000PNU for epidermals/hairs/feathers, 1:1000–1:50 000 w/v or 50–250 PNU for dust mites, and 250–1750 PNU for insects. The findings of this study confirm that even within the same geographical area, intradermal testing continues to lack standardization and consensus. Funding: Self funded. Conflict of Interest: None declared.

P-013 Significant cross-reaction or co-sensitisation is common among related allergens in canine intradermal tests L. M. BUCKLEY, V. M. SCHMIDT, N. A. MCEWAN and T. NUTTALL School of Veterinary Science, University of Liverpool, Neston, Cheshire, UK Intradermal tests (IDT) are commonly used to identify allergens for avoidance and immunotherapy in atopic dogs. This study reviewed IDT results in 563 dogs with atopic dermatitis. The diagnosis was made according to accepted criteria. Intradermal tests were performed with 53 house dust and storage mite, epidermal, insect, tree, weed and grass pollen, and mould allergens (Greer Laboratories; Lenoir, NC) following standard procedures. Pairwise comparisons were used to calculate the odds ratio (OR), 95% confidence intervals (CIs), and statistical significance for the results of each allergen pair, with significance set at P < 0.001 using a Holm-Bonferroni correction to reduce the false-detection rate. Apart 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

from cotton, feathers, cockroach, red clover, grain smut, and Penicillium, the results for all the allergens within each of the above groups were highly correlated (OR 14.4 [CI 3.9–54] to 1204 [CI 371–3909]; P = 0.0009 to P < 0.0001). Excluding red clover, 96% of the results between tree, weed, and grass pollens were also highly associated (OR 7.2 [CI 2.7–19.5] to 54 [CI 14.4–199]; P = 0.0005 to P < 0.0001). In contrast, no results between allergens from unrelated groups were significantly associated (OR 0.3 [CI 0.1–1.1] to 6.5 [CI 0.4–110]; P = 0.07 to P = 1.0). The ORs for related allergens (median 108.1) were significantly higher than the ORs for unrelated allergens (median 1.65) (P < 0.0001). These results suggest that there is significant cross-reaction or co-sensitization between related allergens. This may have important implications for allergen selection in testing and immunotherapy, but further studies are required to differentiate cross-reaction from co-sensitization. Funding: Self funded. Conflict of Interest: None declared.

P-014 Differences in skin test reactivity of 59 allergens tested with two different test concentration in 269 atopic dogs P. HENSEL, S. ZABEL and N. OKUNAKA University of Georgia, Athens, GA, USA Intradermal testing is the preferred test to identify allergens for immunotherapy in atopic dogs. False positive and negative reactions have been reported in atopic and non-atopic dogs. Recent studies have shown that higher intradermal test concentrations (ITCs) do not result in false-positive irritant reactions or adverse effects, and that dust mites should be tested at lower ITCs. This study assessed differences in skin test reactivity between two different ITCs in atopic dogs. Intradermal testing with 59 allergens was performed in 269 atopic dogs. The standard ITC for plant, mould, and insect allergens was 1000 PNU/mL, 500 PNU/mL for animal hair/epithelia, and 250–500 PNU/mL for dust mites. Adjusted ITCs were used for plant, mould, and insect allergens ranging from 1250–8000 PNU/mL, 1250 PNU/mL for animal hair/epithelia, and 50– 200 PNU/mL for dust mites. Results of both ITCs were compared using McNemar’s test, hypothesis tests were two-sided, and the significance level was a = 0.05. Significant increases in positive reactions were found with adjusted ITC for cocklebur (P = 0.0082), red maple (P = 0.0196), and Alternaria (P = 0.0455). Significantly more negative reactions were observed with adjusted ITC for Dermatophagoides farinae (P < 0.0001), Dermatophagoides pteronyssinus (P < 0.0001), and Tyrophagus putrescentiae (P = 0.006). For all other allergens except dust mites, an increased number of positive reactions (without statistical significance) were observed with adjusted ITCs. The findings suggest that adjusted ITC for dust mites may result in fewer false positive reactions, while for the other allergens the use of higher ITC may yield more positive reactions and adjusted ITC may identify allergens missed with standard ITC. Funding: Self funded. Conflict of Interest: None declared.

Abstracts

P-015 Intradermal tests in retrievers: evaluation of reproducibility X. LANGON Clinique Ve´te´rinaire du Bournet, Seyssins, France Intradermal allergy test is the gold standard in identifying allergens for immunotherapy in atopic dogs. This study evaluated the reproducibility of this test in 20 retriever dogs living in urban environments, with or without clinical signs of atopic dermatitis. Intradermal allergy tests were performed twice monthly. All patients met the following criteria: they were more than 6 months old, had a golden-coloured coat, and had not been subjected to therapy for at least 8 weeks prior to the tests. The tests were performed by one practitioner using Stallervet test (Stallergenes SA; Antony, France). The injections were administrated intradermally, without premedication, to the inner/proximal aspect of the thigh. Five allergens were tested: Dermatophagoides farinae, Dermatophagoides pteronyssinus, Blomia tropicalis, grass mixture, and storage mite mixture. The main criteria evaluated were erythema and the diameter of wheal in mm, 20–30 min after injection. Most dogs reacted to house dust mites (85%); D. farinae (58%), and D. pteronyssinus (50%). No reaction to grass mixture was seen. Reproducibility of positive reactions to mites was 88% (the positive predictive value was 89% and negative predictive value was 72%). In a second trial, the mean wheal diameter of histamine decreased by 20% while the mean wheal diameter for all mites remained constant, leading to a reproducibility of 82%. In a few tests, 9% of the reactions became conversely positive or negative. This study show that retrievers frequently react to mites on the intradermal allergy test and that this test has good reproducibility. Funding: Self funded. Conflict of Interest: None declared.

P-016 Patch testing with predigested proteins in sensitized dogs C. JOHANSEN*, C. MARIANI and R. S. MUELLER* *Centre for Clinical Veterinary Medicine, Ludwig Maximilian University Munich, Munich, Germany; Royal Canin, Aimargues, France Hydrolyzed veterinary diets have been introduced for the diagnosis of canine adverse food reaction. During hydrolysis, protein sources are enzymatically broken down into polypeptides, changing and reducing the allergenic properties of the molecules. This study investigated the allergenic capacities of predigested proteins (beef, pork, and salmon). Three types of digestion were chosen: complete digestion (majority of proteins 200– 800 daltons), pepsin and pancreatin digestion (majority of proteins 200–800 daltons), and pepsin digestion (majority of proteins 800–6000 daltons). We tested four dogs with adverse food reaction to beef (n = 2), pork (n = 1), and salmon (n = 1) confirmed with elimination diets and individual provocation. We conducted a patch

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test with relevant allergens (cooked, raw, and predigested in the three ways described above) on the lateral chest 48 h after clipping with and without tape stripping the epidermis 10 times. Reactions were interpreted as positive when an erythematous wheal occurred after 24 h and/or 48 h. The negative control was petroleum jelly. All dogs were positive on the patch test for their relevant raw or cooked proteins. No positive reactions were observed with predigested beef and pork, but the salmon-allergic dog was positive to pepsin-digested salmon. There was no difference between results from tape-stripped and non-tape-stripped skin. In conclusion, predigested beef and pork (< 800 daltons) did not induce positive reactions on patch testing in sensitized dogs, but pepsin-predigested salmon did. Tape stripping of the epidermis did not influence the results. Funding: Royal Canin, Aimargues, France. Conflict of Interest: C Johansen’s residency is supported by Royal Canin, Aimargues, France and Kerstin Orstadius Minnesfond, Sweden. C. Mariani is an employee of Royal Canin. In the last 5 years, R. Mueller has obtained funding, lectured or consulted for Bayer Animal Health, Boehringer Ingelheim, Dechra Veterinary Products, Intervet, Merial, Novartis Animal Health, Pfizer Animal Health, Procter & Gamble, Royal Canin, Selectavet, and Virbac.

P-017 A practical method to identify pollen adhered to canine hair B. WILDERMUTH* and R. REID *Animal Dermatology Clinic, San Diego, CA, USA; Erik and Ese Banck Clinical Research Center, San Diego, CA, USA Since epicutaneous exposure is considered the most important route for allergen sensitization in canine atopic dermatitis, it would be helpful to identify the pollen adhered to the canine coat as a means to characterise individual pollen exposure. We hypothesized that obtaining a clear tape preparation from the forepaw would give sufficient yield for identification of pollen types. Sixty-six dogs were sampled between 1 February and 10 March 2011. Two separate 3.18 cm2 squares of double-sided tape (Scrapbooking Tape, 3M; St. Paul, MN, USA) were adhered to a glass slide. For each dog, one tape square was firmly pressed against the anterior aspect of the third digit of the left forepaw, and the other tape square against the right. Slides were then stained with a glycerin jelly containing basic fuchsin stain, and pollen was identified via bright field microscopy. Eighteen different anemophilous pollen types were identified, and entomophilous and damaged pollens were grouped together and declared unidentified. There was a mean of 4.1 pollen types per dog (range from zero to 10). We counted a total of 1706 pollen grains, with a mean of 25.7 per dog (left and right paw samples combined). The most prevalent pollen types were pine, grass, unidentified, and oak with a mean of 9.97, 8.05, 4.48, and 0.61 grains per dog, respectively. Identification of pollen adhered to the canine coat could be used to aid in allergen selection for allergen-specific immunotherapy. Funding: Self funded. Conflict of Interest: None declared.


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P-018 Patch testing with feather hydrolysate, corn starch and a commercial diet containing corn starch and feather hydrolysate in chicken- and corn-allergic dogs C. JOHANSEN*, C. MARIANI and R. S. MUELLER* *Centre for Clinical Veterinary Medicine, Ludwig Maximilian University, Munich, Germany; Royal Canin, Aimargues, France An elimination diet and subsequent re-challenge detects food allergy and intolerance and therefore remains the most reliable diagnostic test currently available for these conditions. However, patch testing with food allergens recently was reported to have a high negative predictive value. This study evaluated patch testing with a novel diet containing corn starch and low-molecular-weight feather hydrolysate (Anallergenic; Royal Canin, Airmargues, France) in chicken- and corn-allergic dogs. We diagnosed food-adverse reactions against chicken (n = 5) and corn (n = 3, all of which were also allergic to chicken) using an elimination diet and rechallenge with the original food and individual allergens. Subsequently, a patch test was conducted on the lateral chest 48 h after clipping with a negative control (petroleum jelly), raw and cooked chicken, corn and the corn starch and lowmolecular-weight feather hydrolysate diet. After 48 h, reactions were evaluated. Erythema was seen in the chicken patch tests on all dogs with chicken-induced food reactions, on the corn patch test on all dogs with corninduced food reactions and on one dog on the corn starch and low-molecular-weight feather hydrolysate diet. This dog showed a recurrence of clinical signs when fed the novel diet. In conclusion, the novel corn starch and lowmolecular-weight feather hydrolysate (Anallergenic) seems useful in the management of dogs with adverse food reactions to chicken and corn. Funding: Royal Canin, Aimargues, France. Conflict of Interest: C Johansen’s residency is supported by Royal Canin, Aimargues, France and Kerstin Orstadius Minnesfond, Helsingborg, Sweden. C. Mariani is an employee of Royal Canin. In the last 5 years, R. Mueller has obtained funding, lectured or consulted for Bayer Animal Health, Boehringer Ingelheim, Dechra Veterinary Products, Intervet, Merial, Novartis Animal Health, Pfizer Animal Health, Procter & Gamble, Royal Canin, Selectavet, and Virbac.

P-019 Sensitization of dogs to pollens and its evolution in western France during the period 1999–2010 as determined by intradermal testing V. BRUET, A. ROUSSEL and P. BOURDEAU Dermatology, Parasitology and Mycology Unit, ONIRIS, Nantes, France Among aeroallergens, pollens play a significant role in allergic skin diseases of the dog. However, few studies have investigated over extended periods the seasonal aspect of pollen sensitization or its gradual change in dogs with atopic dermatitis. This study evaluated both of these aspects. Studies of canine pollen sensitization were carried out through statistical analysis of 261 intradermal tests 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

performed from 1999 to 2010 at the National Veterinary School of Nantes (Oniris). Allergens tested included eight weed pollens and 17 tree pollens. Intradermal tests were performed in dogs with atopic dermatitis diagnosed based on widely accepted criteria. Chi-squared tests were used for statistical analysis. We found that 213 dogs (81.6%) were sensitized to at least one allergen; 108 (41.4%) to tree pollens, and 91 (34.9%) to weed pollens. No increase in the percentage of positive intradermal tests was recorded over the three periods 1999–2002, 2003–2006, and 2007–2010. No statistical correlation was found between pollen sensitization and sex, age, or month of birth. Moreover, no link was found between sensitization and the season in which the intradermal testing was performed, suggesting that there is no seasonality in the sensitization. However, a significant increase in the percentage of dogs sensitized to pollens was observed between 1999–2002 and 2007–2010 (26% more for weed pollens, 25% more for tree pollens, respectively). This increase was not associated with a temporal increase of pollination in Nantes (Aerobiological Network of Surveillance). Funding: Self funded. Conflict of Interest: None declared.

P-020 Evaluation of Polycheck results used for causal allergen determination in canine atopic dermatitis N. TARPATAKI*, B. BIGLER and J. BOSZNAY* *Department and Clinic of Internal Medicine, Faculty of Veterinary Science, Szent Istvań University, Budapest, Hungary; Labor Laupeneck/Imovet bg, Bern, Switzerland The aims of this study were the following: to evaluate Polycheck test results used for causal allergen determination in canine atopic dermatitis, to collect data about the most common allergens involved in Hungary, and to compare them with existing publications from Europe and the rest of the world. Sera of 75 dogs with a clinical diagnosis of atopic dermatitis were examined by Polycheck test. The most prevalent allergens were Dermatophagoides farinae and Tyrophagus putrescentiae with a prevalence of 94.7%, followed by flea and Acarus siro (88% each), and D. pteronyssinus (82.7%). These results illustrate a major incidence of arthropod allergens in canine atopic dermatitis and are in accordance with many other studies and previous Hungarian results. Correlation analysis showed breed-related variations in response to allergen testing. Golden retrievers had a high number (P < 0.01) of positive reactions to D. farinae and Lepidoglyphus destructor, but reactions to Malassezia was rare (P < 0.01); Labrador retrievers had a high number of positive reactions to sorrel (P < 0.01), and Hungarian Vizslas had a high number of positive reactions to D. pteronyssinus and rye (P < 0.01). In contrast, L. destructor, birch/alder/hazel, plantain/willow/poplar, and grass mix were uncommon allergens in the West Highland white terrier (P < 0.01). Moreover, a certain degree of resistance of mixed-breed dogs to sensitization to flea allergens was noted. Our results suggest breed predispositions not only to atopic dermatitis in general, but also to sensitization to one or several specific allergens in particular. Funding: Self funded. Conflict of Interest: B Bigler is CEO of Labor Laupeneck/ imovet bg

Topic 4: THERAPY OF ALLERGIC DISEASES P-021 Factors affecting ciclosporin concentrations in canine skin A. HILLIER, K. E. MONING and L. L. GRAY The Ohio State University, Columbus, OH, USA Ciclosporin (Atopica; Novartis Animal Health; Greensboro, NC, USA) is approved for treatment of canine atopic dermatitis. We evaluated the effect of duration of treatment, body location, and timing of sampling on skin ciclosporin concentrations. Six healthy research dogs were divided into two groups of three each: group 1 (G1) received ciclosporin 5.0 mg/kg orally once daily for 28 days; group 2 (G2) received ciclosporin 2.5 mg/kg and ketoconazole 2.5 mg/kg orally once daily for 28 days. Skin (8-mm punch biopsy) and whole blood samples were collected weekly, skin samples were collected hourly two to six h after dosing on day 1 and day 28, and skin samples were collected at the same time from four body locations (dorsal neck, lateral neck, ventral abdomen and axilla) on days 15 and 21. Ciclosporin concentrations were quantified by high performance liquid chromatography mass spectrometry. There was no difference between G1 and G2 in the weekly mean skin (2.03 and 2.32 ng/mg, respectively) and whole blood concentrations (313 and 338 ng/mL, respectively) of ciclosporin. Skin concentrations were highly variable (0.29–8.59 ng/mg). We detected the highest 4-h postadministration skin ciclosporin concentrations on days 14 or 21 in G1 and day seven in all dogs in G2. Highest skin concentrations varied between two and six h postadministration, and no clear time of peak skin concentration could be determined. Mean ciclosporin skin concentrations by location showed dorsal neck>axilla>lateral neck>ventral abdomen. Given individual variability, monitoring skin ciclosporin concentrations may not be clinically useful. Funding: Canine Research Funds, The Ohio State University. Conflict of Interest: A. Hillier is a paid consultant, advisor, and speaker for Novartis Animal Health.

P-022 The effect of a spot-on formulation containing fatty acids and essential oils (Essential 6, Dermoscent, LDCA France) on epidermal ceramides of dogs with canine atopic dermatitis M. BLASKOVIC*, J. STAHL, W. BAÜMER and R. S. MUELLER* *Clinic of Small Animal Medicine, Centre for Clinical Veterinary Medicine, Ludwig-Maximilian University, Munich, Germany; University of Veterinary Medicine Hannover, Foundation, Institute of Pharmacology, Hannover, Germany In dogs and humans with atopic dermatitis there is evidence for a decreased skin barrier function. This may be caused by a deficiency of ceramides, especially ceramides of the classes 1 and 9, which are components of the stratum corneum intracellular lipid laminae. One possible mechanism of action of essential fatty acid

supplementation is improvement of the epidermal barrier function by changing the composition of the epidermal lipids. This study evaluated whether treatment with a topical spot-on containing fatty acids (Essential 6, Dermoscent; LDCA, France) applied once weekly for 8 weeks changes the epidermal lipid composition. The study was randomized, placebo-controlled, and doubleblinded. Twenty dogs with atopic dermatitis were treated with the spot-on or placebo applied once weekly directly on the skin of the dorsal neck. Before the study and after 8 weeks, skin samples were taken with a cyanoacrylate resin (VetbondTM Tissue Adhesive, 3MTM; St. Paul, MN, USA), which was placed on a microscope slide. Thin layer chromatography was performed to measure different ceramide classes, cholesterol, and free fatty acids. Using a Kruskal–Wallis test and Dunn post test we compared concentrations of ceramides, cholesterol, and free fatty acids in dogs treated with essential fatty acids or placebo before and after treatment. There was no significant change before and after the study or between groups. In conclusion, based on this study treatment effects with the spot-on containing essential fatty acids are not the result of a change in the lipid composition of the epidermis. Funding: LDCA France. Conflict of Interest: M Blaskovic was financially supported by LCDA France. In the last 5 years. R. Mueller has obtained funding, lectured or consulted for Bayer Animal Health, Boehringer Ingelheim, Dechra Veterinary Products, Intervet, Merial, Novartis Animal Health, Pfizer Animal Health, Procter & Gamble, Royal Canin, Selectavet, and Virbac.

P-023 Effect of a topically applied moisturizer containing physiological lipid granules on dogs with atopic dermatitis J. JUNG, E. NAM, S. PARK, S. HAN and C. HWANG College of Veterinary Medicine, Seoul National University, Seoul, Korea In humans with atopic dermatitis (AD), skin barrier dysfunction has been demonstrated and is thought to be responsible for increased transepidermal water loss and enhanced penetration of allergens. Studies in support of an abnormal skin barrier have shown decreased ceramides and abnormal contents and extrusion of the lamellar bodies. Similarly, canine AD is characterized by disarrangement of corneocytes and disorganization of intercellular lipids in the stratum corneum. In particular, decreased ceramide levels have also been shown in atopic dogs. This study evaluated the effect of a moisturizer containing physiologic lipids in dogs with AD. We measured several parameters, in addition to transmission electron microscopy (TEM) using ruthenium tetroxide post-fixation. Dogs with mild to moderate clinical signs (n = 20, aged 3–8 years) were recruited, and owners were instructed to apply the moisturizer containing physiologic lipids for 4 weeks. We evaluated transepidermal water loss, skin hydration, a pruritus index for canine atopic dermatitis (PICAD), and the canine atopic 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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dermatitis extent and severity index (CADESI) in all dogs. Transmission electron microscopy was performed in five dogs. Values for transepidermal water loss, PICAD, and CADESI decreased after 4 weeks of moisturizer application (P < 0.001). Skin hydration increased dramatically over that time (P < 0.05). In all five dogs for which transmission electron microscopy was performed, electron micrographs showed that the skin barrier was partially restored (P = 0.038). In conclusion, our results showed that the moisturizer containing physiologic lipids was effective in diminishing skin barrier dysfunction and improving the clinical condition of the skin in dogs with AD. Funding: Self funded. Conflict of Interest: None declared.

P-024 Acupuncture in the treatment of canine atopic dermatitis Y. CIMINELLI and A. COSTA-VAL Escola de Veterina´ria, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Canine atopic dermatitis is an itchy skin disease in which genetically predisposed dogs become sensitized to allergens commonly found in the environment. This chronic inflammatory skin disorder affects greatly the animal’s welfare, which is also unfavourably affected by some drugs used to control atopic dermatitis. Acupuncture is a potential alternative treatment, with no adverse effects to the patient. In order to test this hypothesis, three dogs with atopic dermatitis were treated with acupuncture sessions once per week for 8 weeks. The sessions lasted 20 min. Acupuncture needles were placed in multiple locations, according to 25 locations previously selected in traditional Chinese medicine. Before each session, we quantified the main clinical signs of canine atopic dermatitis, such as erythema, lichenification, excoriation, and self-induced alopecia by scoring them using the canine atopic dermatitis extent and severity index (CADESI)-03. The perception of the pruritus level was assessed by owners using the Hill scale. After eight sessions, a major decrease in CADESI-03 scores was achieved: 61.3% in dog 1; 83.4% in dog 2, and 53.4% in dog 3. The pruritus scores declined 19% in dog 1, 48% in dog 2, and 45% in dog 3. In conclusion, acupuncture showed beneficial effects for these three dogs with atopic dermatitis as it decreased their degree of the pruritus as well as clinical signs. Therefore, acupuncture promises to be a potential treatment modality for dogs suffering from this non curable skin disorder. Funding: Self funded. Conflict of Interest: None declared.

P-025 A six-month repeated-dose toxicity study of ciclosporin A oral solution in cats S. KING*, K. A. VAN LARE* and J. K. HEWARD *Novartis Animal Health US, Inc., Greensboro, NC, USA; MPI Research, Inc., Mattawan, MI, USA We examined the safety of ciclosporin (ATOPICA for Cats, Novartis Animal Health; Greensboro, NC, USA) given at 0, 8, 16, 24, or 40 mg/kg/day (0, 1·, 2·, 3·, or 5· the upper dose range) for 6 months in 40 cats (four cats/ 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

sex/group). Cats were approximately 6 months old and 2.0–5.2 kg on day 1. At study end, cats were euthanized and necropsied. One female cat receiving 5· dose was euthanized in extremis on day 14; clinical signs included decreased body weight (approximately18%). At postmortem, a healing rib fracture and bone marrow hypocellularity were observed and judged to be incidental to treatment. Treatment effects included increases in soft faeces, body weight, and food intake. Possible treatment effects included lymphoma involving the kidneys and a mesenteric lymph node in one male cat receiving 5· dose, and intermittent interventricular conduction disturbances in one male (3· dose) and one female (5· dose) during month 6 of treatment. No hepatic or renal toxicity was observed in any cat. Prolonged activated partial thromboplastin time was seen at dose levels >1·, but was considered unlikely to be biologically significant. Maximal plasma drug concentration and area under the curve were not dose-proportional at dose levels >1·. Bioaccumulation values in cats receiving 1· dose indicated a low potential for bioaccumulation with long-term dosing of ciclosporin. Daily oral administration of ciclosporin for 6 months was well tolerated in cats receiving 1· and 2· the upper dose range. These results support the long-term safety in cats of ciclosporin at clinically relevant doses. Funding: Novartis Animal Health US, Inc. Conflict of Interest: K Van Lare and S King are employees of Novartis Animal Health.

P-026 Ciclosporin efficacy in the treatment of feline hypersensitivity dermatitis is not influenced by the feeding status J. STEFFAN, S. KING and W. SEEWALD Novartis Animal Health, Basel, Switzerland Feeding status influences the pharmacokinetic parameters of ciclosporin A (CsA) microemulsion in cats as well as dogs. Maximum bioavailability is achieved when the solution is given to fasted cats or mixed with their food. An approximate 22% reduction in bioavailability occurs when CsA is administered just after feeding. We evaluated the influence of feeding status in a clinical study in which cats received the therapeutic dose of ciclosporin (Atopica for Cats, Novartis Animal Health, Basel) mixed with food or given after feeding. Cats were enrolled when diagnosed with hypersensitivity dermatitis manifested with lesions of excoriations, miliary dermatitis, bilateral alopecia, and/or eosinophilic plaques along with pruritus. Animals received CsA at a target dose of 7.0 mg/kg once daily for 6 weeks, tapering off according to the clinical response. All cats initially received the solution mixed with food. If the cat refused the food for two consecutive days, treatment was administered on the following day directly into the cat’s mouth after feeding. The clinical response was assessed after 3, 6, 10, 14, 16, and 20 weeks. Lesion severity and extent along with pruritus were evaluated at each time point. Mean scores at study exit indicated a similar response independent of administering CsA mixed with food or directly into the cat’s mouth. The time response curve was similar for all other parameters. At the final visit, there was no significant difference for any of the efficacy parameters. The total lesion and pruritus scores were low in cats dosed directly into the mouth or with food.

