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Comparative expression of antimicrobial peptides in lesional and nonlesional skin of dogs with atopic dermatitis

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S. TORRES*, C. LANCTO†, S. KOCH* and M. RUTHERFORD†

* Departments of Veterinary Clinical Sciences andVeterinary and Biomedical Sciences, University of Minnesota, St Paul, MN, USA

Antimicrobial peptides (AMPs) are small cationic peptides expressed in various tissues, including the skin, and function as important components of the innate immune defense against bacteria, viruses, fungi and protozoa. The aim of the study was to compare mRNA expression of AMPs in the skin of atopic dogs, normal dogs and dogs with other inflammatory skin disorders (INFs), including sebaceous adenitis, panniculitis, acral lick dermatitis, pemphigus foliaceus, bullous disease, vasculopthy and nonspecific cutaneous inflammation. Quantitative RT-PCR was used to measure the mRNA expression of seven AMP genes [canine β-defensin-1 (CBD1), CBD102, CBD103, CBD122, CBD124, skin-derived antileukoproteinase (SKALP) and secretory leukoprotease inhibitor (SLPI)] in lesional and nonlesional skin of dogs with confirmed atopic dermatitis (AD). The AMP transcription levels in these dogs were compared with levels in dogs with other INFs and with normal skin (N). Kruskal–Wallis test was used to evaluate AMP expression among the groups, with P ≤ 0.05 considered significant. The expression of SLPI in dogs with AD was found to be significantly higher than in normal dogs (< 0.01). The SLPI expression in lesional skin of dogs with INF was found to be significantly higher than some normal body sites (< 0.05). Moreover, no significant difference was found in the expression of SLPI between nonlesional skin of dogs with INF and normal skin. No significant differences were found in the expression of all other tested AMPs among the groups. These findings suggest that AMP expression does not account for the high prevalence of bacterial skin infection observed in dogs with AD.

Source of funding: Morris Animal Foundation.

Conflict of interest: No conflicts of interest have been declared.

Evaluation of a polyclonal antibody against canine filaggrin in immunohistochemical studies in atopic and normal beagle dogs

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R. MARSELLA*, K. AHRENS*, A. L. THOMAS* and D. SANTORO†

* Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL, USADepartment of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Filaggrin has attracted considerable attention for the impact of its decreased expression on skin barrier dysfunction in human atopic dermatitis (AD). Previous studies evaluating filaggrin expression in canine samples have been hindered by technical challenges linked to the use of reagents developed for human filaggrin. Recently, a polyclonal antibody against canine filaggrin peptide sequence was generated. This antibody is completely blocked by pre-incubation with filaggrin and shows no cross-reactivity with canine epidermal proteins. Thus, the purpose of this study was to investigate filaggrin expression by immunohistochemistry in skin samples taken from normal and atopic beagles, before (day 0), at the peak (day 3) and after allergen challenge (day 10, once clinical signs had resolved) using the new antibody. Eighteen atopic beagle dogs allergic to house dust mites (HDMs) and five normal beagle dogs were challenged for 3 days consecutively with HDMs. Skin biopsies were taken from either the inguinal or the ventral thorax area from lesional areas. Antigenic sites to filaggrin were unmasked with Tris–EDTA, and primary filaggrin polyclonal antibody was detected using BioGenex HRP kit and Nova Red. Six images per section were subjectively scored by three independent viewers for intensity and continuity. Stratum granulosum was traced and the percentage of filaggrin was calculated (ImageJ). For the objective measurements, analysis of variance showed a significant effect of group (P = 0.0414, AD < control dogs), a significant effect of time (P = 0.0066, days 3 and 10 > day 0) and a marginal group × time interaction (P = 0.0606). For the subjective evaluations, there were no significant results.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Does transepidermal water loss correlate with disease severity in canine atopic dermatitis? A compilation of studies

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R. MARSELLA

Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL, USA

In human medicine, the measurement of transepidermal water loss (TEWL) is used for indirect assessment of skin barrier function and has been demonstrated to correlate with disease severity in patients with atopic dermatitis (AD). Preliminary evidence exists that skin impairments exist in dogs with AD, both in cases with naturally occurring disease and in an experimental model of AD. In veterinary medicine, concerns exist regarding the variability of TEWL measurements regardless of the type of device used (open chamber versus closed chamber). The purpose of this study was to investigate the existence of a correlation between clinical severity of disease as measured by the Canine Atopic Dermatitis Extent and Severity Index Score (CADESI) and TEWL measurements. Data collected in three different studies were analysed. The first study used an open-chamber device and involved atopic beagle dogs with experimentally induced disease (n = 24), and the two other studies involved patients with naturally occurring AD and used a closed-chamber device (n = 14 and n = 18). Pearson product–moment correlation coefficient was used for analyses. The TEWL of inguinal, axillary and antebrachial regions was available in all studies. The TEWL of additional areas was available in one study