Abstracts Funding: Novartis Animal Health. Conflict of Interest: All authors are employees of Novartis Animal Health.

P-027 Evaluation of efficacy, adverse effects, and quality of life in atopic dogs treated with ciclosporin A. V. YAZBEK and C. E. LARSSON Faculdade de Medicina Veterina´ria e Zootecnia da Universidade de Saõ Paulo, Saõ Paulo, Brazil Atopic dermatitis is a chronic allergic skin disease. Pruritus is the predominant sign leading to intense suffering of the animal and its owner. ‘Quality of life’ is increasingly requested from owners of animals with allergic skin diseases and other chronic diseases. Ciclosporin (CsA) is considered a good therapeutic option in the treatment of canine atopic dermatitis. The objectives of this study included: analysis of the efficacy of CsA in reducing skin lesions and pruritus of 21 atopic dogs using canine atopic dermatitis extent and severity index (CADESI)-03 and two scales to quantify levels of itching; detection of adverse effects secondary to CsA therapy (Sandimmun Neoral; 5 mg/kg, once daily for 60 days); evaluation and monitoring of quality of life of dogs treated with CsA with a validated scale. Ciclosporin was considered an effective treatment for atopic dogs because it reduced the mean skin lesion scores by 70% (P = 0.032) after 60 days of therapy. During that period, there was a reduction of body itching (numerical scale ranging from zero to 10) of 52.6% (P < 0.001), and there was significant (P < 0.001) reduction on a modified qualitative scale of body itching. Gastrointestinal signs were observed and appeared most often in the first 15 days of therapy. Laboratory abnormalities were not detected. The quality of life of these atopic dogs treated with CsA for 60 days was improved by 32%. Ciclosporin was effective and safe in the treatment of dogs with atopic dermatitis and improved their quality of life. Funding: Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo. Conflict of Interest: None declared.

P-028 Evaluation of CD4+/CD8+ ratio, canine atopic dermatitis extension and severity index, and pruritus visual analog scale in 15 atopic dogs before and after treatment with ciclosporin A L. CORNEGLIANI*, S. COMAZZI, M. BECCATI, V. MARTINI and A. VERCELLI* *Ambulatorio Veterinario Associato, Torino, Italy; Dipartimento di Patologia Animale, Igiene e Sanita` Pubblica Veterinaria, Universita` di Milano, Milano, Italy; Centro Medico Veterinario Adda, Bergamo, Italy Ciclosporin (CsA) is commonly used in veterinary dermatology to treat canine atopic dermatitis, because of its selective ability to block the production of inflammatory cytokines. The CD4+/CD8+ ratio can be useful for evaluating the immune system for the

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indirect correlation with specific lymphocytic immune response (Th1/Th2 ratio). This prospective study evaluated the CD4+/CD8+ ratio, CADESI (canine atopic dermatitis extent and severity index), and pruritus using a visual analogue scale (VAS), in atopic dogs before and after treatment with ciclosporin. Fifteen dogs with canine atopic dermatitis were included. We performed flow cytometry, CADESI, and VAS evaluations on day 0 (V0), day 30 (V30), and day 90 (V90) of CsA therapy. Healthy dogs were used as controls for evaluation of the CD4+/CD8+ ratio. Statistical analysis was performed with SPSS17 software. Analysis of variance (ANOVA) for repeated measures was used to evaluate differences in CADESI and CD4+/CD8+, and the Friedman test was used to analyze differences in VAS. For all analyses, P < 0.05 was considered significant. Results showed that on day 90 CADESI and VAS decreased significantly (P = 0.001); however, there was no change in the CD4+/CD8+ ratio (P = 0.694). Based on these results, CsA significantly decreased CADESI and VAS index improving the clinical condition of affected dogs, but did not modify the CD4+/CD8+ ratio. Funding: Self funded. Conflict of Interest: None declared.

P-029 Multicentre open trial demonstrates efficacy of sublingual immunotherapy in canine atopic dermatitis D. J. DEBOER* and M. MORRIS *School of Veterinary Medicine, University of WisconsinMadison, Madison, WI, USA; Allergychoices, Inc., La Crosse, WI, USA Allergen-specific immunotherapy is commonly administered via the sublingual route in human atopic disease. There is renewed interest in sublingual immunotherapy for atopic dermatitis in humans, especially with recent evidence that it may function by different mechanisms than does injection immunotherapy. A previous pilot study of sublingual immunotherapy in dogs sensitive to house dust mites provided evidence of clinical benefit and coincident immunologic changes. The present study evaluated the clinical efficacy of sublingual immunotherapy in a larger group of dogs. Nine veterinary dermatology specialty clinics enrolled a total of 217 dogs with atopic dermatitis in an open study on the efficacy of sublingual immunotherapy. All dogs received twice-daily administration of an escalating-dose, non-aqueous sublingual immunotherapy formulation devised according to individual-tested sensitivities. The response of each patient after at least 6 months of sublingual immunotherapy was graded by the clinician according to four subjective response categories. Of 124 evaluable cases, 68 dogs (55%) were judged to have a good-to-excellent response to sublingual immunotherapy. Among these 124 dogs, 77 dogs that had received no previous immunotherapy had a response rate of 59%. The remaining dogs (n = 47) had failed injection immunotherapy due to lack of efficacy, adverse reactions, or compliance difficulties. Of these injection failures, 23 dogs (49%) had a good-toexcellent response to sublingual immunotherapy. In this multicentre, open trial, we conclude that sublingual immunotherapy appears to be an effective treatment for canine atopic dermatitis, including in dogs that have failed injection immunotherapy. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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Funding: Self funded. Conflict of Interest: M. Morris has an ownership interest in Allergychoices, Inc.

P-030 Investigations on the effects of sublingual immunotherapy on clinical signs and immunological parameters using a canine model of atopic dermatitis: a double-blinded, randomized, controlled study R. MARSELLA and K. AHRENS University of Florida, Gainesville, FL, USA This prospective, randomized, controlled study evaluated clinical and immunological effects of 1 year of sublingual immunotherapy. Eighteen atopic beagles, sensitized to dust mites, timothy grass, and ragweed were randomly divided into control (n = 6, vehicle) and active (n = 12, three allergens) groups. Allergen challenge and scoring of clinical signs during challenge was done before and at the end of sublingual immunotherapy. Clinical signs (without challenge) were scored 1, 2, 3, 4, and 8 months of sublingual immunotherapy and 2 months after stopping immunotherapy. Blood was drawn at baseline, four, eight, and 12 months of sublingual immunotherapy and 2 months after stopping for allergen-specific IgE, IL-10, and TGF-beta. For clinical scores, analysis of variance (ANOVA) showed significant effect of time (end < beginning). One dog in each group worsened at end of study. Improvements were as follows: in the controls 0 > 80%, 1/6 61–80%, 2/6 41–60%; 2/6 21–40%, 0 < 20%; in the active group 0 > 80%, 1/12 61–80%, 7/12 41–60%, 2/12 21–40%, 0 < 20%. Overall, the percentage of dogs that improved > 40% was 50% in the control and 66% in the active group. For allergen-specific IgE a significant effect of time was found for dust mites (end < beginning) and ragweed (end > beginning). For TGF-beta, significant effects of group (active > control) and time (end > beginning) were found for ragweed. For IL-10, a significant effect of group (active > control) and time (end > beginning) was found for ragweed. Also for IL10, a significant effect of time (end > beginning) and group · time interaction were found for timothy grass. Funding:Partly funded by Nelco and partly self funded. Conflict of Interest: None declared.

P-031 The efficacy of cetirizine hydrochloride on the pruritus and dermatitis of cats with mild to moderate atopic dermatitis: a randomized, double-blinded, placebo-controlled, cross-over study K. WILDERMUTH*, S. ZABEL and R. A. ROSYCHUK* *Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA; Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA, USA This study investigated the efficacy of cetirizine hydrochloride (CTZ, Zyrtec: McNEIL-PPC; Skillman, NJ, 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

USA) on the pruritus and dermatitis of cats diagnosed with mild to moderate atopic dermatitis. We enrolled cats with a canine atopic dermatitis and severity index (CADESI-03) score modified for the feline patient of ‡ 25 and < 125 and a pruritus score of ‡ 3 and < 7. Cats received either 1 mg/kg CTZ (group 1) or placebo (group 2) orally, once daily for 28 days, followed by a 14day wash-out period. Treatments were then crossed over, and cats received either placebo (group 1) or CTZ (group 2) orally for 28 days. Owners marked a pruritus severity scale on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70. Lesions were scored by the clinician based on the modified CADESI-03 before initiating and on the last day of each treatment (days 0, 28, 42, 70). Data of all enrolled cats were included in the analysis (intention-totreat analysis). For each outcome, the effect of CTZ compared to placebo was evaluated using mixed effects linear regression. Twenty-one cats were enrolled and 19 completed the study. One cat was lost to follow up after day 28 and another cat was excluded after day 28 due to severity of clinical signs. There were no significant statistical differences between treatment with CTZ and placebo for modified CADESI-03 or pruritus scores. Based on these results, CTZ is not effective for reducing pruritus and dermatitis in cats with mild to moderate atopic dermatitis. Funding: American College of Veterinary Dermatology resident research award; Royal Canin USA, Inc.; Heska Corporation. Conflict of Interest: None declared.

P-032 Investigations on the effects of a topical ceramide and free fatty acid solution (Allerderm Spot-On) on clinical signs and skin barrier function in dogs with atopic dermatitis: a double-blinded, randomized, controlled study R. MARSELLA, D. GENOVESE, L. GILMER and K. AHRENS Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA The skin barrier is impaired in canine atopic dermatitis, and topical application of lipids has been suggested to be beneficial. This double–blinded, controlled clinical trial investigated the efficacy of Allerderm Spot-On (Virbac, Forth Worth, TX, USA) for clinical signs and skin barrier function in dogs with atopic dermatitis. Thirtytwo dogs diagnosed with atopic dermatitis were divided into two groups: 16 received Allerderm three times weekly on assigned areas for 4 weeks and the rest received a control solution. Areas treated were pinna, antebrachial, axilla, and groin. Skin barrier was assessed by measurement of transepidermal water loss (TEWL) using a closed-chamber evaporimeter and clinical signs by canine atopic dermatitis extent and severity index (CADESI). Dogs were evaluated at days 0, 14, and 28. One investigator did all clinical evaluations and the TEWL measurements were done by two different investigators. Due to the possibility of operator effect on results, TEWL values were analyzed separately. Analysis of variance (ANOVA) was used for statistical analyses, and P < 0.05 was considered significant. For total

Abstracts CADESI, we found a significant effect of time (day 28 < day 0) and group (Allerderm < control). For TEWL, in one subset (n = 10) no differences were found, while in the other subset (n = 22) a significant effect of group (Allerderm < control) in antebrachial and axillary area as well as of time (day 28 < day 0) were found. Interestingly, an effect of laterality was found (right > left). It is concluded that topical application of

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ceramides has beneficial effects in canine atopic dermatitis after several weeks of application. Funding: This abstract discusses two clinical trials conducted at two different times. The first was self-funded and completed in the summer of 2009 (10 dogs), and the second was funded by Virbac (22 dogs). Conflict of Interest: None declared.


Topic 5: BACTERIAL DISEASES AND THERAPY P-033 Preliminary evaluation of the bacterial microbiome of the skin and ear in dogs A. STURGEON, M. COSTA and J. S. WEESE University of Guelph, Guelph, ON, Canada The skin harbours a commensal microbial population, yet this population has not been adequately investigated in dogs. This study used advances in sequencing and bioinformatics to characterize the skin and ear bacterial microbiomes in healthy dogs. Swabs were collected from the ear, mid-back, inguinal region, and interdigital space from two dogs. We performed 16s rRNA analysis (V3– V4 region), followed by next-generation sequencing. We obtained 8739 reads and identified 38–110 different species in skin samples, with only 16 aural species. The Firmicutes phylum predominated (85–97%) except in an inguinal sample that had 58% Actinobacteria (from an abundance of Collinsella aerofaciens). With the exception of that sample, Staphylococcus (mean 49%, range 13– 83%) and Lactobacillus (mean 29%, range 1–59%) predominated. Gammaproteobacteria were uncommon (2.4%), with Escherichia accounting for 1.6% of sequences (range 0–8.1%) and Pseudomonas for 0.6% (range 0–1.6%). Lactobacilli spp. predominated in the ear (81%), followed by Staphylococcus epidermidis (11%). Overall, 10 Staphylococcus spp. were identified. Dermatophilus congolensis was identified in one dog. A wide range of unusual genera were identified, including Agrobacterium, Alcanivorax, Alicyclobacillus, Anaeromyxobacter, Anaerotruncus, Anoxybacillus, Azospirillum, Bibersteinia, Butyricococcus, Butyrovibrio, Chitinophaga, Cylindrospermopsis, Hespellia, Kinetoplastibacterium, Lechevalieria, Methylobacterium, Methylocella, Roseivivax, Sphingomonas, and Terrimonas, most of which have not been previously reported in dogs. These preliminary data indicate a complex and diverse skin (and to a lesser extent ear) bacterial microbiome in dogs. Further study of these microbiomes and what affects them may provide critical information about how they may be manipulated to understand the pathophysiology, prevention, and treatment of disease. Funding: Medi-Cal/Royal Canin Canada Conflict of Interest: None declared.

P-034 In vitro antimicrobial activity of a spot-on containing a mixture of essential oils and a plant extract against Staphylococcus pseudintermedius and Malassezia pachydermatis E. BENSIGNOR*, L. FABRIE`S and C. MARTIN-VO *Veterinary Dermatology Referrals, Rennes-Cesson, Paris, Nantes, France; Veterinary Clinic, Toulouse, France; Laboratoire de Dermo-Cosme´tique Animale (LDCA), Castres, France This study evaluated the antimicrobial potential of a commercial preparation (Dermoscent PYOspot, LDCA; Castres, France) containing a mixture of essential oils and a specific plant extract against Staph 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

ylococcus pseudintermedius (CIP 81.60) and Malassezia pachydermatis (IP 1649.86). We determined the minimum inhibitory concentrations (MICs) using the microbroth dilution technique, and we obtained the minimum bactericidal or fungicidal concentrations (MBCs or MFCs) through subculturing on agar media using a Denley multipoint inoculator. We included positive controls containing a tryptone salt dilution of each microorganism culture and growth medium, and negative controls containing growth medium and the test product. Staphylococcus pseudintermedius was cultured aerobically on Muller Hinton broth or agar at 37C and M. pachydermatis anaerobically on modified Dixon broth or agar at 32.5C. For S. pseudintermedius, the minimum inhibitory concentration was between 1/65536 and 1/ 32568 dilution of the product, and the minimum bactericidal concentration was between 1/512 and 1/256. The minimum inhibitory concentration and the minimum bactericidal concentration for M. pachydermatis were between 1/64 and 1/32 dilution of the product and 1/32, respectively. These preliminary in vitro results suggest that the test product could be useful in association with conventional antimicrobial therapy for the treatment of pyoderma or Malassezia dermatitis in dogs. Clinical trials are necessary to confirm the obtained outcomes. Funding: Laboratoire de Dermo-Cosme´tique Animale, Castres, France. Conflict of Interest: C. MARTIN-VO is an employee of LDCA.

P-035 Meticillin-resistant Staphylococcus aureus: an emerging pathogen in veterinary teaching medical hospitals M. NADEEM ASI*, M. SAQIB*, G. MUHAMMAD* and M. TOUFEER *Department of Clinical Medicine and Surgery, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan; Institute of Microbiology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan Meticillin-resistant Staphylococcus aureus (MRSA) infection is a growing problem that could ultimately result in the death of affected companion animals. Unfortunately, despite the serious health concerns, limited work has been done on MRSA infections in Pakistan. The present study determined the prevalence and epidemiological features of MRSA infections in dogs in a veterinary teaching hospital. Ninety-seven dogs with accidental and surgical wounds admitted to the veterinary medical teaching hospital were recruited regardless of clinical history. Samples were collected from wounds and nares of the dogs. Initial screening of MRSA was done with a series of differential media and the API Staph System. Staphylococcus aureus-positive samples were subjected to polymerase chain reaction (PCR) for the amplification of the nuc and mecA genes. We also tested for sensitivity to a broad range of antibiotics. Seventy-three of 97 samples (75.3%) yielded S. aureus. Of the 73 isolates, 35 (47.9%) were meticillin-sensitive S. aureus (MSSA), whereas 38 isolates (52.1%) were MRSA. We found that high rates

Abstracts of MRSA nasal colonization and wound infections were present in the feral dog population (34.78%, 57.14%, respectively) compared with indoor dogs (7.84%, 5.88%, respectively). All MRSA isolates were resistant to oxacillin, cefoxitin, penicillin, and meticillin. Higher rates of MRSA in dogs may be a result of the increased mobility of resistant genes in the bacterial population, making it a serious emerging veterinary pathogen. Funding: Self funded. Conflict of Interest: None declared.

P-036 Diversity of Staphylococcus spp. strains isolated from canine bacterial superficial pyoderma E. BENSIGNOR*, P. BOURDEAU, A. KODJO, E. THIBAULT§, J. L. PELLERIN– and H. GANTELET§ *Dermatology Referral Service, Rennes-Cesson, France; Laboratoire de Dermatologie, Parasitologie et Mycologie, Oniris, Nantes, France; Laboratoire des Leptospires, VetAgro Sup-Campus Ve´te´rinaire, Marcy l’e´toile, France; §Laboratoire Biovac, Beaucouze´, France; – Unite´ Microbiologie-Immunologie, Oniris, Nantes, France Eleven dogs suffering from multifocal superficial pyoderma were selected to take part in a study to characterize the bacterial flora present in the clinical lesions. Each animal had four samples taken: three from lesions and one from an apparently healthy area. After bacteriological analysis of the samples, we identified Staphylococcus spp strains from all four samples of 10 cases (total of 40 samples). Staphylococcus pseudintermedius was confirmed by molecular biology techniques to be the isolate in a majority of the samples (34/40) taken from the 10 dogs. In further analysis, we compared the isolated strains by pulse field gel electrophoresis (PFGE). In six of 10 cases the strains taken from a single dog presented a clonal relation. In the additional four cases, genetic diversity was observed. In order to understand this diversity, we compared the antibiotic susceptibility among the strains isolated from each of these four cases. Major differences in antibiotic susceptibility were observed between Staphylococcus pseudintermedius strains taken from the same animal. These results show that different staphylococcal strains may be associated with different lesions in the same patient; therefore, it is very important that clinicians sample multiple lesions and perform antibiotic susceptibility of all isolated strains before starting antibiotic therapy. Funding: Biovac. Conflict of Interest: E Thibault and H Gantelet are employees of Biovac.

P-037 Analysing the selection of cefalosporin-resistant staphylococci mutants using cefalosporin antibiotics K. ISHIHARA*, H. FUJITA*, Y. TOYODA, K. IYORI, C. KAMEDA, K. IDE, K. NISHIFUJI, T. IWASAKI and Y. TAMURA* *Rakuno Gakuen University, Ebetsu, Hokkaido, Japan; Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; Pfizer Japan Inc., Tokyo, Japan

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The emergence of staphylococcal isolates resistant to cefalosporins, which had been often used to treat dogs with pyoderma, has been reported. We investigated in vitro the spontaneous appearance of cefalosporins-resistant staphylococcal mutants from susceptible isolates, and tested the susceptibility to cefalosporins of staphylococcal isolates from dogs with pyoderma. To select spontaneous cefalosporins-resistant mutants, we inoculated approximately 1010 CFU of two canine cefalosporins-susceptible Staphylococcus pseudintermedius isolates without the mecA gene in a medium with four times the minimal inhibitory concentrations (MICs) of cefalexin (4 mg/L) and cefovecin (1 mg/L) for each strain. Thus, cefalexin-resistant mutants (MICs, 8–16 mg/L) were obtained from each susceptible isolate at a frequency of 5.1 · 10)9 and 1.9 · 10)8. The cefovecin MICs of these mutants were equal to those of each susceptible (parental) strain. One isolate did not yield a cefovecin-resistant mutant (< 3.6 · 10)11). The cefalexin and cefovecin MICs were determined for 57 S. pseudintermedius isolates from 30 dogs. The mecA gene was tested in all isolates using polymerase chain reaction (PCR). mecA was detected in 29 isolates resistant to cefalexin (MIC, ‡ 8 mg/L) and cefovecin (‡ 8 mg/L), one resistant to cefalexin, and three susceptible to both antibiotics. One isolate resistant to only cefalexin, and 23 susceptible isolates did not harbour mecA. Since almost all the cefalosporins-resistant isolates from dogs harboured mecA, we concluded that this gene contributes to staphylococcal cefalosporins resistance. However, spontaneous cefalexin-resistant staphylococcal mutants were obtained in vitro. The study underscores the importance of characterizing the resistance profile of diagnostic isolates. Funding: Pfizer Japan Inc. Conflict of Interest: C Kameda is an employee of Pfizer Japan.

P-038 Canine leproid dermatitis: a retrospective study of 26 cases from Costa Rica A. BERROCAL Private Laboratory, Heredia, Costa Rica Canine leproid granuloma syndrome or canine leprosy was first described in 1973 in Zimbabwe. Since then, it has been also reported in Australia, Unites States, and Brazil. Clinically, it is presented as a nodular, firm, painless, sometimes ulcerated lesion located on the dorsal fold of the pinnae. Short-coated breeds such as boxers and boxer-crosses are overrepresented. The aetiopathogenesis is unknown. In addition to clinical presentation, the diagnosis is based on demonstrating the mycobacteria within the inflammatory cells with an acid-fast stain. This study reports for the first time this dermatitis in Central America. Between 2005 and 2011, 26 cases of canine leproid granulomas were diagnosed. In all the cases a skin biopsy was taken and processed routinely for histopathological examination. The samples were stained with haematoxylin and eosin (H&E) and later with FiteFaraco. The most affected breeds were boxers (7 [27%]), Rottweilers (4 [15%]), mixed breed (4 [15%]), and American Staffordshire (3 [11%]). Age ranged from 2 to 14 years (average 6 years). In 22 cases (85%) the pinna was affected (13 dogs, both ears; five, one ear; and four, both pinna and other anatomic areas). The clinical presentation was single or multiples nodules, sometimes ulcerated, non responsive to anti-inflammatories and/or 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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Abstracts

antibiotics. Histopathologically, the dermis and subcutis showed a nodular to diffuse presence of macrophages with abundant pale foamy cytoplasm, mixed with neutrophils and lymphocytes. All cases were positive with a Fite-Faraco stain. The epidemiological, clinical, and histopathological findings are very similar to those previously reported in the literature. Funding:Self funded. Conflict of Interest: None declared.

P-039 Efficacy and adverse effects of rifampicin in canine pyoderma M. DE LUCIA*, M. BARDAGI, M. CALDIN*, L. FERRER, M. MONACO, I. RAVERA, F. SCARAMPELLA§, G. ZANNA§ and A. FONDATI* *Clinica Veterinaria Privata San Marco, Padua, Italy; Universitat Auto`noma de Barcelona, Barcelona, Spain; Centro Veterinario Prati, Rome, Italy; §Studio Dermatologico Veterinario, Milan, Italy Rifampicin is an antibiotic recently reported as effective in canine infections due to meticillin-resistant staphylococci (MRS). Medical records of dogs treated with rifampicin for superficial or deep pyoderma due to multidrug resistant and MRS strains were retrospectively selected. Data regarding response to treatment and adverse effects were reviewed. Nineteen patients (20 treatment courses), including 18 large breed (> 20 kg) and one medium breed (15 kg) dogs, from 2 to 13 years of age (median 7 years) were treated with 5–11 mg/kg of rifampicin twice daily for one to 10 weeks (median 4 weeks). Concurrent systemic and topical antibiotics or antiseptics were used in 19/20 dogs. Response to treatment was good in 15/19 and poor in 5/19 dogs. Vomiting (2/20), anorexia (2/20), haemolytic anaemia (1/20), and ultrasonographic hepatic abnormalities (1/20) were observed after 4 weeks of treatment. Elevations of alkaline phosphatase (7/10) and alanine aminotransferase (1/10) were reported within the second week of treatment, and total bilirubin increased in 3/16 dogs by the fourth week of treatment. Blood parameters were rechecked in three dogs and normalized in 6–11 weeks after rifampicin withdrawal. Uninvestigated sudden death was reported in a dog after 4 weeks of treatment and in another dog 4 weeks after treatment withdrawal. Rifampicin associated with topical and systemic antibacterial treatment could be an effective therapeutic option for canine pyoderma caused by multi-drug resistant and MRS infections. Monitoring of complete blood cell count, liver enzymes, and total bilirubin level before, during, and after treatment are recommended. Funding: Self funded. Conflict of Interest: None declared.

P-040 Management of pyoderma in feline eosinophilic dermatitis using oral doxycycline and a topical formulation with fusidic acid and betamethasone valerate: an open-label trial in 14 cats G. MANIGOT Dermlink Buenos Aires, Buenos Aires, Argentina Fourteen cats diagnosed clinically and cytologically with eosinophilic dermatitis and secondary pyoderma were 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

included in the trial. All cats were free of topical, oral, or injectable medication at the time of inclusion. Complete blood cell count, chemistry profile, feline immunodeficience virus (FIV), feline leukemia virus (FLV), feline coronavirus, and toxoplasmosis titres were performed. Nine cats were castrated males and five were spayed females. Ten cats were domestic short hair, two domestic long hair, and two Siamese. All cats were treated with doxycycline and a topical antibacterial/steroid formulation for 3 weeks. Doxycycline was administered once daily with good tolerance by the patients and good owner compliance. Cost was considered low compared to other antibiotics. No adverse reactions were observed. A topical formulation containing 2% fusidic acid and 0.1% betamethasone valerate in an emulsion was also prescribed to be applied once daily over the affected areas, using a finger unit for each area and not more than five finger-unit areas per cat/day, for 3 weeks. The topical formulation was well tolerated and quickly absorbed. All cats completed the treatment protocol. Seven cats (50%) had a good response, and three had a partial response. Four cats didn’t respond. The cats with the weakest response were positive to FLV, FIV, toxoplasmosis or coronavirus (with feline infectious peritonitis signs). In conclusion, oral doxycycline at 10 mg/kg combined with a topical formulation containing 2% fusidic acid and 0.1% betamethasone valerate was a safe and efficient option for cats with secondary pyoderma and eosinophilic dermatitis. Funding: Self funded. Conflict of Interest: None declared.

P-041 In vitro efficacy of pradofloxacin in canine recurrent pyoderma: a prospective laboratory study L. CORNEGLIANI, A. VERCELLI and A. CORONA Ambulatorio Veterinario Associato, Torino, Italy Pradofloxacin is a fluoroquinolone recently introduced in veterinary medicine. It has good antimicrobial activity against Gram-positive bacteria. Canine pyoderma is generally caused by Staphylococcus pseudintermedius and increasing resistance of strains to various antimicrobial drugs, makes the management of pyoderma difficult. This study determined the antimicrobial susceptibility of bacterial strains to pradofloxacin and other commonly used antibiotics in the management of dogs with recurrent pyoderma. Swabs were obtained from 60 dogs with pyoderma and inoculated onto blood agar, mannitol salt agar and MacConkey agar. The plates were incubated for 24–48 h. Isolated bacteria were Staphylococcus (pseud)intermedius (34–57%), Escherichia coli (9–15%), Pseudomonas aeruginosa (6–10%), Proteus vulgaris (5– 8%), S. aureus (3–5%), Enterococcus spp., Klebsiella spp. and Streptococcus b-haemolytic (3–6%). Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method in Muller Hinton agar. We tested amoxicillin-clavulanic acid, cefadroxil, cefalexin, cefovecin, clindamicin, enrofloxacin, marbofloxacin, oxacillin, and pradofloxacin. All S. (pseud)intermedius isolates were sensitive to pradofloxacin (100%), including the strains resistant to oxacillin (12–35%). The data showed high resistance of S. (pseud)intermedius and S. aureus to

Abstracts cefovecin (43%) and cefadroxil and cefalexin (41% each). Only two E. coli and one P. aeruginosa were resistant to pradofloxacin. Pradofloxacin is of great interest and could be considered a new veterinary drug for treatment of difficult or recurrent cases of canine pyoderma resistant to commonly used antibiotics. Funding: Self funded. Conflict of Interest: None declared.

P-042 Comparative in vitro efficacy of antimicrobial shampoos for Staphylococcus pseudintermedius and Staphylococcus schleiferi subsp. coagulans E. WATANDO*, N. MURAYAMA, Y. TERADA, M. OKUAKI, Y. KATAOKA*, M. NAGATA and K. HARADA* *Nippon Veterinary and Life Science University, Tokyo, Japan; ASC Dermatology Service, Tokyo, Japan Topical antimicrobial therapies are used for canine pyoderma with Staphylococcus pseudintermedius (SP) and S. schleiferi coagulans (SS), but the short killing rates of topical biocides against SP and SS have not been

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fully evaluated. This study assessed the in vitro efficacy of antimicrobial shampoos against SP and SS. Four and three strains of SP and SS, respectively, were incubated for 1, 3, 5, 10, and 15 min with 2% chlorhexidine acetate (Nolvasan Surgical Scrub; Fort Dodge Animal Health, Iowa, USA), 2.5% benzoyl peroxide (Paxcutol; Virvac JP., Osaka, Japan), and 10% ethyl lactate (Etiderm: Virvac JP., Osaka, Japan), and were added to soybean casein digest agar with lecithin and polysorbate 80 (SCDLP) broth to inactivate these shampoos. The SCDLP broth was diluted with sterile saline, and the diluted broth was spread-plated on SCDLP agar. After 24 h of aerobic incubation, colonies growing on the media were counted. Minimal bactericidal concentrations (MBCs) of SS and SP showed no differences. The MBCs of 2% chlorhexidine acetate were 1:500 at 1 min and 1:5000 at 5 min. The MBCs of 2.5% benzoyl peroxide was 1:5 at 5 min, but at the concentration of 1:100 at 15 min bacterial growth was noted. The MBCs of 10% ethyl lactate were 1:5 at 3 min and 1:100 at 10 min, but at the concentration of 1:200 at 15 min bacterial growth was noted. We conclude that 2% chlorhexidine acetate is more effective against SS and SP compared to 2.5% benzoyl peroxide and 10% ethyl lactate. Funding: Self funded. Conflict of Interest: None declared.


Topic 6: CASE REPORTS P-043 Alopecia secondary to owner’s topical hormone replacement therapy in six dogs D. BERGER, T. LEWIS and A. SCHICK Dermatology for Animals, Gilbert, AZ, USA Six dogs (from three households) diagnosed with endocrine alopecia secondary to topical hormone exposure are reported. These included three spayed females and three neutered males composed of three pug dogs, two basenji dogs, and one Boston terrier that ranged in age from 1.5 to 11 years with an average body weight of 10.6 kg (6.8– 16.6 kg). Duration of alopecia prior to presentation ranged from 2 months to 2.5 years. All dogs demonstrated alopecia affecting ventral surfaces, proximal lateral extremities, and lateral trunk. The head, distal extremities, and dorsum were spared. Five dogs had physical exam findings suggestive of feminization (mammary development or vulvar enlargement). At the time of diagnosis, 4/6 dogs had complete blood counts and serum chemistry profiles within reference ranges. In all cases serum total thyroxine was within normal reference range. Two dogs had pituitary-adrenal axis testing either by low-dose dexamethasone suppression or adrenocorticotropic hormone stimulation; both were within reference range. Affected skin was biopsied in 5/6 dogs. All biopsies shared four similar histological characteristics: basal melanosis, epidermal and follicular hyperkeratosis, atrophic hairless telogen follicles, and small sebaceous glands. All dogs had elevated estradiol levels, and 4/6 had a concurrent elevation of progesterone values. All three owners were subsequently found to be applying hormone replacement therapy topically to their medial forearms. Average time from the owner beginning therapy to development of clinical signs was 5.5 months (range four to 6 months). Following discontinuation of topical hormone therapy by the owners, all dogs had significant to complete resolution of their alopecia. Funding: Self funded. Conflict of Interest: None declared.

P-044 Paraneoplastic pemphigus foliaceus in a cat A. RECHE JR*, L. WANG, C. A. GERALDO JR, F. SALVAGNI and C. E. LARSSON* *School of Veterinary Medicine and Animal Science, University of Saõ Paulo, Saõ Paulo, Brazil; Vetmasters, Saõ Paulo, Brazil; Lab & Vet, Saõ Paulo, Brazil Feline pemphigus foliaceus (PF) is an autoimmune disease rarely reported in Brazil. To the authors¢ knowledge there are no reports of feline PF associated with internal neoplasia. A female cat, mixed-breed, 20 years old, was previously treated with cefalosporins, fluoroquinolones, and corticosteroids for a skin condition without satisfactory results with decreased appetite and weight loss. On physical examination, the following was seen: asthenia, ambulatory difficulty, hypothermia, and mild dehydration. Skin lesions on the face, ears, neck, and abdomen included alopecia, erythema, and crusts. The paws were alopecic and oedematous with purulent periungueal crusts and were painful on palpation. Parasitological examination of skin scrapings was negative, and the following abnormalities were found in the 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

biochemical profile: urea 85 mg/dL, creatinine 2 mg/dL, albumin 2 g/dL, alkaline phosphatase 33 U/L, alanine aminotransferase 27 U/L. Total blood count revealed normocytic, normochromic anaemia and lymphopenia. Retroviral serology (feline immunodeficiency virus and feline leukemia virus) was negative, and abdominal ultrasound showed hepatomegaly, splenomegaly, enlarged mesenteric lymph nodes, renal atrophy with loss of corticomedullary ratio, and thickening of the lining of the stomach and ileum. Given the suspicion of either an adverse drug reaction or PF a skin biopsy was performed. Histopathological findings included subcorneal pustules with acantholytic cells, neutrophils, and eosinophils suggestive of PF. The owner decided on euthanasia. The necropsy and histopathological findings were compatible with alimentary lymphoma with metastasis to the kidneys and liver. The PF in this cat was likely associated with intestinal lymphoma. Funding: Self funded. Conflict of Interest: None declared.

P-045 Generalized nodular dermatofibrosis in the absence of renal tumours in a Dalmatian dog E. GUAGUE`RE*, A. MULLER* and F. DEGORCERUBIALES *Clinique Ve´te´rinaire Saint Bernard, Lomme, France; Laboratoire d’Anatomie Pathologique Ve´te´rinaire du SudOuest, Toulouse, France Generalized nodular dermatofibrosis and renal cystadenomas or adenocarcinomas (NDRC) are regularly reported in the German shepherd dog. NDRC is probably inherited in an autosomal dominant manner. This report describes a clinical case of generalized nodular dermatofibrosis without renal tumours in a Dalmatian dog. An 8-year-old spayed female Dalmatian dog had at least a 4-year history of cutaneous and subcutaneous nodules. No systemic signs were noticed. Dermatological signs were characterized by innumerable, non alopecic, non ulcerated, firm papules, nodules, and plaques over the entire body (notably, the trunk and legs). Nodules varied in size (2 mm to 15 cm in diameter) and were typically non pruritic and non painful on palpation. No biochemical and haematological abnormalities were detected. Abdominal ultrasound revealed only small renal calcifications. Histopathological findings showed well-demarcated or not, dermal and hypodermal proliferation of mature collagen fibres, sometimes involving the epimysium of panniculus carnosus. Recent lesions contained areas of active fibroplasia and well-differentiated fibroblasts, whereas older lesions contained mature collagen fibres. To our knowledge, this is the second report of nodular dermatofibrosis in a dog without renal cysts, cystadenoma, or cystadenocarcinoma. The BurtHogg-Dube´ (BHD) gene is the human orthologue of the NDRC-associated canine gene. In our case, the nodules could be due to haplo-insufficiency at the BHD locus. The lack of renal neoplasia might reflect the absence of a functional significant second hit mutation in the BHD gene within the kidneys. Funding: Self funded. Conflict of Interest: None declared.

Abstracts

P-046 A feline non-exfoliative thymoma-associated dermatitis: a case report X. LANGON Clinique Ve´te´rinaire du Bournet, Seyssins, France Interface lymphocytic mural folliculitis associated with a thymic tumour in cats is a rare paraneoplastic skin disease named ‘feline thymoma-associated exfoliative dermatitis’. Extensive exfoliation is the clinical hallmark of this syndrome. A 9-year-old male neutered domestic short hair cat was presented for a non pruritic facial alopecia and deterioration of general body condition. Histological features showed discrete interface dermatitis and infiltrative lymphocytic mural folliculitis with apoptotic cells, particularly affecting the isthmus and the inferior portion of hair follicles leading to follicular rupture. Pigmentary incontinence was also noted. Thoracic radiographs revealed a thymoma. The dermatitis resolved when the cat was treated with chemotherapy, then relapsed 8 months later synchronously with thymoma regrowth. This case highlights the similarity and complexity of the pathogenesis of infiltrative/interface lymphocytic mural folliculitis syndrome. Instead of instigating typical interface lymphocytic mural folliculitis associated with characteristic clinical presentation (exfoliation of the head, trunk, and limbs), this thymoma induced infiltrative lymphocytic mural folliculitis with subtle epidermal interface change, discrete lymphocytic exocytosis and moderate apoptosis clinically expressed by alopecia and subtle scales. In conclusion, this case of thymoma-associated dermatitis highlights that interface and infiltrative lymphocytic folliculitis are related entities and the clinical presentation of this entity depends on lymphocyte behaviour. Funding: Self funded. Conflict of Interest: None declared.

P-047 Metastatic cutaneous nodules in five dogs B. HUBERT*, C. MAUREY, T. MARCHAL, J. P. MAGNOL and G. MARIGNAC* *Unite´ de Parasitologie-Mycologie-Dermatologie, Ecole Nationale Ve´te´rinaire d’Alfort, Maisons-Alfort, France; Unite´ de Me´decine, Ecole Nationale Ve´te´rinaire d’Alfort, Maisons-Alfort, France; Unite´ d’Anatomie Pathologique, Vet-Agro Sup Lyon, Marcy l’Etoile, France Cutaneous metastases arising from visceral tumours are rare dermatological events. Five clinical cases seen between 1980 and 2010 are reported. These cases included: a nephroblastoma in a young mixed-breed dog, an ovarian dysgerminoma in an Afghan hound bitch, a mammary adenocarcinoma in a Labrador bitch, an extraskeletal mammary osteosarcoma in a doberman bitch, and a bladder adenocarcinoma in a Briard dog. The last two cases progressed to final stage of Alamartine-Ball-Cadiot syndrome. In all cases, the dogs displayed a group of clinical signs that could not all be clearly related to the organ affected by the primary tumour. The macroscopic aspect of the skin nodules was also not distinctive. When the metastatic cells were welldifferentiated, cytological examination of fine needle

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aspirates and the histopathological findings of biopsies of the nodule(s) were sufficient to attain a diagnosis: nephroblastoma, mammary adenocarcinoma, and ovarian dysgerminoma. When the cells lost their original characteristics, immunohistochemistry was used in order to identify the primary tumour: bladder adenocarcinoma and extraskeletal mammary osteosarcoma. For the bladder adenocarcinoma, tissue markers such as antipankeratin were used in order to identify the epithelial origin of the metastatic lesion. Funding: Self funded. Conflict of Interest: None declared.

P-048 Cutaneous T-cell lymphoma of large granular lymphocytes in a pet rat V. MEIER*, C. GEIGY*, P. GREST and S. WILHELM *Division of Radiation Oncology, Department of Small Animal Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; Vetsuisse Faculty, Institute of Veterinary Pathology, University of Zurich, Zurich, Switzerland; Division of Dermatology, Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland A 1.5-year-old female pet rat was presented due to progressive multifocal alopecia, pruritus, crusts, and erosions. The rat was otherwise in good general condition. We excluded differential diagnoses, such as ectoparasites, dermatophytosis, and barbering using skin scrapings, fungal culture, and separation from other rats. A suspected deep pyoderma with abundant cocci was confirmed by cytological examination. We initiated treatment with enrofloxacin (Baytril; Provet, Lyssach, Switzerland), meloxicam (Metacam; Boehringer Ingelheim, Basel, Switzerland) and chlorhexidine (Chlorexyderm+ Spot Gel; Ufamed, Sursee, Switzerland). Despite treatment, the clinical symptoms worsened and skin biopsies were collected, and these revealed a cutaneous CD3+ CD79) T-cell lymphoma that spared the epidermis, but infiltrated multiple hair follicles. Neoplastic cells were small to rarely medium-sized, and many cells contained small eosinophilic intracytoplasmic granules typically seen in large granular lymphocytes. Oral prednisolone drops (Christoffelapotheke; Bern, Switzerland) 1.3 mg/kg once daily were initiated, and antibiotic treatment continued. Because of disease progression, we added oral lomustine (Ceenu; Bristol-Myers Squibb, Baar, Switzerland) 5 mg total dose every 3 weeks, Lasparaginase (Medac, Hamburg, Germany) 400 IU/kg subcutaneously with the first two lomustine administrations and thalidomide (Christoffelapotheke, Bern, Switzerland) 2.5 mg orally once daily. Erythema was persisting 8 weeks after initiation of chemotherapy; however, a marked clinical benefit with disappearance of crusts and pruritus was noted. Rodents with skin abnormalities are frequently presented to veterinary practice, with parasites or dermatophytosis being the most common findings. This report suggests that lymphoma is a possible differential diagnosis in rats with chronic skin disease. Funding: Self funded. Conflict of Interest: None declared. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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P-049 Skin involvement of chronic lymphocytic leukaemia in a dog T. OSUMI*, I. SEKI*, K. UCHIDA, H. MOCHIZUKI and H. TSUJIMOTO *Pigeon Animal Care Hospital, Kawaguchi, Japan; Department of Veterinary Pathology, University of Tokyo, Tokyo, Japan; Department of Veterinary Internal Medicine, University of Tokyo, Tokyo, Japan We examined a dog with multiple skin nodules on the trunk and found that the lesions resulted from an infiltration of neoplastic lymphocytes of chronic lymphocytic leukaemia. This 13-year-old neutered female miniature schnauzer was examined because of a history of dermatologic problem localized to the trunk for 3 months. At presentation, the dog had erythema, pustules, and crusts on the dorsal aspect of the trunk. Three months after the initial presentation, multiple small nodules on the trunk, severe pruritus and enlargement of peripheral lymph nodes developed. A homogeneous population of small lymphocytes was observed in the aspirates of enlarged lymph nodes. Histopathological examination of the skin lesions showed infiltration of sheets of small lymphocytes in the dermis. There was no epitheliotropic infiltration of the lymphocytes. Immunohistochemical examinations of the skin lesions revealed a homogenous small lymphocyte population positive for CD3 and negative for CD20. Haematologic examination revealed a marked lymphocytosis (20 000 cells/lL) with a homogenous population of small mature lymphocytes. Flow cytometric analysis of peripheral blood revealed a clonal expansion of mature T cells with an aberrant immunophenotype (CD3+, CD4+, abTCR+, CD8), CD21), cdTCR), CD45), CD34)). These findings indicated a possible skin involvement of T cell chronic lymphocytic leukemia in this dog. The dog was treated with corticosteroid (0.25–1 mg/ kg, PO, q24h) and chlorambucil (2 mg/m2, PO, once daily) for 4 weeks, resulting in a remarkable improvement with disappearance of the multiple skin nodules and pruritus. Funding: Self funded. Conflict of Interest: None declared.

P-050 A new canine skin disorder resembling granular parakeratosis: clinical and pathological features of three cases P. PRELAUD*, N. COCHET-FAIVRE*, F. DEGORCE-RUBIALES, A. POUJADE and A. ROSTAHER* *Clinique Advetia, Paris, France; LAPVSO, Toulouse, France A new canine skin abnormality characterized by refractory pruritus, marked erythema, and papules covered with large scales was seen in three privately owned dogs (7-year-old female neutered dwarf spitz, 3-year-old male French miniature poodle and 6-year-old female neutered whippet). The lesions were limited to the ventrum and perianal area. Two dogs also exhibited fissures on the abdomen. In all dogs we observed worsening of signs despite treatment using different topical formulations containing chlorhexidine, glycerine, boric acid, zinc 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

oxide, and glucocorticoids. The major histopathologic findings involved the epidermis. There was a severely thickened stratum corneum with parakeratosis and marked diffuse retention of keratohyaline granules. In all dogs a spontaneous cure was observed within 2– 4 weeks after discontinuation of the topical treatment. A 1–4-year follow up did not reveal further skin changes. The clinical and histopathologic findings in these dogs represent those seen in granular parakeratosis, a human keratinization disorder of unknown aetiology. Typical clinical signs in humans include brown or red keratotic or scaly, occasionally pruritic papules which often coalesce into plaques and are generally confined to intertriginous areas, as in our cases. A defective profilaggrin-filaggrin pathway is hypothesized to be involved in the pathophysiology of this disease. Therapeutic responsiveness is ambiguous; however, spontaneous resolution appears to be the rule in most cases. It remains unclear whether granular parakeratosis is a disease entity or whether it is just a reaction pattern to unknown stimuli. Funding: Self funded. Conflict of Interest: None declared.

P-051 Lymphoma of the tympanic bulla in a feline leukemia virus-negative cat G. GHIBAUDO*, A. TOMBA, G. ABBIATI, M. MAZZUCCHELLI*, M. CAPPELLETTI and A. VERCELLI§ *Clinica Veterinaria Malpensa, Samarate, Italy; Norad Diagnostics, Samarate, Italy; Ambulatorio Veterinario, Monza, Italy; §Ambulatorio Veterinario Associato, Torino, Italy Feline lymphoma can occur in virtually every organ, and the intestine is reported to be the most common site in older feline leukemia virus (FeLV) negative cats. Diseases affecting the tympanic bullae in cats are rare, with the exception of inflammatory nasopharyngeal polyps frequent in young subjects. Reports of tumours involving the middle ear are generally rare. A 12-year-old European short hair neutered male cat was presented to the neurology service for Horner’s syndrome localized to the right aspect of the face. Magnetic resonance imaging showed the presence of material in the tympanic bulla and myositis of the right medial pterygoid muscle. Otoendoscopic examination revealed a neoplasm protruding into the right ear canal and occupying the tympanic bulla. A biopsy and histopathological examination of the mass indicated the presence of a malignant lymphoma of medium-sized cells. Immunohistochemistry confirmed a poorly differentiated mainly T-cell lymphoma. Staging confirmed the presence of the tumour only in the tympanic bulla. After intravenous chemotherapy with doxorubicin (Doxorubicina Ebewe, Rome, Italy), three doses of 20 mg/m2 administered every 3 weeks, the animal developed urinary incontinence and paralysis of the hind legs, and the owners requested euthanasia. Autopsy was not permitted. In conclusion, in the last two decades, the overall incidence of lymphoma in cats has decreased, and the relative frequency of different anatomical sites has changed. This case report is, to the authors’ knowledge, the first description of lymphoma of the tympanic bulla in a cat in Italy and the third case reported in the literature. Funding: Self funded. Conflict of Interest: None declared.

Abstracts

P-052 A case of histoplasmosis limited to the skin in Europe N. FISCHER*, C. FAVROT*, M. MONOD, P. GREST, K. RECH§ and S. WILHELM* *Department of Dermatology, University of Zurich, Vetsuisse Faculty, Zurich, Switzerland; Department of Dermatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; Department of Pathology, University of Zurich, Vetsuisse Faculty, Zurich, Switzerland; §Small Animal Practice, Rech, Zurich, Switzerland A 6-year-old outdoor cat was presented to the dermatology service with a history of skin lesions developing in the past month and consisting of several papules and nodules on the head and neck. Some lesions were ulcerated, and a serosanguinous discharge was observed. General examination was unremarkable. Differential diagnoses included skin tumours, deep bacterial and fungal infections, and sterile pyogranulomas. Cytological examination of the ulcerated nodules revealed a pyogranulomatous infiltrate with numerous macrophages containing oval yeast-like cells, 2–5 lm in size, with a central, lightly basophilic core surrounded by a clear halo. A tentative diagnosis of fungal infection was made, and skin biopsies were taken. Histological examination confirmed the cytological findings, and Grocott staining showed numerous organisms suggesting Histoplasma or Sporothrix within macrophages. Thoracic X-rays, abdominal ultrasound, and routine laboratory testing were unremarkable. Fungal culture of a nodule was negative. Polymerase chain reaction (PCR) of total DNA extracted from the infected tissue and subsequent sequencing confirmed the diagnosis of Histoplasma capsulatum var. capsulatum. Surgical excision of the other nodules was performed, and the cat was treated with itraconazole 5 mg/kg orally, daily. Twelve weeks after initial consultation, no lesions were visible. No recurrence was observed upon a 4-month follow-up period. Histoplasma capsulatum has a worldwide distribution but reports in Europe remain rare. This is the second report of histoplasmosis in a cat in Europe (the first was a case report of feline disseminated histoplasmosis in Italy), the first described case in Switzerland, and the first case worldwide of feline histoplasmosis infection limited to the skin. Funding: Self funded. Conflict of Interest: None declared.

P-053 Combination therapy for canine generalized demodicosis and severe pruritus of chronic atopic dermatitis with 10% imidacloprid and 2.5% moxidectin spot-on, milbemycin oxime, and ciclosporin: a case report J. WANNAWONG Skin and Allergy Clinic, Thonglor Pet Hospital, Bangkok, Thailand A 12-year-old, spayed female shih tzu dog presented with generalized demodicosis and severe pruritus of chronic atopic dermatitis. Deep skin scrapings were positive for several Demodex canis mites. Cytology revealed suppu-

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rative inflammation. Atopic dermatitis was diagnosed by typical history and clinical signs, and exclusion of differential diagnoses. Serum allergen-specific IgE was positive for Dermatophagoides farinae, D. pteronyssinus, Acarus siro, and Morus alba. The dog was treated with a spot-on formulation containing imidacloprid 10% and moxidectin 2.5% (Advocate; Bayer, Kiel, Germany), once weekly for 24 weeks. Marbofloxacin at 4 mg/kg/day was prescribed to treat a secondary bacterial infection. Improvement was mild after 2 weeks. Milbemycin oxime (1 mg/kg/day) was added to the treatment regimen in the second week, for approximately 22 weeks. Partial response was observed with the combination of milbemycin oxime daily and once weekly Advocate. Ciclosporin (5 mg/kg/day) was added to the treatment regimen at week 11, for approximately 13 weeks. Moderate improvement in the pruritus level and skin lesions were reported following the combination therapy of Advocate once weekly and, milbemycin oxime, marbofloxacin, and ciclosporin once daily. Deep skin scrapings were negative for the first time after ciclosporin was added to the treatment regimen. Haemogram and blood chemistry were unremarkable. In this case, the addition of ciclosporin was needed to control the atopic dermatitis; however, the combination of milbemycin oxime and Advocate prevented the worsening of demodicosis. The dog has been maintained on Advocate once weekly and milbemycin oxime twice weekly and no relapse of demodicosis has occurred. Funding: Thonglor Pet Hospital. Conflict of Interest: None declared.

P-054 A case of advanced histiocytic mural folliculitis, thrombocytopenia, and anaemia in a dog S. SADEGHI* and R. EMAMI *Vellore Village Pet Hospital, Vaughan, ON, Canada; School of Veterinary Medicine, Mashad, Iran Granulomatous mural folliculitis is rarely reported in dogs, resulting in a lack of sufficient data for further subclassification of the disease. The present case report provides more information regarding the aetiology of granulomatous mural folliculitis in dogs. A 5-year-old Chihuahua dog was presented with a 5-month history of non pruritic progressive patchy alopecia, scaling, and erythema involving the forehead, ventral abdomen, and medial side of the limbs. This was followed by development of concurrent severe thrombocytopenia and moderate anaemia at a later stage. External parasites, dermatophytes, Lyme disease, and endocrinopathies were ruled out. Histopathology revealed severe nodular to diffuse granulomatous dermatitis characterized by histiocytic cell infiltration in the perifollicular areas, which partially to completely obliterated follicular structures, including sebaceous glands. Periodic acid Schiff, Gomeri methenamine silver, and acid fast stains did not reveal any infectious organisms. Thoracic and abdominal ultrasonography showed no internal abnormalities. The following differential diagnoses were considered: sterile pyogranulomatous/granulomatous syndrome, alopecia areata, pseudopelade, and mature histiocytic mural folliculitis. Based on the history, clinical signs, blood tests, and the histopathological findings, we made a diagnosis of advanced or mature granulomatous (histio 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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cytic) mural folliculitis with a possible immune-immune aetiology. Treatment with prednisone 1 mg/kg twice daily (Apo-prednisone, Apotex; Toronto, Canada) resulted in resolution of the blood abnormalities and skin lesions, and re-growth of the hair. This is the first report of the disease with concurrent thrombocytopenia and anaemia. The present case suggests an immune-mediated response as the main aetiology of this skin syndrome. Funding: Self funded. Conflict of Interest: None declared.

P-055 Clinical study of canine juvenile cellulitis in a university small animal practice in Chennai, India B. NAGARAJAN, S. KAVITHA, U. PREETHI and A. P. NAMBI Small Animal Dermatology Clinic, Madras Veterinary College Teaching Hospital, Chennai, Tamilnadu, India This study described the prevalence and clinical presentation of canine juvenile cellulitis over a period of 1 year (June 2010 to June 2011). Eleven puppies were presented with the history of pustular dermatitis, not responding to antibiotic. Among those, 6/11 were males and 7/11 were Labrador retrievers. Six cases (55%) were 30–45 days old, and five cases (45%) were between 45 and 60 days old. Seven cases (64%) were presented before the first vaccination and three cases (27%) after vaccination, and one case had no correlation with vaccination. In all of the cases we observed anorexia, depression, lethargy, lameness, swollen face with pustular lesions around the lips, muzzle and periocular area along with submandibular and prescapular lymphadenopathy. Pustular otitis was observed in seven cases (64%) and generalized lymphadenopathy in five cases (45%). Only four cases had draining fistulous tracts. Skin scrapings revealed no parasites, and dermatophyte culture of hairs was negative in all 11 cases. Impression smear of exudates showed numerous degenerated neutrophils with few macrophages. Bacterial culture yielded Staphylococcus spp in six cases (55%). Haematology revealed leucocytosis and neutrophilia. All cases were treated initially with prednisolone 2 mg/kg twice daily orally and then tapered based on clinical response. Those with secondary bacterial infection were treated with cefalexin 30 mg/kg twice daily. The puppies recovered within three to 6 weeks of treatment. Funding: Self funded. Conflict of Interest: None declared.

P-056 Canine sterile neutrophilic dermatosis (Sweet’s syndrome): two new cases E. GUAGUE`RE*, N. COCHET, O. MEJIA PONCE, F. DEGORCE-RUBIALES and A. MULLER* *Clinique Ve´te´rinaire Saint Bernard, Lomme, France; Facultad de Medicina, Veterinaria y Zootecnia, Hospital Veterinario de Especialidades, Universidad Nacional Autońoma de Me´xico, Coyoacań, Me´xico; Laboratoire d’Anatomie Pathologique Ve´te´rinaire du Sud-Ouest, Toulouse, France Sterile neutrophilic dermatosis is an uncommon skin condition in dogs. In humans clinical and histopatho 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

logical lesions might be the consequence of an unusual hypersensitivity reaction to bacterial, viral, or tumour antigens. Case 1 was a 3-year-old female cocker spaniel that was presented for lameness, fever, and generalized intense pain. Two days after the onset of systemic signs, dermatological signs appeared and were characterized by sudden development of erythematous, purpuric, and pustular lesions involving the face, legs, and feet. Case 2 was a 3-year-old spayed female Chihuahua dog that was presented with a 5-month history of painful papular, pustular, ulcerative, and crusted dermatitis, progressively involving the face, dorsal trunk, and the thighs. A sudden papular/pustular rash with hyperthermia and lymphadenomegaly was noticed during the previous 2 days. In both cases, no other disease preceding the cutaneous eruption was reported, but a severe leukocytosis with absolute neutrophilia was noticed. Cytologic findings showed numerous non degenerated neutrophils. Histopathologic findings revealed a diffuse neutrophilic dermatitis with a heavy infiltrate of mature neutrophils throughout the dermis and extending to the subcutis (case 1) and variable multifocal leukocytoclasis without true vasculitic lesions. In case 2, some eosinophils were admixed. There was marked oedema of the superficial dermis leading to subepidermal vesiculation in case 1. The overlying epidermis and hair follicles were either spared (case 2) or involved (case 1) with neutrophilic exocytosis (neutrophilic spongiotic vesicles) and luminal infundibular folliculitis. Bacterial culture from intact pustules was sterile. A gradual, spontaneous cure was observed in both cases. Funding: Self funded. Conflict of Interest: None declared.

P-057 Lupus-like dermatitis associated with visceral poly-parasitism in a dog B. HUBERT*, E. LALOY, G. MARIGNAC*, R. CHERMETTE* and J. J. FONTAINE *Unite´ de Parasitologie-Mycologie-Dermatologie, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France; Unite´ d’AnatomiePathologique, Ecole Nationale Ve´te´rinaire d’Alfort, Universite´ Paris-Est, Maisons-Alfort, France A 14-month-old German shepherd female dog, unvaccinated, was presented for hyperthermia (40C). The dog was underweight and had chronic diarrhoea, bilateral purulent conjunctivitis and lameness. Cefalexin (15 mg/ kg, twice daily) had been given for 2 weeks. Dermatological examination revealed numerous plaques, erythema, erosions, ulcers, and crusts on the nasal planum, forehead, pinnae, legs, and ventral abdomen with a symmetrical distribution. Joints were painful, and ulcers were present on all four feet. Complete blood count and serum biochemistry revealed leukocytosis with neutrophilia, increased total serum protein (98 g/L) and serum ß2-gamma globulin. Cytological examination of impression smears showed numerous neutrophils with intracellular and extracellular cocci bacteria. Additional tests (skin scrapings, polymerase chain reaction) ruled out demodicosis, canine distemper, and leishmaniosis. A tentative diagnosis of systemic lupus erythematosus was proposed. The dog died spontaneously the same night. Dermatohistopathology revealed severe chronic granu-

Abstracts lomatous multifocal adnexial dermatitis. Necropsy and histopathology revealed a severe pulmonary angiostrongylosis, with both interstitial pneumonia associated with the parasite eggs and larvae, and pulmonary arteritis with thrombosis and the presence of adult forms of Angiostrongylus vasorum. In the kidneys, multiple superficial, plasmocytic, and sparsely eosinophilic granulomas with few macrophages were observed and attributed to migrating ascaris larvae. Haemorrhagic colitis with numerous parasites (Trichuris vulpis) was also present. Severe, multiple, chronic parasitic infestation affecting several visceral organs was the probable cause of the observed clinical signs. The skin lesions and systemic signs that mimicked lupus erythematosus were unusual. Funding: Self funded. Conflict of Interest: None declared.

P-058 Pemphigus-like drug reaction in a dog after a single application of Vetra 3D K. MORIELLO* and M. BEHR *School of Veterinary Medicine, University of WisconsinMadison, Madison, WI, USA; Wisconsin Veterinary Diagnostic Laboratory, Madison, WI, USA A 41-kg 10-year-old male neutered Labrador retriever dog developed a pemphigus foliaceus-like focal drug reaction after a single application of a spot-on flea control product containing 4.95% dinotefuran, 0.44% pyriproxyfen, and 36.08% permethrin (Vectra 3D; Ceva Animal Health, USA). Lesions developed at the site 10 days post-application. Clipping of the hair coat revealed diffuse erythema, erosions, and pustular lesions. Skin cytology revealed rafts of acantholytic cells; skin biopsy was compatible with pemphigus foliaceus, and bacterial culture was negative. Five days after skin biopsy, erythema, swelling, and pustules developed on the dog’s muzzle and nose; clipping had allowed the dog contact the previously treated skin site. Erythema and pustules also developed at formerly unaffected skin areas after contact with the clippers used at the original lesion site. Systemic signs of illness were absent. Lesions responded to topical triamcinolone spray. For 10 months, lesions at the original and clipper contact sites waxed and waned, and acantholytic cells were always found. Pemphigus foliaceus-like drug reactions have been reported with Promeris; this dog had no prior exposure to Vectra 3D, Promeris or permethrin flea control products. The development of similar lesions in previously normal skin only after exposure to the clippers used at the lesion site is interesting and may be a ‘Koebner phenomenon-like reaction’. The timing of the drug application and development of skin lesions make it

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probable that this was a pemphigus foliaceus-like drug reaction. This case emphasizes the unpredictable and idiosyncratic nature of drug reactions. Funding: Self funded. Conflict of Interest: None declared.

P-059 Non uremic non fatal idiopathic calciphylaxis in a kitten N. THOM*,, E. ER* and M. REINACHER *Small Animal Clinic, Veterinary Faculty, Justus Liebig University, Giessen, Germany; Tierklinik Hofheim, Hofheim, Germany; Department of Veterinary Pathology, Veterinary Faculty, Justus Liebig University, Giessen, Germany A 10-week-old male domestic short hair kitten was presented in state of shock and peracute skin lesions. He had been treated for coughing with amoxicillin and penicillin/streptomycin one week earlier, but had been healthy otherwise. Initial dermatological signs were characterized by sharply demarcated erosions and ulcerations, distributed over the left side of the face, including nasal planum and lips. Shock was successfully treated with fluid replacement, but despite a thorough work-up, no cause was detected. Cutaneous lesions rapidly progressed into thick crusts covering ulcerated skin, involving the chin, cheek, lips, eyelid, and pinna on the left side of the face. Histopathology revealed severe epidermal necrosis and calcification, multifocal pannicular calcification, and calcified subcutaneous vessels, supporting diagnosis of calciphylaxis. Treatment consisted of systemic and topical antimicrobials, analgesics, pentoxifylline, sodium thiophosphate, and vitamin K. After initiation of therapy no further progression was noticed. All medications were discontinued and no relapse was seen in the following year. Calciphylaxis is a rare cutaneous disorder, characterized by vascular calcification and progressive skin necrosis, not yet described in cats. It is scarcely reported in animals, due to either iatrogenic or uremic disturbances of the calcium/phosphorus balance. In humans, it is most commonly seen with end-stage kidney disease, but several non-uremic patients, including inherited dysfunctions of tissue-calcification inhibitors, are also described. This cat showed no abnormality in calcium/phosphorus balance, renal disease, primary hyperparathyroidism, or any iatrogenic cause. The young age of onset is suggestive of an inherited disorder, which is incompatible with the transient character seen in this cat. Funding: Self funded. Conflict of Interest: None declared.


Topic 7: LARGE ANIMALS, SMALL ANIMALS AND EXOTICS P-060 Pilot investigation on skin barrier in equine atopic dermatitis: observations on electron microscopy and measurements of transepidermal water loss R. MARSELLA, D. SAMUELSON, C. JOHNSON and K. AHRENS University of Florida, Gainesville, FL, USA Skin barrier impairment plays an important role in human and canine atopic dermatitis. Equine atopic dermatitis is recognized in clinical practice but is not well investigated, and it is currently unknown whether skin barrier is impaired. This pilot study investigated skin barrier in horses with atopic dermatitis both ultrastructurally, using transmission electron microscopy, and functionally, measuring transepidermal water loss. Two normal and two atopic horses were selected. Diagnosis of atopic dermatitis was based on compatible history, clinical signs (seasonal pruritic dermatitis despite treatment for Culicoides), and positive results on skin test and serology. All measurements of transepidermal water loss were taken in triplicate using a closed chamber device in a temperature/humidity-controlled room where horses were acclimatized for at least 30 min. To assess whether transepidermal water loss can measure skin barrier damage in horses, we tape stripped a site (lateral thorax, control) 15 times and measured transepidermal water loss before and after. Tape stripping doubled the transepidermal water loss, reflecting skin damage. Sites selected for further investigation in all horses included areas predisposed to atopic dermatitis (periocular, antebrachial, inguinal) and non atopic sites (lateral thorax and lateral thigh). No differences were found for transepidermal water loss between normal and atopic horses and between sites. Biopsies were taken from atopic (antebrachial, inguinal) and non atopic (lateral thorax) sites and processed with ruthenium tetroxide. Stratum corneum was compacted with continuous lipid lamellae in normal samples while irregularities and abnormal amorphous lipids were found in the atopic horses. These changes were most prominent in atopic sites. Funding: Self funded. Conflict of Interest: None declared.

P-061 Clinical characteristics of dermatophytosis in guinea pigs (Cavia porcellus) and importance of fungal culture: results from 333 cases P. J. BOURDEAU and C. MICHOT-ANADON Veterinary School, Oniris, Nantes, France Dermatophytosis is a relatively common condition in companion guinea pigs. However, there is little information available on the accurate mycological identification of the aetiological agents. This study describes the most important characteristics of this infection in dermatophytosis cases confirmed by fungal isolation (Mycology Unit DPM Veterinary School, Oniris, Nantes). The samples were cultured and identified using reference methods. The clinical parameters analyzed were obtained 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

from a specific form that contained the type of lesions (with scoring) and their distribution. Infected and non infected animals were compared using chi square test (P £ 0.05). Of 776 samples analyzed, 333 (46.4%) were positive for dermatophytes (94% Trichophyton mentagrophytes, 5.7% Microsporum canis). The most frequent lesions were (in decreasing order): alopecia, crusts, pruritus, scaling, and erythema. However, only crusts (more frequent) and pruritus (less frequent) in infected animals were significantly different from non infected animals (P = 1.9 10)5 and P = 0.011, respectively). In 76% of infected and 29% of non infected cases, lesions were found on the head (P = 2.2 10)8) and, in 17% of infected and 40% of non infected cases on the dorsum (P = 1.13 10)5). When present, head lesions were mainly observed on ears, nose, and periocular area but, there was no significant difference compared to non infected cases (P > 0.05). Asymptomatic carriage was more frequent with Microsporum canis (12%) than with Trichophyton mentagrophytes (3%). The negative predictive value of trichoscopy was 0.38 and sensitivity was 0.2. We conclude that an appropriate diagnosis of dermatophytosis in guinea pigs should systematically include accurate mycological cultures. Funding: Self funded. Conflict of Interest: None declared.

P-062 Efficacy of a spot-on formulation containing selamectin as an active ingredient against the cat flea Ctenocepalides felis and the ear mite Otodectes cynotis in ferrets T. FUKASE* and Y. NAKAMURA*, *Hayashiya Institute of Life Sciences, Uji-shi, Kyoto, Japan; Hayashiya Animal Hospitals, Uji-shi, Kyoto, Japan We evaluated the efficacy and safety of a spot-on formulation containing selamectin as the active ingredient (Revolution 6%, Pfizer Japan Inc.; City, Japan) against the cat flea Ctenocephalides felis and the ear mite Otodectes cynotis on ferrets. Twenty-four ferrets naturally infested with fleas were assigned to four groups (n = 6 each): one non medicated control group and three medicated groups. Medicated groups received selemectin at a dose of 6, 12, and 18 mg/kg. Fleas were completely eliminated from the ferrets of all three medicated groups within 3 days after administration. To study the effectiveness of selamectin against ear mites, 30 ferrets naturally infested with Otodectes cynotis were treated with selamectin administered to the dorsal neck at a dose of 6 mg/kg. Thereafter, the drug was applied directly to the pinna and the external auditory canal at a dose of 6 mg. Treatment was repeated at weekly intervals until no mites could be detected. The ear mites were eliminated from all 30 ferrets after a single systemic treatment or one to two additional treatments applied directly to the ears. No adverse effects of the drug were observed in any of the medicated ferrets. It was concluded that the spot-on formulation of selamectin was safe and fully effective against C. felis and O. cynotis infestations in ferrets. Funding: Hayashiya Institute of Life Sciences. Conflict of Interest: None declared.

Abstracts

P-063 An outbreak of red leg disease in a colony of African clawed frogs (Xenopus laevis) M. L. STREBER and E. MARAVILLA INCMNSZ, Mexico, Mexico One of the first warning signs of red leg disease is the reddening of the skin, particularly on the belly and inner aspect of the legs. This abnormal redness on the legs can be difficult to detect because several frog species have naturally a red hue on their legs. The reddening related to this condition is the result of ruptured blood vessels causing blood to accumulate under the skin. Due to the light colour of the African clawed skin, the red colour of the blood shows through the skin and is most visible on the belly and legs. Frogs with red leg disease often become lethargic and show a loss of appetite, swelling of the legs, accumulation of fluid in the abdomen, and open sores on the areas of blood accumulation. These signs are brief and seen before the start of convulsions and sudden death. Our colony of frogs suffered spontaneous death, especially after surgical incisions to obtain eggs. Necropsy findings showed some animals to have ascites. The skin lost its bright colour and was congested, and skin hemorrhage was present on the legs, feet, and ventrum. Samples were collected for culture. The organisms isolated were Aeromonas spp, Pseudomonas spp, and Proteus spp. The histopathology showed skin oedema and colonies of Gram-negative bacteria. A complete depopulation was done. Funding: institutional funded. Conflict of Interest: None declared.

P-064 Prevalence of dermatomycosis (ringworm) due to Trichophyton verrucosum in dairy farms in Shahriar county of Tehran, Iran S. OZMAIE and A. ANOSHEHPOUR Islamic Azad University, Science and Research Branch, Tehran, Iran Dermatophytosis is a common cosmopolitan disease. In cattle, it is most commonly caused by Trichophyton verrucosum and less frequently by T. mentagrophytes, T. equinum, and Microsporum gypsum. The disease is considered a public health problem all over the world. This study determined the prevalence of bovine dermatophytosis in dairy farms in Shahriar county, Iran. A total of 710 cattle were examined from March 2011 to August 2011. Skin scrapings were performed on all animals clinically suspected to have dermatophytosis. The samples were examined microscopically for fungal elements after adding potassium hydroxide (KOH). Fungal cultures were performed on selective agar for pathogenic fungi and culture plates were kept at approximately 28C for 4 weeks. Of the 710 cattle, 109 (15.21%) were clinically suspected of having dermatophytosis. Trichophyton verrucosum was isolated in 100% of the samples of animals with clinical signs of dermatophytosis. This study revealed that T. verrucosum was the cause of dermatophytosis in the tested cattle in Shahriar, Iran. Funding: Islamic Azad University Foundation. Conflict of Interest: None declared.

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P-065 Comparative activity of antifungal topical treatments on dermatophytes in an in vivo bovine model with Trichophyton verrucosum P. BOURDEAU, A. ROUSSEL and L. IMPARATO Dermatology, Parasitology, and Mycology Unit, Veterinary School, ONIRIS, Nantes, France The growth of Trichophyton verrucosum in culture is difficult, and thus epidemiologic studies and evaluation of treatment efficacy in cattle with dermatophytosis is very difficult. This study evaluated the efficacy of topical antifungals through the count of arthrospores in a young bull with spontaneous dermatophytosis. The trunk and neck were divided into nine equivalent areas and treated three times a week during 1 month with 3% chlorhexidine shampoo, 2% chlorhexidine + 2% miconazole shampoo (CMS), 3% chlorhexidine + 0.5% climbazole shampoo (CCS), and diluted enilconazole at 1/50 (label), 1/100, or 1/25 (E, E/2, EX2, respectively) or lime sulphur at 1/32 (label) or 1/64 (LS or LS/2, respectively). One area was left as control (water application), and the rest of the body (i.e. head and legs) was treated with enilconazole. Periodically on each area, 500 mg of hairs and scales obtained from skin scrapings were crushed in water and filtered, and a drop was spread on a slide and stained. The arthrospores were counted on five and then 10 microscopic fields (x100) in a blinded manner, and results were statistically analysed. From day 0 to day 32 we measured a highly significant reduction of arthrospores on treated sites as compared to the control (in decreasing order: CCS (92.6%), EX2 (80.6%), LS (73.9%), LS/2 (73.7%), E (72.3%), CMS (63.4%), E/2 62.3%), except for 3% chlorhexidine shampoo, which increased by 63.9%. Counting arthrospores could be a valuable method for evaluating the efficacy of antifungal agents for dermatophytosis, although fungicidal effect cannot be directly demonstrated by this method. Funding: Self funded. Conflict of Interest: None declared.

P-066 Genome-wide association study in Eringer cows with alopecia areata K. TIMM*, F. BUERGISSER*, C. FLURY, T. LEEB, P. ROOSJE* and C. DROGEMULLER *Division of Clinical Dermatology, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland; Bern University of Applied Sciences, Swiss College of Agriculture, Zollikofen, Switzerland; Vetsuisse Faculty, Institute of Genetics, University of Bern, Bern, Switzerland Alopecia areata appears to occur with increased frequency in Eringer cattle, a small indigenous Swiss breed, while it is only sporadically reported in other cattle breeds. In a recent study we described the clinical phenotype and histopathological patterns of alopecia areata in Eringer cattle. Genetic predispositions for the development of alopecia areata are studied extensively in different species, and a genome-wide association study in humans with alopecia areata provided evidence for the involvement of both innate and acquired immunity in the pathogenesis of the disease. The breed-specific increased 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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prevalence of alopecia areata in Eringer cattle suggests a genetic predisposition. The limited genetic diversity within a closed domestic animal population should facilitate the identification of the causative mutation(s). We performed a genome-wide association study to identify possible alopecia areata associated genomic regions in the cattle genome. Twenty affected and 118 healthy control Eringer cows were analyzed for 54 000 single nucleotide polymorphisms (SNP) evenly distributed across the entire bovine genome. We observed association signals to genomic regions containing genes controlling the immune response. As no genome-wide significant association was found, the number of cases was increased, and the recently released high-density cattle 777k SNP chip was used for further analysis to confirm the influence of genetically triggered autoimmunity in the pathogenesis of alopecia areata in Eringer cattle. Funding: Self funded. Conflict of Interest: None declared.

P-067 Stephanofilarial dermatitis in cattle population of West Bengal, India S. K. MUKHOPADHAYAY, K. S. SINGH, S. GANGULY and S. DHANALAKSHMI West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, India In the present study, prevalence, transmission and characteristics of the parasite Stephanofilaria assamensis, commonly known as ‘humpsore’ was studied in two ecoclimatic zones of West Bengal, India. The prevalence of the disease in South Bengal was found to be 17.12% in which the parasite affected the hump, dewclaw, hooves, and abdomen. In contrast, in North Bengal, the prevalence was higher (38.37%), and infection was mostly limited to the hump region causing the so called real ‘humpsore’. We found 22.60% infection in indigenous breeds and 36.64% infection in cross-bred and exotic cattle. The prevalence was highest among the animals of 4–5 years of age (28.01%). The infection rate in males (29.25%) was higher than in females (21.84%). Influence of season on prevalence indicated that it was highest during the rainy season (47.13%). Funding: Self funded. Conflict of Interest: None declared.

P-068 Diseases with abnormal keratinization in goats: histopathology in the light of differential diagnoses L. R. M. SA´*, J. M. GUERRA*, V. GOMES, A. M. M. P. DELLA LIBERA, V. G. FERREIRA, M. B. R. ALVES and F. J. BENESI *Department of Pathology, School of Veterinary Medicine and Animal Health, University of Saõ Paulo, Saõ Paulo, Brazil; Bovine and Small Ruminants Clinic, Department of Internal Medicine, School of Veterinary Medicine and Animal Health, University of Saõ Paulo, Saõ Paulo, Brazil


Disorders of keratinization are characterized by the abnormal accumulation of keratin on the superficial epidermis and hair follicles. Chronic skin disorders can occur in goat herds, and usually the clinical presentation includes generalized hyperkeratosis with or without pruritus. Several aetiologies have been associated with keratinization disorders such as ectoparasitisms, seborrhoea, zinc responsive disease, dermatophytosis, and dermatophilosis among others. Six adult goats with skin disorders underwent thorough physical examination and appropriate diagnostic tests were performed. Clinical exam showed severe generalized hyperkeratosis, hypotrichosis, skin folding, and lichenification with focal exudative and crusting lesions distributed on the head, ears, tail, hind limbs, ventral abdomen and dorsal thoracic region. Four goats had variable pruritus, and one had horn and roof abnormal growth. We performed skin biopsies, skin scrapings for parasitic infestation, cytology, fungal and bacterial cultures, and zinc blood levels on all goats. The diagnosis was established on the basis of clinical signs and results of the various diagnostic tests. Four goats had mange characterized histologically by hyperplastic and hyperkeratotic perivascular dermatitis with neutrophilic pustules and luminal folliculitis with intralesional mites. The histopathological diagnosis in one case was allergic disease characterized by hyperplastic, eosinophilic perivascular dermatitis with secondary bacterial infection. Another goat had histopathological findings of ichthyosis characterized by severe laminated orthokeratosis involving the epidermis and follicular infundibula associated with atrophic epidermis. In conclusion, hyperkeratosis seems to be a common and non specific feature of chronic dermatitis in goats; therefore, a systematic approach to the diagnosis of hyperkeratotic conditions in goats is necessary. Funding: Self funded. Conflict of Interest: None declared.

P-070 Vitiligo in a black-ear-tufted marmoset (Callithrix penicillata): first description L. R. SA´ Department of Pathology, School of Veterinary Medicine and Animal Health, University of Saõ Paulo, Saõ Paulo, Brazil Vitiligo is an acquired depigmenting skin disorder characterized by idiopathic, progressive, circumscribed hypomelanosis of the skin and hair, with total absence of melanocytes. It occurs worldwide in humans, but is considered uncommon in domestic pets or exotic animals. An adult female black-ear-tufted marmoset (Callithrix penicillata) showed, after 2 years of captivity, at an approximate age of 3 years, a progressive macular depigmentation with leukotrichia on the face, pawpads, ears, and distal portions of hind and forelimbs for 10 months until its death. The skin lesions were bilateral and symmetric, and involved non haired and haired regions. Despite being paired and in good condition, the female did not reproduce. At necropsy, the animal was thin and no specific gross lesions were found, and cardiorespiratory failure was established as causa mortis. The microscopical analysis of depigmentated areas was characterized by complete absence of epidermal melanocytes

Abstracts and melanin in the epidermis. The adjacent pigmented skin, besides having small numbers of melanophages in the dermis, was unremarkable. To the best of the author’s knowledge this is the first documented description of vitiligo in a primate in the western hemisphere. Funding: Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo, Brazil. Conflict of Interest: None declared.

P-071 Cutaneous horn in a hamster: a case report M. L. STREBER* and O. SANTANA *INCMNSZ, Mexico, Mexico; Small animal practice, Mexico, Mexico Cutaneous horn is a rare lesion consisting of a conical projection of cornified material that varies from a few

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milimeters to several centimeters in length. The diagnosis of cutaneous horn is based on the characteristic clinical presentation; however, different lesions at the base of the keratin mound have been described histologically. A 1.5year-old male golden hamster (Mesocricetus auratus) was presented with a large horn of a few months duration, arising from its left flank. The horn had a conical shape with a total length of 2.5 cm. A nodule measuring 4 · 3.5 · 0.7 cm had also developed at the base of the horn. Surgical excision was performed and the tissue was routinely processed for histopathology and stained with hematoxylin and eosin, toluidine blue, and chromotrope 2B. The final diagnosis was squamous cell carcinoma associated with an abscess at the base of the horn. Only one report of cutaneous horn in a rat could be found in the English literature. Funding: Self funded. Conflict of Interest: None declared.


Topic 8: FUNGAL DISEASES AND THERAPY P-072 One-year surveillance of the isolation of dermatophyte spores from risk areas in a veterinary medical teaching hospital W. OLDENHOFF and K. MORIELLO School of Veterinary Medicine, University of WisconsinMadison, Madison, WI, USA This study determined the frequency of isolation of dermatophytic fungal spores from a veterinary medical teaching hospital, identifies high-risk areas, and determined whether cleaning practices need to be changed. Over a 1 year period, we sampled high animal traffic areas (waiting room; primary care receiving area; oncology, medicine, surgery, anaesthesia, and student surgery ward; dermatology examination room; critical care unit) and administrative office (negative control) on random days before institutional cleaning using a commercially available electrostatic cleaning wipe (Swiffer Sweeper Cloths; Proctor and Gamble, USA). Four fungal cultures were inoculated with samples from each site (n = 1604) onto mycobiotic agar and incubated for 21 days at 27C. Dermatophytes were isolated from 23/ 401 site samplings (5.7%). Microsporum canis was isolated from 22/401 (5.5%) and Microsporum gypseum was isolated from 1/401 (0.2%) site samples. The most frequent site with fungal spore contamination was the dermatology examination room, which accounted for 10/ 23 (43.4%) of positive site samplings. Positive site samplings were seasonal and clustered around time periods when an infected animal was diagnosed with dermatophytosis. Excluding the dermatology examination room, these results reveal that contamination of the hospital with dermatophyte spores is infrequent and that current sanitation practices are adequate (sweeping, mopping, and disinfection with a quaternary ammonium based cleaner). These findings highlight the need for routine surveillance for spore contamination of risk areas where animals with dermatophytosis are most likely to be examined. Surveillance post-examination of known infected animals is particularly important along with periodic reviews of sanitation practices. Funding: Gifts to the Dermatology Research Laboratory. Conflict of Interest: None declared.

P-073 Itraconazole for the treatment of cutaneous candidiasis in dogs: a pilot study of eight cases E. BENSIGNOR Dermatology Referral Service, Paris, France Cutaneous candidiasis is rare in dogs. Its treatment has not been standardized and azole derivatives have been recommended. This pilot study evaluated the use of itraconazole, a triazole derivative, for cutaneous candidiasis. We included eight dogs of various breeds and ages presenting with candidiasis; diagnosis was based on the presence of compatible skin lesions and the demonstration of characteristic fungal elements by cytological examination and fungal culture. Dogs were treated with


itraconazole (Itrafungol; Janssen Animal Health, Beerse, Belgium), 5 mg/kg/day orally given with food. Follow-up visits were scheduled after 15, 45, and 90 days. Lesions (erythema (8/8), erosions (8/8), creamy exudate (8/8), lichenification (6/8), alopecia (6/8), and hyperpigmentation (5/8)) were localized to the interdigital area and/or the carpal/metacarpal area (8/8 cases). Candida spp. were identified on cytological examination (7/8 cases) and on fungal culture (8/8 dogs). A clear improvement was noted in 6/8 dogs after 2 weeks of treatment and in 8/8 dogs after 6 weeks. Resolution of skin lesions was noted in 5/8 dogs after 6 weeks and in 8/ 8 dogs after 10 weeks of itraconazole treatment. Mild adverse effects were noted in two dogs (vomiting and diarrhoea). Results of this pilot study support the use of itraconazole for candidiasis in dogs. Further studies are needed to compare the efficacy of itraconazole with other azole derivatives for the treatment of cutaneous candidiasis. Funding: Self funded. Conflict of Interest: None declared.

P-074 Retrospective evaluation (1993–2011) of itraconazole therapy for canine and feline sporotrichosis C. ROSSI, J. ODAGUIRI and C. E. LARSSON School of Veterinary Medicine and Animal Science, University of Saõ Paulo, Saõ Paulo, Brazil This study retrospectively evaluated the response to itraconazole treatment for cases of canine and feline sporotrichosis admitted to the dermatology service of a veterinary teaching hospital over a 19-year period (1993– 2011). The animals were treated with itraconazole (Sporanox oral capsules 100 mg, Jansen-Cilag, Saõ Paulo, Brazil) at 10 mg/kg once daily. Medical records were evaluated and the following efficacy criteria were used: therapeutic response, side effects, duration of therapy, and relapse. From the evaluated data, 20 animals were selected: 17/20 cats and 3/20 dogs and most animals were males (16/20) and of mixed breed (15/ 20). The localized cutaneous form was the most prevalent (12/20). Complete remission was noted in all animals regardless of the clinical form, and no side effects were reported. The average length of therapy until resolution was 3.4 and 11.3 months in cats and dogs, respectively. The maximum time required for resolution of clinical signs was 16 months, regardless of species. Recurrence was observed in 3/20 (one cat and two dogs) as a result of glucocorticoid therapy or other concomitant disease. Itraconazole is an effective treatment for sporotrichosis in dogs and cats with no apparent adverse effects resulting from its use. The association of sporotrichosis with another illness and the use of drugs with potential for immunosuppression appeared to contribute to recurrence of disease. Funding: Self funded. Conflict of Interest: None declared.

Abstracts

P-075 Aetiology of dermatophytosis in dogs and cats in the clinical routine of the veterinary hospital of Castelo Branco University, Rio de Janeiro, Brazil F. CLARE*,,, P. VIANA§, C. VIDEIRA–, D. TEIXEIRA**, M. MOURA and J. VILELA, *College of Veterinary Medicine, Severino Sombra University, Vassouras, Rio de Janeiro, Brazil; College of Veterinary Medicine, Castelo Branco University, Rio de Janeiro, Rio de Janeiro, Brazil; Center for Health Surveillance, Vassouras, Rio de Janeiro, Brazil; § Veterinary, Rio de Janeiro, Rio de Janeiro, Brazil; – Veterinary Medicine, Department of Dermatology, Pet Vet Veterinary Clinic, Rio de Janeiro, Rio de Janeiro, Brazil; **University Castelo Branco, Rio de Janeiro, Rio de Janeiro, Brazil; College of Veterinary Medicine, Rural Federal University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil Dermatophytosis, a superficial fungal infection caused by the genera Trichophyton sp., Microsporum sp., and Epidermophyton sp, has great importance in public health as an anthropozoonosis. Clinically evident lesions lead the owner to seek professional help for diagnosis and treatment. This study evaluated the occurrence of dermatophytes in dogs and cats seen at the Clinical School Doctor Paulo Alfredo Gissoni, Castelo Branco University, Rio de Janeiro, Brazil, from 2001 to 2011. We analysed 847 fungal cultures of hairs, of which 202 (23.85%) were positive for dermatophytes (53 cats and 149 dogs). Of the 202 positive cultures, 130 (64.4%) yielded Trichophyton sp., 65 (32.2%) Microsporum sp., and seven (3.5%) Epidermophyton sp. Samples from 102 dogs (68.5%) were positive for Trichophyton sp., from 40 (26.8%) for Microsporum sp., and from seven (4.7%) dogs for Epidermophyton sp. In cats we found 28 (52.8%) samples positive for Trichophyton sp. and 25 (47.2%) for Microsporum sp. None of the cats were positive for Epidermothyton sp. Trichophyton metagrophytes had a high occurrence in both dogs (18.1%) and in cats (30.2%). Most infected dogs were adults or geriatric and most infected cats were young. Higher incidence of dermatophytes was observed in animals with long hair. There was no statistic difference between the genders (P > 0.05). The genus Microsporum was found in only 32.2% of cases, contradicting the literature that reports Microsporum sp. as the most prevalent dermatophyte in dogs and cats. Seasonality did not affect the incidence of dermatophyte infection in this study. Funding: Castelo Branco University; Severino Sombra University. Conflict of Interest: None declared.

P-076 Electron microscopic study of skin changes in dogs with Malassezia dermatitis C. GANGULY, A. P. NAMBI, S. R. SRINIVASAN and S. KAVITHA Madras Veterinary College, Chennai, Tamil Nadu, India This study investigated the expression of epidermal lipids ultrastructurally in dogs suffering from Malassezia dermatitis. Of 72 dogs with Malassezia dermatitis presented

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to the Small Animal Dermatology Clinic of Madras Veterinary College Teaching Hospital, 30 dogs were randomly selected and subjected to a treatment trial with oral ketoconazole or oral itraconazole each combined with topical acetic acid. For electron microscopic studies punch biopsy specimens (6 mm) were obtained from both lesional and non lesional skin of six affected dogs (before treatment) and six normal dogs. The stratum corneum of lesional skin of affected dogs exhibited long, thin corneocytes with intercorneocyte spaces almost empty and incomplete and disorganized intercorneocyte lipid lamellae. The stratum corneum of non lesional skin of affected dogs exhibited absence of structured intercorneocyte lipid lamellae and very little vertical cohesion between the corneocyte strata. The stratum corneum of lesional skin of dogs that did not respond to therapy exhibited long and thin corneocytes with almost vacant intercorneocyte spaces. The deposition of stratum corneum lipid lamellae appeared markedly heterogeneous and disorganized in these dogs compared with normal canine skin. Premature separation of corneocytes was also evident. The stratum corneum of non lesional skin of dogs that did not respond to therapy exhibited absence of structured intercorneocyte lipid lamellae and very little vertical cohesion between the corneocyte strata. The stratum corneum changes of non responding cases of Malassezia dermatitis had features similar to that of atopic dogs suggesting that Malassezia sp could be causing an allergic reaction in these dogs. Funding: Self funded. Conflict of Interest: None declared.

P-077 Molecular analysis of Malassezia pachydermatis from canine ear and skin in Korea S. H. HAN, E. H. NAM, S. H. PARK, J. Y. JUNG, Y. U. LEE, C. Y. SONG and C. Y. HWANG College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Korea Malassezia spp. are commensal organisms of dog skin, and are mostly found in sebum-rich and humid areas of the body such as the external ear canal. This study investigated the prevalence of Malassezia sp. according to the body distribution (external ear canal vs. other body sites such as nasal fold, axillary area, inguinal area; interdigital spaces, and perianal area) in healthy dogs and dogs with skin disorders, and compared their genotypes determined by the DNA sequences of intergenic spacer 1 region (IGS-1) and internal transcribed spacer 1 (ITS-1) region. We recovered 201 samples from four groups including: (i) ear of dogs with otitis (68%); (ii) ear of healthy dogs (43%); (iii) skin of dogs with dermatitis (56%) and (iv) skin of healthy dogs (47%). Malassezia pachydermatis was identified by PCR-RFLP in all 201 samples. Both ITS-1 and IGS-1 loci had three main sequence genotypes (A, B, C) representing all Malassezia isolates, which were designated as seven subgenotypes for ITS-1 and 10 subgenotypes for IGS-1. Genotype A was the most predominant type (60–92%), and genotype C was more frequently isolated from the ear canal (39– 40%) than other body parts (4–10%) regardless of presence of lesions. Our results suggest that specific genotypes of M. pachydermatis are more frequently 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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found on specific parts of canine skin, thus body distribution has a greater influence than health status of the skin on distribution of M. pachydermatis. Funding: Self funded. Conflict of Interest: None declared.

P-078 MalDESI, an index for the clinical evaluation of dogs with Malassezia dermatitis O. CROSAZ*, C. HADADJE*, H. HAHN*, B. HUBERT*, G. MARIGNAC*, L. DESQUILBET, R. CHERMETTE* and J. GUILLOT* *Parasitology, Mycology, Dermatology, CHUVA, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France; Animal Production and Public Health, Ecole Nationale Ve´te´rinaire d’Alfort, UPE, Maisons-Alfort, France The yeast Malassezia pachydermatis may become pathogenic when the skin surface microclimate or host defense is altered. Various therapeutic options for Malassezia dermatitis have been tested in dogs, but, in many studies, clinical improvement was evaluated only subjectively by clinicians or owners. This study developed an index called the Malassezia Dermatitis Extent and Severity Index [MalDESI] to clinically evaluate the extent and severity of Malassezia dermatitis in dogs. MalDESI is based on the degree of erythema, lichenification, excoriation, alopecia, and seborrhoea on 36 anatomical sites with a severity scale ranging from 0 (none) to 5 (severe). The new scoring system was assessed in 19 privately owned dogs in the Paris area that had a local or generalized dermatitis associated with cytological detection of yeasts and a large number of Malassezia colonies in culture. Dogs were treated with a 2% miconazole - 2% chlorhexidine shampoo twice a week for 3 weeks. A significant decrease of MalDESI was observed between day 0 (mean value 86.9) and day 30 (mean value 19.0) with a direct correlation with the degree of pruritus and the number of yeast colonies in culture. This new scoring system could be recommended as a tool for the assessment of disease severity in clinical trials testing the efficacy of interventions in dogs with Malassezia dermatitis. Funding: Dechra Veterinary Products SAS, Suresnes, France. Conflict of Interest: None declared.

P-079 Topical vs. systemic treatment of Malassezia dermatitis in dogs: a comparative, blinded, randomized trial E. BENSIGNOR*, H. HAHN and J. GUILLOT *Dermatology Referral Service, Paris, France; Alfort Veterinary School, Alfort, France Recommended treatment for Malassezia dermatitis in dogs includes topical therapy with azole derivatives and/ or systemic therapy with ketoconazole or itraconazole. This study compared these treatments. Thirty dogs with compatible clinical signs of Malassezia dermatitis and high numbers of yeast on cytological examination were 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

randomly assigned to three groups: A, once daily oral administration of ketoconazole at 10 mg/kg; B, twice weekly application of a shampoo containing chlorhexidine 2% and miconazole 2%; and C, combination of the topical and systemic treatments. Follow-up visits were scheduled after 3 and 6 weeks for clinical and cytological evaluations. After 3 weeks, a marked improvement was noted for all the dogs. Improvement of clinical and cytological scores was respectively: group A, 48.2% and 46.9%; group B, 57.3% and 61.8%; and group C, 69.6% and 68.7%. The improvement in each these scores was more noticeable after 6 weeks (group A, 82.7% and 75.0%; group B, 84.5% and 91.2%; group C, 89.6% and 93.7%, respectively). Significant differences (P < 0.05) were noted in favour of group C for clinical and microbological parameters. Our results confirmed that all three treatment options led to clinical and cytological improvement of Malassezia dermatitis in dogs. However, the combination of a topical and a systemic antifungal treatment was more effective than systemic therapy used alone, confirming the value of the tested antifungal shampoo. Further studies are necessary to compare the cost, efficacy, and adverse effects of therapeutic options in localized vs. generalized or mild vs. severe cases. Funding: Self funded. Conflict of Interest: None declared.

P-080 In vitro activities of antifungal agents against clinical isolates of dermatophytes from animals R. KANO*, S. ITOI*, A. HASEGAWA and H. KAMATA* *Nihon University School of Veterinary Medicine, Kanagawa, Japan; Teikyo University Institute of Medical Mycology, Tokyo, Japan The great majority of canine, feline, and rodent dermatophytosis are caused by Microsporum canis, Trichophyton mentagrophytes, or Microsporum gypseum. The susceptibility of these dermatophytes to antifungal drugs is well documented for human isolates but not for animal isolates. We measured the in vitro susceptibility of 54 dermatophyte isolates from dogs, cats, rabbits and guinea pigs with dermatophytoses to ketoconazole, itraconazole, and terbinafine using the microdilution assay (CLSI M38-A2 test) and the E-test and compared with the reported human data. All three drugs showed antifungal activity. The minimum inhibitory concentrations (MICs) of ketoconazole and itraconazole were almost the same for human and animal isolates of T. mentagrophytes and T. rubrum. Using the microdilution assay, we found that the MIC of terbinafine was almost the same for dermatophytes isolated from humans and animals (0.031–16). However, the MICs of ketoconazole and itraconazole for animal isolates of M. canis were higher than those for human isolates. Ketoconazole displayed the broadest MIC range (0.125–16) against M. canis. The MICs of ketoconazole and itraconazole were almost identical for animal isolates of the T. mentagrophytes complex, which were divided into Arthroderma benhamiae- and A. vanbreuseghemii-related genotypes by internal transcribed spacer (ITS) region analysis. The Etest was confirmed to be suitable for testing the antifungal susceptibility of dermatophytes to itraconazole (0.0625–4). The higher MICs of ketoconazole and

Abstracts itraconazole for some animal isolates of M. canis compared to human isolates suggest possible therapeutic failure in practice. Funding: Grants-in-aid from the Academic Frontier Project of the Japanese Ministry of Education, Culture, Sports, Science and Technology; and Nihon University. Conflict of Interest: None declared.

P-081 Efficacy of eight commercial disinfectants against Microsporum canis spores on textile swatches D. KUNDER and K. MORIELLO School of Veterinary Medicine, University of WisconsinMadison, Madison, WI, USA This study screened eight commonly used disinfectants for fungicidal activity against Microsporum canis. We inoculated 16 cm2 gauze swatches with M. canis spores from contaminated hairs in a manner that simulated an area a client would deem ready for disinfectant application. Each disinfectant was tested by applying one and five spray volumes onto separate contaminated swatches. After a 10-min contact time, samples were pressed to dermatophyte test medium (Remel, Thermo Fisher Scientific; Lenexa, KS) plates, incubated at 30C, and monitored for growth. Studies were done in replicates of eight. Untreated controls grew > 300 colony forming units per plate. There was no growth on plates from swatches treated with undiluted bleach. The following disinfectants were ineffective with one spray application: sodium hypochlorite 0.0095% (Clorox Anywhere; The Clorox Company, Oakland, CA, USA), ethoxylated alcohol mixture 3% (Simple Green; Sunshine Makers, Huntington Beach, CA, USA), quaternary ammonium 0.22% (Fantastik; SC Johnson & Son, Racine, WI, USA), and potassium peroxymonosulfate 21.41% and sodium chloride 1.5% (Trifectant; Vetoquinol, Fort Worth, TX, USA). However, these four disinfectants had no growth with the five spray application. The following disinfectants had no growth at either one or five sprays: quaternary ammonium 0.3% (409; The Clorox Company, Oakland, CA, USA), sodium hypochlorite 1.84% (Clorox Clean-Up; The Clorox Company; Oakland, CA, USA), lactic acid 3.2% (Lysol; Reckitt Benckiser, Parsippany, NJ, USA), and hydrogen peroxide 0.5% (Accel TB; Virox Technologies, Oakville, ON, Canada). The four disinfectants that inhibited dermatophyte

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growth with both one and five sprays warrant further investigation. Funding: Unrestricted gift from Maddie’s Fund. Conflict of Interest: None declared.

P-082 Changes in serum chemistries in shelter cats treated with 21 days of oral itraconazole for dermatophytosis K. MORIELLO, M. VERBRUGGE and J. GINN School of Veterinary Medicine, University of WisconsinMadison, Madison, WI, USA A common protocol for treating cats with dermatophytosis is itraconazole 5 mg/kg orally once daily on a week on/week off rotating treatment schedule. Hepatopathy has been associated, albeit idiosyncratically, with itraconazole administration, particularly with longer treatment protocols or anecdotally in cats with preexisting illnesses. In some shelters, a daily treatment protocol may be needed to minimize lapses of treatment. Shelter cat populations are unique because often treatment decisions need to be made with little or no knowledge of prior history. This study looked for evidence of hepatopathy in shelter cats (n = 21) being treated for dermatophytosis with 5 mg/kg itraconazole orally once daily for 21 days. Blood samples were collected before and after 21 days of itraconazole treatment and analyzed and compared pre- and posttreatment. Data were analysed using a Mann–Whitney U test (P < 0.05). No significant differences were found in changes in total bilirubin (P = 0.60), serum alkaline phosphatase (P = 0.56), or aspartate aminotransferase (P = 0.26). A significant increase was seen in alanine aminotransferase (P = 0.025), but none of the cats had values outside the normal reference ranges (20–108 U/ L). Pre-treatment mean, median, and range of alanine aminotranferase were 41.7, 42.5, and 25–67 and posttreatment were 50, 48.5, and 32–83, respectively. None of the cats became ill or anorexic on this protocol. Based upon these results, neither clinical nor biochemical evidence of hepatopathy were documented in cats undergoing continual treatment with itraoconazole. However, biochemical monitoring is indicated if cats become ill or anorexic or if longer treatment is needed. Funding: Unrestricted gift from Maddie’s Fund. Conflict of Interest: None declared.


Topic 9: DERMATOLOGICAL PRACTICES P-083 The early days of the Canadian Academy of Veterinary Dermatology (1984–1992) V. E. DEFALQUE Canada West Veterinary Specialists, Vancouver, BC, Canada Canada is the only country to have organized the World Congress of Veterinary Dermatology (WCVD) twice. The Canadian Academy of Veterinary Dermatology (CAVD) was instrumental in the organization of the second WCVD in Montreal in 1992. This earlydays conference attracted over 500 delegates. The history of the CAVD can be traced back to 1984. It evolved from the Ontario Veterinary Dermatology Interest Group based on the model of the British Veterinary Dermatology Interest Group. Three Ontario veterinarians, Drs. Ackerman, Schroeder, and Pukay, observed the widespread interest in the booming specialty and later considered expanding their group nationally. The first official meeting took place at the annual meeting of the Society of Ontario Veterinarians on 1 February 1985. The following summer, the ‘founding fathers’ gathered in Fenelon Falls in Ontario to discuss a tentative constitution and by-laws that were modeled after those of the American Academy of Veterinary Dermatology. There were over 140 members from across Canada by year’s end. The constitution was officially ratified at the annual Society of Ontario Veterinarians meeting held in Toronto on 1 February 1986. The CAVD was born with Dr. Ackerman as first president. The first annual symposium was held as a satellite conference to the 23rd World Veterinary Congress in Montreal on 15 August 1987. Lecturers included Drs. Ackerman, Schroeder, Scott, Keaney, Yager, and Paradis. Annual seminars in subsequent years helped establish the reputation of the association in the country. Funding: Self funded. Conflict of Interest: None declared.


P-084 Cross-sectional study of skin disorders in 225 canine patients presented at a dermatology practice in Buenos Aires, Argentina (2010–2011) G. MANIGOT Dermlink Buenos Aires, Buenos Aires, Argentina This study documented the demographics of skin disease in dogs in the area of Buenos Aires, Argentina. We studied 225 dogs presented for skin consultation at a private dermatology service in Buenos Aires. All cases constituted first-time visits to a specialty practice and were consecutively selected as they were received between April 2010 and July 2011. All procedures were performed and data obtained by the same specialist (the author). Cytology, skin scraping, and acetate tape tests were performed in all cases. Several parameters were analysed, including: breed, sex, age, use of a flea control, use of ivermectin, and previous procedures performed by referring veterinarians (cytology, skin scrapings, biopsy, culture, blood tests, thyroid profile, allergy testing, and food trials). Other information considered relevant was also recorded and analysed (previous episodes of otitis, homeopathic treatment). Bacterial folliculitis, canine atopic dermatitis, and flea-bite dermatitis accounted for 22%, 20%, and 9%, respectively, of all the dogs examined at our private dermatology service during a 15-month period. The most common breeds of dogs seen at the practice during the period of the study were Labrador retriever, toy poodle, and beagle, which reflect popular breeds in Buenos Aires. The preferred tests performed by referring veterinarians were complete blood count, chemistry, and thyroid profiles. The tests least used before specialty consultation were cytology, biopsy, and skin scraping. Fipronil was the most used flea-control product (24%), and 4–12 weeks was the most frequent interval for spot-on application (30%). Funding: Self funded. Conflict of Interest: None declared.

Topic 10: IMMUNE-MEDIATED DISEASES AND THERAPY P-085 Canine pemphigus foliaceus: a retrospective study of 102 cases (1986–2011) in Saõ Paulo, Brazil J. ODAGUIRI*, L. LUCARTS*, N. MICHALANY, C. E. LARSSON JR* and C. E. LARSSON* *School of Veterinary Medicine and Animal Science, University of Saõ Paulo, Saõ Paulo, Brazil; School of Medicine, Federal University of Saõ Paulo, Saõ Paulo, Brazil Since a limited number of retrospective studies of canine pemphigus foliaceus are available in South America, we reviewed clinical records of dogs attended by the dermatology service over a 25-year period. The diagnosis was based on clinical signs as well as cytologic and histopathologic examinations. One hundred and two cases of canine pemphigus foliaceus were identified (4.1 cases/year); 74% were purebred dogs with the Akita (9/102) being the most prevalent breed. Females accounted for 58/102 of the cases, and 61/102 of the dogs were 4–9 years old. Pruritus was present in 78/102 of the dogs (severe pruritus, 38/102). Lesions were observed mainly at abdominal (79/102), head (76/ 102) and thorax (72/102) areas. The most frequent lesions were crusts (94/102), pustules (76/102), erythema (56/102), and papules (54/102). A good clinical response was observed in 58/102 patients that received sole treatment with prednisone at 2 mg/kg/day per os. The remaining 44 dogs required the addition of azathioprine (2 mg/kg/day) to the treatment regimen in order to control the disease and reduce the glucocorticoid dose. Funding: Self funded. Conflict of Interest: None declared.

P-086 Serum antibody determination by indirect immunofluorescence in dogs affected with pemphigus foliaceus, before and during treatment L. LUCARTS*, C. LARSSON*, V. AOKI and N. MICHALANY *Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Saõ Paulo, Saõ Paulo, Brazil; Faculdade de Medicina, Universidade de Saõ Paulo, Saõ Paulo, Brazil; Escola Paulista de Medicina, Universidade Federal de Saõ Paulo, Saõ Paulo, Brazil Pemphigus foliaceus (PF) is characterized clinically by the presence of pustules, histopathologically by acantholysis, and immunologically by the presence of autoantibodies (IgG). It is believed that these autoantibodies are related to disease activity. This study evaluated the feasibility of indirect immunofluorescence (IFI) in the diagnosis of PF in dogs, and identified a possible correlation between serum antibodies titres and treatment responses. For the indirect immunofluorescence, canine pawpads fragments were used as substrate, and intercellular fluorescence pattern was considered positive. Sera from seven dogs with clinical and histo-

pathological diagnosis of PF were stored at )70C. Indirect immunofluorescence was first performed at the time of diagnosis and, in one animal, at the time of severe relapse. In all dogs with a positive test, further indirect immunofluorescence tests were performed in every recheck visit. The clinical score was determined by pemphigus foliaceus extent and severity index (PEFESI). These animals were followed up for 101–341 days. Positivity was obtained in five (71.4%) of the dogs. From the positive animals, those with the highest PEFESI score showed the greatest autoantibody titres. During the treatment period, the IgG titres decreased in all dogs as did the PEFESI score. We conclude that indirect immunofluorescence is a feasible diagnostic method for canine PF, with a predictive value of 71%. The canine pawpad proved to be an effective substrate, and IgG autoantibody titres, as well as the clinical signs of the disease, proved to be useful for monitoring the progression of PF. Funding: Self funded. Conflict of Interest: None declared.

P-087 Randomized, controlled trial of methylprednisolone sodium succinate pulse therapy in canine pemphigus foliaceus: pilot study of 20 dogs (2005–2011) E. BENSIGNOR Dermatology Referral Service, Rennes-Cessone; Dermatology Referral Service, Paris; Dermatology Referral Service, Nantes, France Treatment of pemphigus foliaceus (PF) involves the use of immunomodulating drugs such as glucocorticoids. This study determined whether pulse therapy using intravenous high doses of methylprednisolone (IMPT) helped to induce remission and/or decrease glucocorticoid dose long term. We randomly assigned 20 dogs diagnosed with PF on the basis of clinical, cytological, and histopathological findings to either receive IMPT (group A) or not receive this treatment (group B). All dogs received conventional treatment with methylprednisolone orally, 1–2 mg/kg/day, tapered as soon as clinical lesions improved, and azathioprine orally, 1– 2 mg/kg/day for 1 month and then every other day. However, dogs in group A also received three consecutive doses of IMPT at 10 mg/kg/day. Follow-up visits were conducted every month for clinical evaluation. For each group we calculated the number of patients in remission, time and duration of remission, and cumulative methylprednisolone dose. At the end of the study, 14 dogs were in remission (8/12 in group A and 6/8 in group B). Four dogs were still presenting active disease (two in each group); two dogs were lost for follow-up (one in each group); and two dogs were euthanized due to treatment adverse effects (one in each group). Mean time to remission was 57.5 days in group A and 63.2 in group B (P > 0.05). Mean dose of medication was not statistically different (P > 0.05) between the groups at any time point. Dogs in group A did not present more adverse events. Funding: Self funded. Conflict of Interest: None declared. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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P-088 Juvenile generalized pyogranulomatous dermatitis and lymphadenitis treated with ciclosporin: a report of five cases E. GUAGUE`RE*, A. MULLER* and F. DEGORCERUBIALES *Clinique Ve´te´rinaire Saint Bernard, Lomme, France; Laboratoire d’Anatomie Pathologique Ve´te´rinaire du Sud-Ouest, Toulouse, France Juvenile generalized pyogranulomatous dermatitis and lymphadenitis (JGPDL) is a skin condition of sudden onset seen in puppies. This report describes five cases of JGPDL associated with severe systemic signs that were refractory to oral corticosteroids, but successfully treated with ciclosporin. We describe three German shepherd puppies (10 weeks of age) belonging to the same litter and two golden retriever puppies (10 weeks of age), diagnosed with JGPDL unresponsive to oral prednisolone (1–2 mg/kg/day for 2 weeks). In all cases, systemic signs included a severe febrile syndrome. Dermatological signs were dominated by bilateral symmetrical facial and pedal erythematous, crusty and oedematous dermatitis. Similar lesions were observed on the prepuce and anus. A marked generalized peripheral lymphadenopathy was consistently present. Histopathological findings of biopsies from intact skin revealed granulomatous to pyogranulomatous inflammation centred on sebaceous and apocrine sweat glands. Superficial cytological examination revealed bacterial colonization (cocci). Bacterial culture from non draining lesions was sterile. Antibacterial shampoos were used every 2–3 days. Ciclosporin was given (5 mg/kg/day orally) for 1 month, then at the same dosage every 2 days for a further 3 weeks, and finally every 3 days for a further 3 weeks. No adverse effects were reported. Clinical improvement was progressively seen within 2–3 weeks. Time needed for resolution was between 6 and 9 weeks on average. Ciclosporin appears to be a good alternative to corticosteroids for treating dogs with JGPDL; however, a randomized controlled trial must be performed to confirm this belief. Funding: Self funded. Conflict of Interest: None declared.

P-089 Effects of ciclosporin for sebaceous adenitis and comparative pathology of serial skin biopsies of an Akita dog Y. TSUCHIDA Gori Veterinary Clinic, Hirosaki, Japan A 3 year old, male Akita dog was presented with generalized alopecia and a hair coat with rice bran-like scaling. At presentation, skin biopsies revealed granulomatous inflammation and destruction of sebaceous glands. The granuloma cells tested strongly positive for


anti-lysozyme (macrophage), and CD3 and CD20 antibodies (T and B lymphocytes, respectively) by immunohistochemistry. After 4 weeks of presentation, the dog was treated with ciclosporin at a dosage of 5 mg/kg/day for 4 months. Skin biopsies were performed at 10, 34, 49, 63, and 120 days after treatment was discontinued. Thirty 4 days after discontinuation of ciclosporin, granulomatous inflammation was decreased, and the numbers of hair follicles with immature sebaceous glands was increased suggesting regeneration. These results suggest that ciclosporin is useful for the treatment of sebaceous adenitis in Akita dogs by not only reducing the associated inflammatory process but also inducing the regeneration of sebaceous glands. Funding: Self funded. Conflict of Interest: None declared.

P-090 Treatment of canine idiopathic sebaceous adenitis with topical ciclosporin (0.4%): nine cases R. LUCAS*,, D. BEVIANI*, C. PELEGRINI*, K. CANTAGALLO*, R. ROLAN* and F. ZERBINI* *University Anhembi Morumbi, Saõ Paulo, Saõ Paulo, Brazil; Dermatoclinica Veterinary Dermatology Referrals, Saõ Paulo, Brazil Granulomatous sebaceous adenitis is a primary idiopathic inflammatory disease that targets and destroys sebaceous glands. Although it occurs in many breeds, there is an apparent prevalence in Japanese Akita dogs, standard poodles, Vizslas, and Samoyeds. This retrospective study evaluated the clinical presentation and efficacious treatment of sebaceous adenitis with topical ciclosporin. Sebaceous adenitis was diagnosed in nine dogs: six Japanese Akita dogs, one golden retriever, one Australian shepherd and a mixed-breed dog. Seven dogs were male and two were female. Onset of clinical signs occurred during youth and adulthood. The average age of the dogs was 6.8 years. Follicular casts were present in 100% of affected dogs. Other common clinical signs included alopecia and hypotrichosis. The trunk, head, and ears were commonly affected. Secondary pyoderma was seen in 80% of dogs. In all dogs histopathology revealed absent sebaceous glands and a lymphoplasmacytic periadnexal infiltrate. The dogs were treated with topical ciclosporin (Sandimmun Neoral; Novartis, Basileía, Suı´ ça), by mixing four 100 mg capsules in 100 mL vegetable oil. The solution was applied twice per week. Clinical improvement was noticed in all dogs, and total hair regrowth occurred in 4 months. Topical (0.4%) ciclosporin A applied twice a week was well tolerated and efficacious in the symptomatic treatment of sebaceous adenitis in dogs. Funding: Self funded. Conflict of Interest: None declared.

Topic 11: NEOPLASTIC DISEASES AND THERAPY P-091 Experimental study on the protective effect of Nigella sativa oil on mice skin exposed to ultraviolet B radiation O. NASRULLAH, S. MUHAMMAD and A. AHMAD Sulaimani University, Kurdistan Region, Sulaimani, Iraq Many studies have examined the antibacterial effects of Nigella sativa oil (black seed oil); the present study evaluated the protective effects of this oil against ultraviolet B radiation (UVB) on mouse skin. Unprotected exposure to UVB radiation leads to skin-related changes including inflammation, hyperpigmentation, photoaging, and cancer. The primary aim of this study was to identify an inexpensive and natural protection from UVB exposure. We used age- and weight-matched BALB/c mice (Mus musculus), which are hairless along the dorsum. The mice were divided into three groups. Group A included 15 mice directly exposed to UVB light for 15 min daily (six times per week); group B consisted of 15 mice exposed to UVB light using the same protocol as for group A, after topical treatment of the dorsal hairless area with the oil of Nigella sativa (Hemani; Pakistan). Group C was the control group and included five mice that received no treatment with Nigella sativa oil or UVB exposure. Our results showed that histopathological changes such as skin thickening occurred in all mice in group A (53% severe, 40% moderate, and 7% mild). In group B, 67% of the treated mice showed mild skin thickening and 33% did not show any histopathological changes. These results indicate that black seed oil has good protective effect against skin damage induced by UVB radiation. Funding: Self-funded. Conflict of Interest: None declared.

P-092 Immunohistochemical expression of transforming growth factor beta 1 (TGF-b1) in canine cutaneous tumours A. R. MOVASSAGHI*, M. REZAEE OGHAZI, J. KHOSHNEGAH and M. RAD* *Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran; Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran; Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran Cutaneous neoplasms are the most common neoplasm in dogs. The transforming growth factor (TGF-b) family has an ambivalent role in cancer progression, as it has been shown to have both tumour-suppressing and tumour-enhancing effects. In this study we aimed to detect TGF-b1 expression in canine spontaneous cutaneous tumours by immunohistochemistry. We examined 16 samples of canine spontaneous cutaneous tumours including: sebaceous gland tumours (3), basal cell tumours (2), transmissible venereal tumours (3), hepatoid gland adenoma (1), trichoblastoma (1), reticular cell

carcinoma (1), fibrosarcoma (1), melanoma (1), spindle cell tumour (1), leiomyosarcoma (1), and myxoma (1). These were routinely processed and immunostained with anti-TGF-b1 antibody using the avidin biotin method, horse radish as enzyme reagent, and diaminobenzidine as chromogene. Canine normal skin and mouse ovary tissues were used as negative and positive controls, respectively. Immunoscoring was done according to the distribution of positive cells and intensity of positive signals in comparison with positive control. Slides stained by immunohistochemistry were scored as negative ()), weak (+), moderate (++), and strong (+++). TGF-b1 was variably expressed by the 16 cutaneous tumours in comparison to normal skin. Three tumours showed no specific labelling, five tumours were scored as weakly immunoreactive, five moderately, and three strongly, compared to controls. In summary, we observed that 13 of 16 tumours expressed TGF-b1 to some degree. These data suggest that TGF-b1 may be a potential target for treatment of canine skin tumours. However, more investigation is required to understand its function in cutaneous neoplasms in dogs. Funding: Ferdowsi University of Mashhad, Mashhad, Iran. Conflict of Interest: None declared.

P-093 Immunohistochemical study of genital and extragenital forms of canine transmissible venereal tumours M. MASCARENHAS, T. FRANÇA, R. RAMADINHA, T. COSTA, E. YAMASAKI and P. PEIXOTO Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil Canine transmissible venereal tumours (CTVT) are a naturally occurring contagious round-cell neoplasia located mainly in the external genital mucosa of male or female dogs. However, extragenital CTVT not associated with genital lesions has been reported in the oral and nasal cavities, conjunctiva, eye, skin, tonsils, and oral and anal mucosa. In these atypical cases, it is usually difficult to differentiate CTVT from other canine roundcell tumours such as lymphomas, histiocytomas, poorly differentiated carcinomas, mast cell tumours, and amelanotic melanomas. To provide insight and discuss the problems related to the diagnosis and differential diagnosis of CTVT, especially in its extragenital form, 10 genital and 13 exclusively extragenital CTVTs previously diagnosed by histopathology were compared by immunohistochemistry using various antibodies. CTVT samples were incubated with biotinylated antibodies including anti-macrophage (clone LN-5, Invitrogen), anti-lysozyme (Zymed-Invitrogen), anti-S-100 protein (Zymed-Invitrogen), anti-vimentin (Dako), and antiCD18 (CA 163C10, University of California). The avidin-biotin-peroxidase complex technique was used. A strong reactivity with the anti-vimentin antibody was found in 100% of the tumours tested (22/22). No reactivity was found for the anti-lysozyme, antimacrophage, anti-S-100 protein, and anti-CD 18. No 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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histopathological and immunoreactivity differences between genital and extragenital CTVTs were found. These findings do not corroborate the hypothesis of histiocytic origin of CTVT (no reactivity to anti-lysozyme, antimacrophage, and anti-CD 18 antibodies). The antibody panel used was very useful for narrowing the differential diagnoses of lymphomas, histiocytic tumours, amelanotic melanomas, and poorly differentiated epithelial neoplasias, among others. Funding: Fundaçaõ de Amparo a Pesquisa do Estado do Rio de Janeiro Capes; Coordenaçaõ de aperfeiçoamento de pessoal de nı´vel superior. Conflict of Interest: None declared.

P-094 Comparative study of histopathology and immunohistochemistry results of indefinite round-cell cutaneous tumours and characterization of canine lymphoma M. PALUMBO*, R. LAUFER-AMORIM*, M. FARIAS, J. WERNER, R. TORRES-NETO*, J. C. RODRIGUES*, F. C. OLIVEIRA* and L. H. A. MACHADO* *Saõ Paulo State University, Saõ Paulo, Brazil; Catholic University of Parana´, Curitiba, Parana´, Brazil; Werner & Werner Center of Diagnosis in Veterinary Pathology, Curitiba, Parana´, Brazil To help improve diagnosis in veterinary oncology, this study compared the results of histopathology and immunohistochemistry in cases of canine non specific round-cell tumours. We determined the immunophenotype in cases of canine lymphoma using anti-CD3 and anti-CD79a antibodies and determined the degree of cell proliferation and apoptosis in cutaneous lymphomas using KI-67 (MIB-1) antibodies and caspase-3, respectively. Of the non specific round-cell tumour cases, 4/15 were not confirmed to be lymphoma by immunohistochemistry, and 11/15 presented positive immunohistochemical diagnosis of lymphoma. From those 11 positive cases, only five were suspected of lymphoma by routine histopathological examination using haematoxylin-eosin stain, and the other six cases were previously classified as non specific round-cell neoplasia. All lymphoma cases confirmed by immunohistochemistry were of T-cell origin. These findings suggest that immunohistochemistry is essential to a reliable diagnostic differentiation of round-cell tumours, which will be valuable in determining the therapeutic approach, whether surgical, chemotherapeutic, radiotherapeutic, or a combination of these treatment modalities. Spearman correlation test demonstrated positive correlation between cell proliferation and apoptosis, indicating the growth capacity of lymphomas and their tendency to ulceration and necrosis. The present study has highlighted the importance of the use of more specific techniques to improve diagnosis in veterinary oncology, although further studies in canine lymphoma are still necessary to determine the most frequently involved cell types, their proliferative and apoptotic patterns, and the association of those findings with the disease progression. Funding: Saõ Paulo Research Foundation; Saõ Paulo State University – UNESP. Conflict of Interest: None declared. 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

P-095 Clinical and pathological evaluation of dogs with cutaneous lymphoma treated with lomustine: is there a prognostic factor? A. R. DUARTE, J. A. MARQUES, F. ELSTON, C. A. F. ALVES, R. LAUFER-AMORIM, M. J. SUDANO and L. H. A. MACHADO Saõ Paulo State University, Saõ Paulo, Brazil The continuous progress in veterinary medicine and the improvement of pet care have allowed clinicians to introduce useful practices to improve the diagnosis and prognosis of pet diseases. The present study investigated whether there is any prognostic marker for canine cutaneous lymphoma in haematological and urinary parameters and evaluated any changes in those parameters during lomustine therapy. Complete blood count, serum biochemistry, and urinalysis were performed in 15 dogs with cutaneous lymphoma before, during, and after lomustine therapy. All the tumours were classified as Tcell lymphoma using anti-CD3 and anti-CD79a antibodies. Since breed and age were highly variable among the dogs, it was not possible to verify their influence on the studied parameters. Mean survival time was 59.3 days. Pearson correlation test between survival time and the evaluated parameters did not identify any prognostic factor (P > 0.05). Evaluation of repeated measures before, during, and after treatment by analysis of variance (ANOVA) demonstrated that haemoglobin, haematocrit, total protein, leukocytes, creatinine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and urine specific gravity worsened over the course of the treatment (P < 0.05). On the other hand, red blood count, lymphocyte count, and albumin were not significantly influenced by treatment but tended to worsen. These results did not allow us to identify any prognostic factors for canine cutaneous lymphoma and demonstrated that treatment with lomustine worsened the clinical condition of the animals. These findings demonstrate that therapeutic approaches for this disease need to improve and that further studies investigating prognostic factors are necessary. Funding: Saõ Paulo Research Foundation, Saõ Paulo State University – UNESP. Conflict of Interest: None declared.

P-096 Elbow dysplasia as a cause of interdigital cysts in 20 dogs S. PATERSON Rutland House Veterinary Hospital, St Helens, UK In this study, fragmentation of the medial coronoid process and medial compartment disease was identified in 20 dogs that presented with chronic interdigital cysts to the dermatology department between January 2010 and August 2011. Bulldogs (four), Labradors (four), Dogue de Bordeaux (three), and Staffordshire bull terriers (three) accounted for 70% of the cases. Diagnostic tests included allergy tests (12/20), food trials (7/20), and thyroid function tests (9/20). An underlying dermatological cause could not be identified in any dog. Histopathology revealed perifolliculitis, folliculitis, and

Abstracts furunculosis often with a nodular to diffuse pyogranulomatous inflammation. In 18/20 cases, secondary infection was identified on tissue culture and subsequently treated with 6 weeks of an appropriate antibiotic. Orthopaedic assessment revealed elbow swelling and pain. Computer tomography was performed on both elbows. All dogs had bilateral disease. In cases of fragmentation of the medial coronoid process, a subtotal coronoidectomy was performed arthroscopically. In medial compartment disease, conservative therapy with physiotherapy, exercise restriction, and appropriate analgesia was prescribed. In all cases, appropriate manage-

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ment of their elbow disease and antibiotic therapy when required, led to dramatic improvement or resolution of their interdigital cysts. It is suggested that disease of the medial compartment of the canine elbow may lead to an abnormal posture, forcing dogs to bear weight on haired skin. The ensuing foreign body reaction associated with furunculosis leads to interdigital cyst formation. Where the dog’s gait can be improved by orthopaedic intervention, the interdigital cysts will resolve without specific therapy. Funding: Self funded. Conflict of Interest: None declared.


Topic 12: OTITIS AND THERAPY P-097 Comparison of rigid otoscopy and computed tomography for tympanic membrane assessment in dogs M. DE LUCIA, G. BERTOLINI, M. CALDIN, S. FINESSO and A. FONDATI Clinica Veterinaria Privata San Marco, Padua, Italy Tympanic membrane evaluation is mandatory for therapeutic and prognostic reasons in the workup of dogs with chronic otitis or clinical signs suggestive of middle ear disease. This study compared rigid otoscopy and 16multidetector computed tomography examination for tympanic membrane assessment. Medical records of 22 dogs that had undergone computed tomography of the splanchnocranium and rigid otoscopy were retrospectively selected and reviewed. There were 12 dolichocephalic, five mesaticephalic, and five brachicephalic dogs suffering from chronic otitis (14/22), head tilt (4/22), peripheral vestibular syndrome (2/22), recurrent aural haematoma (1/22), and head shaking (1/22). Administration of systemic or topical glucocorticoids before examination was reported in 3/22 dogs. Unilateral total ear canal ablation and lateral bulla osteotomy was performed in 2/22 dogs. Computed tomography allowed tympanic membrane evaluation in all the examined ears (42/42). Rigid otoscopic examination, performed during the same anaesthetic episode, did not allow visualization of the tympanic membrane in 19/42 ears due to ear canal stenosis (11/30), purulent material (2/30), bleeding (2/30), and proliferative lesions (4/30). Rigid otoscopy allowed tympanic membrane evaluation in the three dogs pretreated with glucocorticoids. Computed tomography seems to represent a more useful procedure, compared to rigid otoscopy, to visualize tympanic membrane in dogs with ear canal changes due to inflammatory or proliferative diseases. Funding: Self funded. Conflict of Interest: None declared.

P-098 Reduction of relapses of recurrent otitis externa in atopic dogs with twice-weekly topical application of hydrocortisone aceponate in the ear canal: a randomized, blinded, controlled study E. BENSIGNOR*, J. PATTYN* and C. REME *Dermatology Referral Service, Rennes-Cesson, France; Virbac SA, Carros, France Frequent relapses of otitis externa in canine atopic dermatitis may be related to residual inflammation in the ear canal. This study evaluated the efficacy of topical intermittent application of a pure dermocorticoid to prevent relapses of otitis externa in canine atopic dermatitis. Twenty atopic dogs with a relapsing (> three episodes/year) bilateral otitis externa were included. After successful treatment of otitis externa with a topical antibiotic-antifungal-corticoid combination, dogs’ left and right ears were each randomly allocated to either an ear cleansing maintenance regimen once weekly (group A) or the same regimen followed by application 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

of three drops of 0.0584% hydrocortisone aceponate (HCA, Cortavance, Virbac) in the ear canal two consecutive days per week (group B). Follow-up visits were scheduled after 1 month and then every 2 months, or until relapse. Clinical (video-otoscope) and microbiological (cytology) parameters were monitored. The time to relapse and total clinical and microbial scores were compared between groups over time. The investigator was blind to treatment allocation. Intention-to-treat analyses were performed. The probability of remaining free of relapse was 95% in group B (median time to relapse: 180 days) compared to 50% in group A (median time to relapse: 90 days) (P < 0.01) after 6 months. The clinical and microbiological parameters were significantly lower in group B compared to group A starting at 1 month of the maintenance treatment and at all subsequent time points. These results suggest that twice weekly hydrocortisone aceponate application in the ear canal provides an effective maintenance regimen to control canine allergic otitis. Funding: Virbac SA, Carros, France. Conflict of Interest: E. Bensignor is consultant for Virbac; C. Reme is an employee of Virbac

P-099 The in vitro diffusion of several antimicrobial otic preparations through canine cerumen J. STAHL, S. MIELKE and M. KIETZMANN Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Foundation Hannover, Hannover, Germany This study evaluated the diffusion of several antimicrobial otic preparations through canine cerumen in vitro and quantified and compared the lipid composition of canine cerumen of normal dogs and dogs with otitis externa. Cerumen of dogs (n = 12) was analysed by high-performance thin-layer chromatography after lipid extraction with ethanol/hexane to obtain a detailed list of several lipid classes. The lipid analysis of canine cerumen showed significant differences between normal dogs and dogs with otitis externa concerning sterol esters. A standardized synthetic cerumen (SSC) based on the lipid composition of major lipids of dogs with otitis externa (methylpalmitate, squalene, sterol esters, and triglycerides) was therefore used to study the effect of different antimicrobial otic preparations on the diffusion of a marker substance (oil red) through canine cerumen. Capillary tubes were filled with SSC, and eight ear products (six antimicrobial otic preparations and two ear cleansers) were applied on the lipid layer after mixing with oil red. Diffusion activity was assessed by measuring the diffusion pathway of oil red out of the ear products in the capillary tubes. Pure oil red in SSC served as the control. The final diffusion pathways of oil red out of different formulations ranged from 0.4–0.9 cm (six products) to 2.4–2.5 cm (one product and control) and up to 5.1 cm for one product (P < 0.001). It was concluded that under the experimental conditions used in the present study, only 1/8 products showed a significant higher and faster diffusion than the control. Funding: Ve´toquinol GmbH, Germany. Conflict of Interest: The authors declare no conflict of interest.

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P-100 Field comparison of the efficacy of two ear products in the treatment of acute otitis externa in dogs

P-101 Comparative study on formulation properties of three topical products for the treatment of otitis externa in dogs

E. GRANDEMANGE and F. WOEHRLE´ Ve´toquinol SA, Lure, France A multi-centre randomized field study was conducted to compare the efficacy of two ear products in the treatment of dogs suffering from a newly diagnosed uni-or bilateral acute otitis externa. The products were administered according to their marketing authorization: Aurizon with a flexible (seven or 14 days), and Easotic with a fixed (5 days) treatment duration. One hundred and sixty dogs were enrolled in the study from which 157 were evaluated (73 Aurizon-treated animals and 84 Easotictreated animals). According to the randomization, dogs received either Easotic (Virbac SA, France) one treatment per day for 5 days or Aurizon (Ve´toquinol SA, France) one treatment daily for seven or 14 days. General and local signs were scored. Results were compared using chi-square tests. The main pathogens isolated before treatment were Staphylococcus pseudintermedius and Malassezia pachydermatis in 39.5% and 60.5% of the dogs, respectively. Clinical cure rates at the end of treatment were 56.3% and 48.8% (P = 0.35) in the Aurizon and Easotic groups, respectively, and 81.2% and 74.7%, respectively, 1 week after the end of treatment (P = 0.34). Final clinical cure rates on day 21 were 84.3% and 73.8%, respectively (P = 0.12). Both treatments were well tolerated. A correlation between severity of clinical signs and treatment duration was observed. In conclusion, the flexible labelling of Aurizon allowed to adapt the treatment duration to the severity of clinical signs and tended to reach better clinical results in the whole population compared to a fixed short treatment duration labelling. Funding: Ve´toquinol SA, Paris, France. Conflict of Interest: All authors are employees of Ve´toquinol SA.

I. BOUTOR and M. MOREAU Ve´toquinol SA, Paris, France Topical products for the treatment of canine otitis externa usually contain anti-inflammatory, antibiotic, and antifungal molecules. The clinical efficacy also relies on the vehicles used. Selecting the appropriate excipients is the challenge to get a satisfactory dispersed system sufficiently homogeneous for the period of time necessary to administer the required dose after shaking the container. This study compared Aurizon (Ve´toquinol, Paris, France), Easotic (Virbac, Carros, France) and Posatex (MSD, Boxmeer, NL, USA) using several standard formulation tests. All products are dispersed systems: they contain a continuous phase, liquid, and a dispersed phase made of insoluble particles. The formulations’ texture was assessed in terms of viscosity, spreadability, and homogeneity of active ingredient distribution, using the following materials: MettlerToledo balance, Nachet microscope, and Malvern viscometer (Bohlin CVO100). Aurizon and Easotic presented a smaller viscosity index (0.162 and 0.099 PaÆs, respectively, at 75 Pa; 0.108PaÆs and 0.082PaÆs, respectively, at 125 Pa) than Posatex (0.360 PaÆs at 75 Pa; 0.296 PaÆs at 125 Pa). These characteristics may ensure better coverage of the external ear canal during treatment. The viscosity index for Posatex was consistent with a weaker spreadability capacity of the daily dose compared to Aurizon or Easotic. The microscopic observations revealed crystals in Easotic with a less homogeneous profile than Aurizon and Posatex in terms of active ingredients’ particle size distribution. We recognize that the excipients used in the formulation may impact the clinical efficacy of a drug registered for otitis externa therapy. Aurizon presented the most interesting profile in terms of formulation texture. Funding: Ve´toquinol SA, Paris, France. Conflict of Interest: All authors are employees of Ve´toquinol SA.


Topic 13: PARASITIC DISEASES AND THERAPY P-102 Canine visceral leishmaniosis in Argentina: description of dermatological clinical signs and its correlation with cytological findings C. NEVOT*, A. COROMINAS, P. RUSSO*, C. VASSILIADES*, A. WOLBERG and O. ESTEVEZ* *Veterinaria del Oeste, Posadas, Misiones, Argentina; Universidad de Buenos Aires, Buenos Aires, Argentina In 2006, the first case of canine visceral leishmaniosis was diagnosed in Posadas City, Argentina. Shortly after, the disease spread throughout the northeast region of the country. Parasitological diagnosis of 3000 symptomatic dogs via cytology allowed us to correlate the different clinical presentations with the cytological findings of samples taken from different tissues. We evaluated 214 symptomatic dogs with cutaneous lesions using bone marrow and/or lymph node cytology, skin scrapings, and impression smears. Leishmania sp was not identified in all the cases by skin cytology. The most important cutaneous signs were ulcers, diffuse exfoliative dermatitis, alopecia, and onychogryphosis. In patients with chronic disease, hyperkeratosis and crusts were seen. Malassezia sp and a hyperplastic reactive pattern were common findings. Corroborating the experience of many researchers, parasites were found less frequently in cytology of skin samples than in samples from other organs such as lymph nodes and bone marrow. Skin cytology is also important for the diagnosis of other cutaneous diseases that contribute to worsening of skin lesions caused by Leishmania sp in addition to requiring specific treatment. Funding: Self funded. Conflict of Interest: None declared.

P-103 Epidemic Sarcoptes scabiei infestation in England: data from 225 cases in a 20-year longitudinal study C. J. CHESNEY Mott Clinic, Tiverton, Great Britain Scabies has been known to occur in epidemic form in both foxes and humans and, in 1994 the fox population of Bristol (UK West Country) was severely affected by an epidemic of scabies. Epidemiologic studies on canine sarcoptic mange are surprisingly limited, being based on anecdote or small studies. This study provides information from a large database. The author’s records (1991– 2011) cover three geographical areas. In most cases, the diagnosis was confirmed by finding any combination of mites, larvae, eggs, or faecal material (192 cases). In some cases, however, the diagnosis was made based on compatible history, clinical signs, and response to therapy (33 cases). The data showed that canine scabies occurred in two regions at epidemic level (1991–2000). At peak, almost 10% of the referred dermatology cases had scabies. In South Wales, across the river Severn, only 2.5% dogs were infested. Thirteen cases (6.7%) were ‘Norwegian scabies’ and highly infectious, with counts of 90–240 mites in one skin scraping. In 36.4% cases, incontact humans had signs compatible with scabies. Mites 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

were found in a much higher proportion of scabies suspect dogs (85.3%) than previously reported. This is attributed to pattern recognition in both microscopy and clinically (typically, clusters of 1–2 mm diameter dark red crusts on the outer aspect of the elbow, or areas of dense yellowish scale, varying from isolated patches a few millimetres across, to covering most of the patient’s body). This study provided good evidence that canine scabies also occurs in an epidemic form. Funding: Self funded. Conflict of Interest: A few cases were identified when the author partook in a clinical trial of selamectin (Stronghold, Pfizer).

P-104 Canine demodicosis treated with a chewable formulation containing milbemycin oxime as an active ingredient and beef as an additive ingredient Y. NAKAMURA*,, S. HAYASHIYA*,, M. HAYASHIYA* and T. FUKASE *Hayashiya Animal Hospitals, Uji-shi, Kyoto, Japan; Hayashiya Institute of Life Sciences, Uji-shi, Kyoto, Japan Demodicosis is a parasitic skin disease caused by mites belonging to the genus Demodex. Long-term treatment is usually necessary to cure canine demodicosis but, treatment may not be successfully completed because of the low palatability of oral acaricidal medications. In the present study, we evaluated the efficacy and convenience of a chewable formulation containing milbemycin oxime as the active ingredient and beef as the additive agent (Milbemycin Tablets ‘Fujita’: Fujita Pharmaceuticals Co., Ltd.; Japan), in the off-label treatment of canine demodicosis. The chewable product was administered to three dogs diagnosed with demodicosis at a dose of 0.5– 2.0 mg/kg once a day until no mites were found on skin scrapings. Milbemycin oxime was fully effective to eliminate the demodex mites in all cases. In addition, it was readily palatable; therefore, long-term treatment could be continued without difficulty, thus, satisfying dog owners. Based on these findings, the chewable formulation of milbemycin oxime was helpful in improving compliance or adherence of clients to the treatment of canine demodicosis because of its high palatability and ease of administration. Funding: Hayashiya Animal Hospitals. Conflict of Interest: None declared.

Abstracts

P-105 Influence of systemic antibiotics on the treatment of dogs with generalized demodicosis E. S. KUZNETSOVA*, S. BETTENAY, L. V. NIKOLAEVA*, M. MAJZOUB and R. S. MUELLER§ *Clinic of Veterinary Medicine ‘‘Beliy Klik’’, Moscow, Russian Federation; Tierdermatologie Deisenhofen, Deisenhofen, Germany; Institute for Animal Pathology, Centre for Clinical Veterinary Medicine, Ludwig Maximilian University Munich, Munich, Germany; §Small Animal Medicine Clinic, Centre for Clinical Veterinary Medicine, Ludwig Maximilian University Munich, Munich, Germany Canine demodicosis is a skin disease with distinct breed predispositions. Secondary bacterial infections are common. Dogs typically receive miticidal therapy in combination with antibacterial treatment. Whether antibiotic therapy influences the duration of acaricidal therapy is unknown at the moment. Moreover, there is debate over how common short-tailed Demodex mites occur in demodicosis. This study evaluated the influence of systemic antibiotic therapy on the course of canine generalized demodicosis, the occurrence of short-tailed Demodex mites in demodectic dogs, the influence of furunculosis on treatment outcome and the breed predisposition for generalized demodicosis in Moscow. Fifty-eight dogs were randomly distributed in two groups. Both groups were treated with ivermectin 0.6 mg/kg once daily orally and benzoyl peroxide shampoo weekly. The dogs in one group were additionally treated with systemic antibiotic for at least 1 month. Physical examinations, skin scrapings, and impression smears were performed monthly. Prior to the study, there was no difference in clinical severity, presence of pyoderma, and mite numbers between groups. There was no significant difference in treatment duration until two negative skin scrapings were obtained between dogs treated or not treated with antibiotic (P = 0.2976). The number of the mites was significantly higher in dogs with furunculosis compared to dogs without furunculosis (P = 0.0059); however, the duration until microscopic remission, albeit longer, was not significantly different (P = 0.0542). Short-tailed Demodex mites were found in 25% of the cases. Pugs and English bulldogs were predisposed. Based on these results, systemic antibiotic therapy may not impact on the actual success of demodicosis treatment. Funding: Self funded. Conflict of Interest: There is no conflict of interest to report for S. Bettenay and M. Majzoub. In the last 5 years E. Kuznetsova, L. Nikolaeva and R. Mueller have lectured for different companies.

P-106 Treatment of feline sarcoptic mange with topical moxidectin and imidacloprid H. P. HUANG*, Y. H. LIEN and J. P. SUN *Institute of Veterinary Clinical Science, Veterinary School, National Taiwan University, Taipei, Taiwan; Azu Clinic for Animals, Taipei, Taiwan Sarcoptes scabiei infestation in cats is rarely reported. Five cats diagnosed with S. scabiei that did not respond

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to subcutaneous ivermectin (0.2 mg/kg every 2 weeks for 8 weeks) and spot-on selamectin (8 mg/kg once monthly for two applications) were included in this study. The diagnosis of sarcoptic mange was based on finding S. scabiei from deep skin scrapings. The major dermatological manifestations recorded were hyperkeratosis/ crusts on both the concave and convex aspects of the pinna (5/5) and dorsal aspect of the nose (5/5), crusty pododermatitis (3/5), crusty dermatitis on the tail (1/5), and itchy erythematous papules on the arms and/or thighs of the owners (5/5). All five cats were living indoors, but two were allowed outdoor access. None of these five cats was living in the household with a dog. Spot-on 10% imidacloprid and 1.0% moxidectin (Advocate; Bayer HealthCare AG, Leverkusen, Germany) at the dose of 0.1 mL/kg was applied every 2 weeks for three applications. All cats were re-evaluated every 2 weeks. All five cats and their owners improved after the first application and reached clinical remission after the third application. Deep skin scrapings were negative after the first application in all cats. No clinical adverse effects or abnormalities on routine blood tests were noted during the period of the study. No re-infestation was reported over a follow-up period of 6 months after treatment discontinuation. In this study, moxidectin/ imidacloprid was effective against the feline cases of S. scabiei infestation that were resistant to ivermectin and selamectin. Funding: Self funded. Conflict of Interest: None declared.

P-107 Efficacy of a spot-on formulation containing moxidectin 1% and imidacloprid 10% for the treatment of fur mite infestations in cats C. P. SOUZA*, S. S. RIBEIRO*, T. A. NUNES* and F. B. SCOTT* *Universidade Federal Rural do Rio de Janeiro, Serope´dica, Brazil; Universidade Estaćio de Sa´, Rio de Janeiro, Brazil This study evaluated the efficacy of a spot-on formulation containing moxidectin 1% and imidacloprid 10% (Advocate; Bayer Animal Health) for the treatment of naturally acquired Lynxacarus radovskyi infestation. A total of 28 infested cats were included. Thirteen cats were assigned to the control group and the remaining 15 received a topical moxidectin and imidacloprid preparation, at a dose providing a minimum of 1 and 10 mg/kg, respectively. The preparation was applied to the skin in a single site. The presence of live mites was assessed before treatment by physical examination and plucking hairs from six body areas (right and left hind limbs, right and left front limbs, cranial abdominal area, and sacral area). Collected hairs were examined under a stereomicroscope, and infestations were noted in all cats [mild (n = 8) and heavy (n = 7)]. Examinations of the same body regions were repeated seven and 14 days after treatment. At each assessment only dead mites were observed on the hair of previously heavily infested cats. No parasites were found on cats with mild infestation. The hair coat of all cats had the characteristic salt-and-pepper appearance before and after treatment. Only one animal presented hair loss and mild pruritus, but it was also under treatment for sporothricosis. In this study, moxidectin 1% and 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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imidacloprid 10% solution was safe and 100% effective against naturally acquired fur mite infestation in cats. Funding: Self funded. Conflict of Interest: None declared.

P-108 Insecticidal effect of fipronil and its spot-on formulation on larvae of the cat flea Ctenocephalides felis T. FUKASE Hayashiya Institute of Life Sciences, Uji-shi, Kyoto, Japan A phenylpyrazole class compound, fipronil, has been effectively used as an adulticide against fleas on dogs and domestic cats, and is expected to be also sufficiently effective against larvae. The present study evaluated the insecticidal effect of fipronil and its spot-on formulation (My Free Guard for Dogs, My Free Guard for Cats: Fujita Pharmaceuticals Co., Ltd.; Tokyo, Japan) on larvae of the cat flea Ctenocephalides felis. Three isolates of the C. felis larvae were collected from the surroundings of dogs and cats, and each was brought into contact with 5–500 lg of fipronil in a petri dish. The isolates were also brought into contact with the hair samples obtained from the surroundings of dogs and cats on day 7 after administration of the spot-on formulation of fipronil in a dose of 6.7 mg/kg for dogs and 10 mg/kg for cats. Flea larvae in contact with fipronil were all knocked down and dead within 24 h of exposure to the chemical at all tested amounts. Time until knock-down was shorter in petri dishes containing a higher amount of fipronil. Larvae were knocked down and dead when in contact with some hair samples, but survived when in contact with other samples showing dose-dependent efficacy. These results indicate that fipronil is fully insecticidal against cat flea larvae. The fact that the hair samples collected from the surroundings of treated animals had a killing effect on the larvae shows that the spot-on formulation is effective for prevention of flea infestation. Funding: Hayashiya Institute of Life Sciences. Conflict of Interest: None declared.


P-109 Efficacy of a spot-on solution containing imidacloprid 10%/moxidectin 2.5% in treating dogs with dermatitis associated with natural microfilariaemia by Dirofilaria repens D. TRAVERSA*, A. DI CESARE*, M. DI TOMMASO, F. ROCCONI, G. ASTE, B. PAOLETTI*, E. DI GIULIO, F. PAMPURINI§ and A. BOARI *Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy; Department of Clinical Veterinary Sciences, University of Teramo, Teramo, Italy; Ambulatorio Veterinario San Francesco, Castelnuovo Vomano, Teramo, Italy; §Bayer Animal Health, Milano, Italy New studies on therapy of Dirofilaria repens infection in dogs are warranted given the current increase of clinical cases in different areas of the world. This study evaluated the efficacy of single application of a spot-on formulation containing imidacloprid 10%/moxidectin 2.5% (Advocate; Bayer Animal Health) in nine dogs naturally infected with D. repens. The microfilariaemia level was 110–560 microfilariae/mL. Dogs were kept in a shelter and diagnosed with D. repens infection in September 2009. All dogs presented with non pruritic skin lesions consisting of subcutaneous nodules, alopecia, erythema, and lichenification. Animals were treated in October 2009 with a single administration of Advocate and evaluated monthly for microfilariaemia and the presence of skin lesions until the next mosquito season. Eight dogs were maintained negative for D. repens during the entire study period by both, Knott’s method and PCR, while one animal became again microfilariaemic with low microfilariaemia levels 2 months post-treatment. In seven dogs the skin lesions disappeared within 3 months after treatment. Interestingly, the other two dogs with persistent lesions also had leishmaniosis. This study showed that a single administration of Advocate to dogs infected by D. repens was effective in eliminating larval D. repens for up to 6 months and the associated dermatitis in most cases. Further studies are necessary to investigate the primary pathogenic role of this nematode in causing canine allergic dermatitis and to evaluate the number of treatments with Advocate required to achieve 100% efficacy in suppressing D. repens microfilariaemia and in treating skin lesions. Funding: Bayer Animal Health, Milano, Italy. Conflict of Interest: Pampurini is an employee of Funding Bayer Animal Health, Milano, Italy.

Topic 14: SKIN BIOLOGY AND GENETIC DISEASES P-110 Feline lanceolate hair phenotype: clinical, histological, and ultrastructural features A. ROSTAHER*, C. S. POTTER, J. CHEN, S. BETTENAY§, L. SPECHT* and R. S. MUELLER* *Centre for Clinical Veterinary Medicine, Ludwig Maximilian University Munich, Munich, Germany; The Jackson Laboratory, Bar Harbor, ME, USA; Department of Dermatology and Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado at Denver Aurora, CO, USA; § Tierdermatologie Deisenhofen, Deisenhofen, Germany This comparative study shows a detailed structural investigation of a new feline hairless phenotype, which closely resembles the lanceolate [Desmoglein 4 (Dsg4) gene] mutation of mice, rats, and humans. It is also similar to mice with a mutation in the keratin 75 gene. This phenotype occurred in a litter of domestic short hair cats. In order to evaluate the cellular and molecular structure and integrity of the hair shafts and follicles, we performed ultrastructural analysis, immunohistochemistry, and immunofluorescence (keratins 14, 71, and 75) on samples from diseased and control cats and mice. Light microscopy of the hairs revealed loss of the normal cuticle pattern and bulbous defects resembling a spear point or lance head. The most striking histologic findings in the skin were prominent hair matrix defects featuring a swelling above the melanogenic zone. This was associated with individual or small clusters of cells in the precortex and premedulla that were enlarged, rounded, and had abundant pale cytoplasm suggesting degenerative changes. Scanning electron microscopy revealed oval defects with a thin pointed protrusion or blunt end where the hair broke. Xray-based element analysis showed a significant decrease in sulphur content just below the defects. Transmission electron microscopy of extracted cat hairs revealed cells in the medulla which were filled with amorphous and globular deposits, and a loss of the septulated architecture. The Dsg4 gene is suspected to be the candidate gene responsible for this novel clinical disease in cats. Funding: Supported in part by mentorship and research grants from the North American Hair Research Society and the American College of Veterinary Dermatology. Conflict of Interest: None declared.

P-111 Immunohistochemical staining of lymph vessels in canine skin N. SLEECKX*, L. VAN BRANTEGEM, G. VAN DEN EYNDEN, S. MAES§, E. FRANSEN– and C. VAN GINNEKEN* *Laboratory of Applied Morphology, Department of Veterinary Sciences, University of Antwerp, Wilrijk, Antwerp, Belgium; Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; Department of Pathology and Cytology, GZA Hospitals, Wilrijk, Antwerp, Belgium; §Valuepath, Hoensbroek, The Netherlands; –StatUa Center for Statistics, Edegem, Antwerp, Belgium

Skin and subcutaneous tumours are the most common tumours in dogs. These tumours consist of tumour cells and tumour-associated stroma. An important component of the stroma is the tumour vasculature (blood and lymph vessels). Angiogenesis and lymphangiogenesis (i.e. the formation of new blood and lymph vessels from existing ones), is induced in tumours. These newly founded vessels facilitate tumour growth and metastasis. In contrast to angiogenesis, to date no information on lymphangiogenesis in canine skin tumours is available. The main reason for this is the absence of reliable immunohistochemical markers for visualizing canine lymph vessels. In human medicine, different markers for lymphatic endothelium are available: Prox-1, Lyve-1, podoplanin, and D2-40. In this study, these primary antibodies were screened for staining lymph vessels in normal canine skin. Since immunohistochemistry with antibodies against podoplanin and D2-40 showed no immunoreactivity, Prox-1 and Lyve-1, the markers with positive immunoreactivity, were used in further research. For five skin samples, haematoxylin-eosin (H&E), antiProx-1, and anti-Lyve-1 immunohistochemical stainings were performed on serial sections. On the H&E slide, three hotspots (highest vascular density) were identified. In these hotspots a comparative analysis of the stainings was done, and the differences in performance were statistically tested. Prox-1 staining performed significantly better. In conclusion, human lymph vessel markers are able to stain lymph vessels in canine skin, with Prox-1 being the marker of choice. This may facilitate the evaluation of lymphangiogenesis and lymph vessel infiltration in canine skin tumours and provide important information for staging and prognosis. Funding: University of Antwerp. Conflict of Interest: None declared.

P-112 Cutaneous pH measurements from various anatomic locations of 61 healthy German shepherd dogs S. ZABEL and P. HENSEL Department of Small Animal Medicine and Surgery, University of Georgia College of Veterinary Medicine, Athens, GA, USA Cutaneous pH plays a role in barrier function and keratinization and may play a role in protection against invasion by microorganisms. Cutaneous pH varies among anatomic sites and there may be an influence of breed. Cutaneous pH measurements have not been reported for German shepherd dogs. This study compared the cutaneous pH of different anatomic locations in 61 healthy German shepherd dogs. Two cutaneous pH measurements were performed on the pinna, axilla, lateral thorax, inner thigh, and interdigital space of each animal. Measured pH differences between dogs were compared to differences in repeated measurements in the same dogs by analysis of variance. Cutaneous pH measurements were averaged for each dog and tested for correlation with age, temperature, and humidity by Pearson’s correlation. Differences in pH between anatomical locations were tested by a repeated measures 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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analysis of variance. Reference ranges were calculated from the 2.5 and 97.5th percentiles. All hypothesis tests were two-sided, and the level of significance was a = 0.05. Sixty-one intact (34 males, 27 females) dogs were evaluated. We found the following: significant differences in pH between dogs for all anatomic sites except the thorax; significant differences in pH between each anatomical location, significant negative correlation between humidity and pH of the inner thigh, and significant positive correlation between environmental temperature and pH of the interdigital space. Reference ranges established for pH for each location were: axilla, 5.88–8.51; inner thigh, 5.84–8.41; interdigital space, 5.49– 8.36; pinna, 5.53–8.33; and thorax, 6.12–8.39. Funding: Self funded. Conflict of Interest: None declared.

P-113 Influence of hair coat in measuring transepidermal water loss in cats Y. MOMOTA, K. SHIMADA, A. TAKAMI, H. NONAKA-AKAOGI and T. SAKO Nippon Veterinary and Life Science University, Musashino, Tokyo, Japan The purpose of this study was to establish a standardized clipping method for measuring transepidermal water loss (TEWL) with a VapoMeter in domestic short hair cats and to investigate TEWL values in different anatomic sites. TEWL values were obtained from three skin sites on the inguinal region of six cats, including an unclipped site, one trimmed with scissors, and one clipped with an electric hair clipper. TEWL values were measured on the three sites at 5 min, 1, 3, 6, 12, 24, and 48 h after clipping. No significant difference was noted among the time points; therefore, the 5 min time point was used for measuring the TEWL in the study. The TEWL values for the unclipped, scissor-trimmed, and clipper-trimmed sites were 16.78 ± 3.19, 10.55 ± 2.17 and 8.54 ± 1.20 g/m2/ h, respectively. The clipper-trimmed method showed the lowest TEWL value and least variation (P < 0.05). Five anatomical body sites (upper back, lower back, lateral upper thigh, axilla, and groin) were investigated after being trimmed with a clipper. The TEWL values were 6.51 g/m2/h for the upper back, 5.26 g/m2/h for the lower back, 5.50 g/m2/h for the lateral upper thigh, 8.07 g/m2/h for the axilla, and 6.07 g/m2/h for the groin. The value for the upper back was significantly higher than the other sites (P < 0.05). The clipping method should be standardized in order to minimize the experimental variation in TEWL measurement in cats. Moreover, as we found that TEWL values varied in different anatomical sites, anatomical location should be considered in comparative investigations of feline TEWL. Funding: Self funded. Conflict of Interest: None declared.


P-114 Comparison of three different sampling methods for canine skin lipids M. ANGELBECK-SCHULZE*, J. STAHL, K. ROHN, M. KIETZMANN, R. MISCHKE* and W. BAÜMER *Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany; Institute of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany; Institute for Biometry and Epidemiology, University of Veterinary Medicine Hannover, Hannover, Germany Epidermal lipids are of major interest in dermatological research, especially in canine allergic skin diseases. Several sampling methods are described in the literature and differ in their invasiveness. This study compared three different sampling methods in order to identify a preferable, minimally-invasive method for collecting canine skin lipids. Samples taken from the croup and inguinal region consisted of three skin scrapings, three skin scrubs, and heat-separated epidermis of three punch biopsies. The latter procedure was adapted from a method of collecting skin microflora modified by using an organic solution (n -hexane/ethanol 2:1). After lipid extraction with chloroform/methanol, lipids were analysed by high-performance thin-layer chromatography. Identification of ceramides and other lipid fractions was verified by corresponding standards, retention time, and mass spectroscopy. To compare the reproducibility of the methods, we assessed the variance components of between-patient and within-patient for each method at each location (basic values referring to lg/cm2). The reproducibility of the scrapings and skin scrubs was comparably high, whereas the variance components of the heat-separated epidermis differed to a certain extent. Lipid and ceramide profile (lipid/ceramide percentages) of scraping and skin scrub were comparable. The locations sampled (croup and inguinal region) seemed to have only marginal influence on skin lipid profile. In conclusion, scraping and skin scrub are comparably suitable methods for skin lipid sampling. By contrast, analysing heat-separated epidermis may not be the method of first choice. Funding: Gesellschaft zur Fo¨rderung Kynologischer Forschung e.V. Conflict of Interest: None declared.

P-115 Expression of different receptors possibly involved in pruritus in canine dorsal root ganglia K. ROSSBACH and W. BAÜMER Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany Several sensory receptors and their ligands have been implicated in the induction of itch in humans and laboratory animals (mice, rats), but little is known about receptors that are involved in the induction of itch in dogs. This study analysed the expression of receptors for various pruritogenic mediators in canine dorsal root ganglia (DRG). Three adult dogs (two beagle dogs, one French bulldog) were euthanized for reasons not related

Abstracts to this study. Multiple dorsal root ganglia per dog were dissected, and, after homogenization of the dorsal root ganglia tissues, total RNA was isolated, reverse-transcribed to cDNA, and amplified with custom-synthesized primers. Apart from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a strong expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) as well as interleukin 31 receptor A and oncostatin M receptor (the IL-31 receptor complex) and histamine H3 receptor (H3R) were detected in the canine DRG. Moreover, toll-like receptor 7 (TLR7) and endothelin receptor type A mRNA were detected in the DRG. Low expression of H2 receptor mRNA was found in the DRG of the beagle dogs. Interestingly, H1 receptor mRNA was low (French bulldog) or undetectable (beagle dogs), and H4 receptor mRNA could not be detected under our conditions. Given that DRG neurons contain cell bodies of sensory afferents, these findings led to the hypothesis that these receptors are involved in neural transmission of itch in dogs, as has been demonstrated for other species. However, functional studies are required to test this hypothesis. Funding: Self funded. Conflict of Interest: None declared.

P-116 C-reactive protein concentration in dogs with skin diseases Y. TERADA, N. MURAYAMA, M. OKUAKI and M. NAGATA ASC Dermatology Service, Tokyo, Japan Serum C-reactive protein is used as a marker of inflammation and tissue damage in humans and dogs, but its clinical investigation in veterinary dermatology is extremely limited. This study assessed the relationship between C-reactive protein and inflammatory skin diseases in dogs. C-reactive protein was measured in 222 dogs with a progressive skin disease using latex coagulating nephelometry (Mitsubishi Chemical Medience Animal Laboratory, Tokyo, Japan). The reference range of C-reactive protein (1.06–0.10 mg/dL) was determined in 70 clinically healthy beagles and used for comparison. We found high C-reactive protein levels in all cases of juvenile cellulitis (median = 9.3 mg/dL, range = 6.8– 11.6 mg/dL, n = 3), pemphigus foliaceous (7.6 mg/dL, 5.2–15.9 mg/dL, n = 3), and epitheliotropic lymphoma (3.1 mg/dL, 1.1–9.8 mg/dL, n = 5), and in 86% of panniculitis (6.3 mg/dL, 0.4–16.2 mg/dL, n = 7), 83% of deep pyoderma (3.4 mg/dL, 0.4–6.3 mg/dL, n = 5), 67% of demodicosis (1.7 mg/dL, 0.2–14.9 mg/dL, n = 9), 59% of scabies (1.2 mg/dL, 0.2–8.1 mg/dL, n = 17), 40% of ischemic disorders (1.0 mg/dL, 0.2– 6.7 mg/dL, n = 5), 33% of Malassezia dermatitis (0.4 mg/dL, 0.2–4.2 mg/dL, n = 18), 26% of superficial pyoderma (0.4 mg/dL, 0.2–4.1 mg/dL, n = 31), and 25% of non specific dermatitis (0.2 mg/dL, 0.2–1.1 mg/ dL, n = 4). It was confirmed that C-reactive protein could return to the normal range after treatment in juvenile cellulitis, pemphigus foliaceus, panniculitis, deep pyoderma, demodicosis, and scabies. C-reactive protein was not elevated in trauma, otitis, intertrigo, atopic dermatitis, and sebaceous adenitis. It is suggested that C-reactive protein is a valuable marker for juvenile cellulitis, pemphigus foliaceus, panniculitis, and deep

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pyoderma. However, further study is warranted before any conclusion can be made. Funding: Self funded. Conflict of Interest: No conflict of interest

P-117 Genetic analysis of acral mutilation syndrome in purebred dogs J. PLASSAIS*,, M. PARADIS, B. HEDAN*,, A. POMMIER*,, E. GUAGUERE§, L. LAGOUTTE*, , C. HITTE*, and C. ANDRE*, *CNRS, UMR 6061, Institut de Geńe´tique et De´veloppement de Rennes, Rennes, France; Universite´ Rennes 1, UEB, IFR140, Faculte´ de Me´decine, Rennes, France; Department of Clinical Sciences, Faculte´ de Me´decine Ve´te´rinaire, University of Montreal, St Hyacinthe, QC, Canada; §Clinique Ve´te´rinaire Saint Bernard, Lomme, France Acral mutilation syndrome is a rare hereditary sensory neuropathy in dogs reported in the literature for 50 years. As already reported, clinical signs consist of acral analgesia, with or without sudden intense licking, biting, and severe self-mutilation of the feet, while proprioception, motor abilities, and spinal reflexes remain intact. In cases for which a spinal ganglia has been analysed, a reduction of the neuron number was observed in some instances. The average age of onset is 4 months, and dogs severely affected are generally euthanized. Despite its rare occurrence among all dog population, several breeds clearly appear to be predisposed. This ongoing work consists in a thorough clinical, histological, and genetic analysis of affected dogs presenting with acral mutilation and/or nociceptive loss. We collected blood samples from several affected breeds: French spaniels, German short-haired pointers, English springer spaniels, English pointers, and miniature pinschers, including 35 affected dogs. The genetic study performed in the French spaniel breed allowed us to construct and analyse pedigrees and confirm a recessive transmission mode. We then performed a genetic analysis with 20 cases and 26 control samples. A genome-wide association study using the recently available Illumina Canine HD single nucleotide polymorphism (SNP) array, consisting of 170 000 SNPs, was performed and identified a locus carrying the responsible gene. This work, in addition to allowing the development of genetic tests for veterinary medicine in the predisposed breeds, has the potential to provide new genes for the human homologous syndromes. Funding: CNRS; the European Commission Companion Animal Health Fund; Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montreál. Conflict of Interest: None declared.


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P-118 Palmar keratoderma in Dogue de Bordeaux dogs: histopathological and genetic analyses J. PLASSAIS*,, E. GUAGUERE, C. DUFAURE DE CITRES§, P. QUIGNON*,, F. DEGORCERUBIALES–, L. LAGOUTTE*,, C. HITTE*,, E. BENSIGNOR**, C. KAERLE§, M. DELVERDIER, A. THOMAS§ and C. ANDRE*, *CNRS, UMR 6061, Institut de Geńe´tique et De´veloppement de Rennes, Rennes, France; Universite´ Rennes 1, UEB, IFR140, Faculte´ de Me´decine, Rennes, France; Clinique Ve´te´rinaire Saint Bernard, Lomme, France; §Antagene, Animal Genetics Laboratory, Limonest, France; –Laboratoire d’Anatomie Pathologique Ve´te´rinaire du Sud-Ouest LAPVSO, Toulouse, France; **Clinique Ve´te´rinaire de la Boulais, Cesson-Se´vigne´, France; Service d’Anatomie Pathologique, Ecole Ve´te´rinaire de Toulouse, Toulouse, France Palmar keratoderma is a canine genodermatosis that segregates mainly in two breeds, Dogue de Bordeaux and Irish terrier. This disease affects almost 1% of the Dogue de Bordeaux breed, and the onset usually occurs in puppies of 4 months of age or less. Clinical signs consist of dermatological lesions affecting footpads and/or nose showing thickening with severe keratinous proliferations and fissures. Histopathological analyses reveal a papil-


lated epidermal hyperplasia, diffuse orthokeratosis, and hypergranulosis, with focal parakeratosis and superficial swollen keratinocytes in the stratum corneum. Some affected dogs walk with a limp, sometimes with pain that can lead to euthanasia. This ongoing work consists of a thorough clinical, histopathological, and genetic analyses of affected dogs presenting footpad hyperkeratosis. We collected blood samples from 100 dogs of the Dogue de Bordeaux breed, including 30 affected dogs. The construction and analysis of a pedigree led us to hypothesize a recessive transmission mode. To identify the locus responsible for this disease, we performed a genome-wide association study on 30 affected dogs and 70 controls using the recently available Illumina Canine HD single nucleotide polymorphism (SNP) array consisting of 170 000 SNPs. We found that most of the genes known to be involved in human palmoplantar keratoderma are not responsible for palmar keratoderma in dogs of the Dogue de Bordeaux breed. This work, in addition to allowing the development of genetic tests for veterinary medicine in this predisposed breed, has the potential to provide new genes for the human homologous palmoplantar keratoderma disease. Funding: CNRS; the European Commission; Socie´te´ Centrale Canine (the French Kennel Club). Conflict of Interest: None declared.

Topic 15: SKIN BIOPSY AND WOUND HEALING P-119 Observation of potential lidocaine toxicity during skin biopsy in dogs N. LEMO, D. VNUK and F. BANOVIC Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Punch biopsy is a widely used diagnostic method in veterinary dermatology. During a biopsy, it is necessary to use local anaesthesia or sedation. This study assessed the potential toxic effects of local anaesthesia during and after a biopsy and measured lidocaine serum concentrations post-biopsy and their association with established toxicity values. Nine dogs were randomly divided into two groups: group L (lidocaine) and group LA (lidocaine + adrenaline). Animals in group L were injected with 6 mL total (1 mL administered subcutaneously for each of the six biopsy sites) of a 2% lidocaine solution, regardless of body weight. Animals in group LA were injected with the same volume of a 2% lidocaine solution plus a 1:20 000 adrenaline solution. Six skin biopsies were performed on each dog. Blood samples were collected 0, 10, 30, 60, 90, and 120 min after lidocaine administration. The highest mean value of serum lidocaine in group L was observed 30 min after administration (0.48 lg/mL) and the lowest 10 min after administration (0.27 lg/mL). After 120 min, the lidocaine mean serum level was 0.35 lg/m. In group LA, the lidocaine serum concentration increased linearly, reaching peak concentration 60 min after administration (0.72 lg/mL) and then decreased slightly, reaching a mean value of 0.53 lg/m 120 min after administration. According to the results of a general regression model, none of the predicted variables (age, gender, weight, and type of treatment) had any significant influence (P < 0.05) on lidocaine blood concentration. Finally, none of the serum concentrations reached known toxic values. Funding: Self funded. Conflict of Interest: None declared.

P-120 The wound healing potential in rats of hydroalcoholic-extract-based ointment from Phoenix dactylifera S. VARZANDIAN*, A. A. ABEDIAN*, N. HAJIPOUR and A. ALIABADI* *Department of Clinical Science, School of Veterinary Medicine, Islamic Azad University, Kazeroun Branch, Kazeroun, Fars, Iran; Department of Histopathology, School of Veterinary Medicine, Islamic Azad University, Kazeroun Branch, Kazeroun, Fars, Iran The present study reports, for the first time, the woundhealing potential of the date (Phoenix dactylifera). Phoenix dactylifera has long played an important role in the economy and social life of the people of arid and semi-arid regions of the world. Dates may have extractable high-value-added components. It has been shown that dates present a high antioxidant capacity due to their richness in polyphenols and tocopherol compounds. We formulated 5%, 20%, and 50% methanolic-extract-based

ointments and evaluated them for their wound-healing ability in 80 Wistar rats. Wounds were induced in rats divided into four groups as follows: group 1 was composed of rats treated with a simple ointment base (control); groups 2–4 were composed of rats treated with a 5%, 20%, and 50% methanolic-extract-based ointment, respectively. The ointment was applied on excised wounds once per day for 10 consecutive days. Skin samples were taken on days 3, 7, 14, and 21 after wound induction following euthanasia. A statistically significant difference was observed between the control and treated groups with respect to reduction in the wound surface area grossly, and in histomorphometric and histhopathologic evaluations after 3 weeks of treatment. The higher concentrations of ointment significantly enhanced the wound contraction and reduced the period of epithelialization as assessed by mechanical (contraction rate, tensile strength), biochemical (increase of collagen, DNA, and protein synthesis), and histopathological characteristics. Phoenix dactylifera accelerated wound healing in rats and thus has the potential for use as an effective herbal remedy, which supports its traditional use. Funding: Self funded. Conflict of Interest: None declared.

P-121 Study on histomorphologic and macroscopic effect of Commiphora opobalsomum on burn wound healing in rats A. DEZHIMAND and S. VARZANDIAN Department of Clinical Science, School of Veterinary Medicine, Islamic Azad University, Kazeroun Branch, Kazeroun, Fars, Iran Modern medicine is rooted in ethnobotanical traditions using indigenous flora to treat symptoms of human diseases or to improve specific aspects of the body condition. Today, researchers are searching for ways to promote faster wound healing. This study investigated histopathologically the effect of Commiphora opobalsomum on the healing of burn wounds in rats. Forty male Wistar rats were randomly assigned to a control and experimental groups of 20 animals each. All animals received ketamin and diazepam anesthesia and had their backs shaved. Grade III burn wounds were induced on the shaved area of all animals. The rats in the experimental group were treated with extract of Commiphora opobalsomum prepared as an ointment, twice per day. Animals in the control group were treated solely with the ointment without Commiphora opobalsomum. The rats were euthanized on days 3, 7, 14, and 21, and photographs and skin samples were taken on these days. We performed histopathological studies and compared the groups for the following wound healing parameters: proliferation of fibroblasts, angiogenesis, re-epithelialization, and collagen organization in healing tissue. Significant differences among the groups were determined by one-way analysis of variance followed by Bonferroni post-test. Statistical significance was considered at P < 0.05. The area of the wounds was significantly smaller (P < 0.05) in the experimental group compared 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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to the control group on all days, showing that Commiphora opobalsomum had a strongly positive effect on the healing of burn wounds. Funding: School of Veterinary Medicine, Islamic Azad University, Kazeroun Branch. Conflict of Interest: None declared.

P-122 Using platelet-rich plasma on wound healing in rabbits M. BAZAIE*, Z. HOSSEINY, B. SABOOKI*, L. HOMAFAR, E. FEREIDOONPOOR and N. VARAA *Department of Clinical Studies, Ariyavet Pet Clinic, Kish Island, Iran; Department of Clinical Studies, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Chronic wounds are complex medical problems and are often difficult to heal because they lack necessary growth factors to maintain the healing process and are frequently complicated by superinfection. Platelet-rich plasma is a fraction of the plasma of autologous blood that has a


platelet concentration above baseline. Platelet-rich plasma contains and releases at least seven growth factors and cytokines that stimulate bone and soft tissue healing. Platelet-rich plasma is used for various purposes in surgical procedures, and is a new treatment modality in tissue regeneration and a developing area for clinicians and researchers. Two 1 · 1 cm full-thickness sterile skin wounds were created on the backs of eight New Zealand white rabbits. One wound of each animal received 0.5 mL platelet-rich plasma. The animals were euthanazed after 10 days, and appropriate skin samples were collected and fixed in 10% buffered formalin for histopathological examination. We observed the following in both groups: complete skin re-epitheliazation, thicker epidermis in comparison to the surrounding tissue, regular collagen fibers and large blood vessels in the dermis. In both groups healing was well underway at 10 days; however, in the group treated with PRP tissue regulation was more evident and we observed more large blood vessels. The PRP also seemed to have the most positive effect on healing rate, tissue fill, and volume fraction of fibroblasts. Funding: Ariya Vet Pet Clinic. Conflict of Interest: None declared.

Abstracts from the International Society of Veterinary Dermatopathology (ISVD), Wednesday, 25th July ISVD – 1 Reactive histiocytosis in a 10-year old Australian shepherd dog J. PETERS-KENNEDY Department of Biomedical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, NY, USA A 10 year-old, spayed female Australian shepherd dog presented with diarrhoea, weight loss, and bilateral chemosis. The dog responded to a course of oral doxycycline 10 mg/kg/day and neomycin/polymyxin/ dexamethasone ophthalmic drops twice daily. Two months later the dog presented with similar signs but the same treatment yielded minimal improvement. One month later the dog developed multiple variably sized alopecic, crusted, pink, irregular, raised lesions on the trunk. Histopathology revealed marked multifocal coagulative necrosis extending from epidermis into dermis and panniculus. Spared areas were infiltrated by CD18+ histiocytes, fewer CD3+ lymphocytes, and smaller numbers of neutrophils that were most intense around and infiltrating into medium sized blood vessels in the mid to deep dermis and panniculus. Multifocally there were several discrete granulomas and pyogranulomas scattered through the deep dermis. Special stains were negative for microbial organisms. Although multiple differential diagnoses were considered, including pyogranulomatous panniculitis, drug induced vasculitis, and lymphomatoid granulomatosis, the tentative diagnosis was reactive histiocytosis. Systemic histiocytosis was considered because of conjunctival lesions, diarrhoea, and weight loss. The dog received 5 mg triamcinolone acetonide (Vetalog) subcutaneously at the time of biopsy, and skin lesions improved clinically 80%. Three months after biopsy, the dog represented with similar but worse skin lesions, continued weight loss, and conjunctivitis. Treatment with 40 mg (approximately 2 mg/kg/ day) oral prednisone and 500 mg each of tetracycline and niacinamide three times daily was initiated. Six weeks later there was significant improvement in the skin and ocular lesions. The dog died four and one half months later. Funding: Self funded. Conflict of interest: None declared.

ISVD-2 Equine generalised granulomatous disease (cutaneous sarcoidosis): a case report with long term follow-up J. D. LITTLEWOOD Veterinary Dermatology Referrals, Cambridge, UK A 9-year old Irish hunter gelding was presented with a three-week history of weight loss, sweating, intermittent pyrexia, superficial lymphadenopathy, respiratory signs, and development of patchy alopecia and scaling. Examination revealed poor body condition, nasal discharge, superficial lymphadenopathy, and generalised hypotrichosis to alopecia, crusting, scaling, and multifocal erosions and ulcerations. Initial tests revealed: no

parasites or fungi on microscopy of hair and skin scale; neutrophilia, hypoalbuminaemia and hypergammaglobulinaemia; a ‘reactive lymph node’ on fine needle aspirate cytology; mainly neutrophils with bacteria in mucus on tracheal wash and a negative fungal culture. Histopathology revealed granulomatous dermatitis, with occasional intraepidermal pustules containing a few cocci. Special stains were negative for mycobacteria and fungi. Giant cells were not seen in rectal biopsy samples. A diagnosis of generalised granulomatous disease (sarcoidosis) was made and treatment initiated with prednisolone 1.25 mg/kg orally once daily. Clinical signs resolved within six weeks and dosing was reduced to alternate day therapy with gradual tapering and discontinued use after six months. The horse returned to full work, and was disease free for one year, but coincident with returning to training, clinical signs of lethargy and skin lesions recurred. Granulomatous cutaneous inflammation was reconfirmed histopathologically. Glucocorticoid treatment again secured remission, but further relapses were observed when in full work. The condition was kept in remission by administration of 2 mg/kg oral prednisolone on alternate days during the hunting season and discontinued in the summer. The horse was retired four years after diagnosis, turned out, ridden occasionally, and was off therapy. Funding: Self funded. Conflict of interest: None declared.

ISVD – 3 Long-term management and apparent seasonal recurrence of generalised granulomatous dermatitis (cutaneous sarcoidosis) in a horse S. L. MYERS Prairie Diagnostic Services, Saskatoon, SK, Canada A 10 year-old Arabian gelding presented with a onemonth history of weight loss followed by non pruritic, bilaterally symmetrical, patchy, scaly skin, which initially affected nostrils, dorsal neck and ventral tail and spread to involve most of the face, trunk, and abdomen. Tufted hairs were easily epilated with dry white scale at the base. Complete blood cell count and serum biochemistry panel were normal. Results of skin biopsy evaluation included granulomatous dermatitis with multinucleated giant cells. Histochemical and immunohistochemical staining for fungi and mycobacteria were negative. A diagnosis of granulomatous dermatitis (cutaneous sarcoidosis) was made. Although the skin did not improve with short-term systemic antibiotic and dexamethasone treatment, appetite, energy level, and body condition did improve. Skin lesions subsequently resolved with oral prednisone at 2 mg/kg/day. Prednisone was gradually tapered and discontinued three months later. The horse returned to full function and condition. Disease recurred in March 2002 (12 months later) and responded to oral prednisone treatment at 2 mg/kg every other day for 12 weeks. For the next four years, prednisone treatment was subsequently initiated as a preventative measure each spring at 2 mg/kg every other day for approximately six weeks 2012 The Authors. Veterinary Dermatology 2012 ESVD and ACVD, Veterinary Dermatology, 23 (Suppl. 1), 2–104

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between February and April. Disease recurred in July 2003 and responded to oral prednisone treatment at 2 mg/kg every other day for 10 weeks. The horse was retired from dressage competition in 2003 and the skin disease has not recurred since. Mild scarring remains


below the tail and around both eyes and nostrils, and scale persists on the mane. Funding: Self funded. Conflict of interest: None declared.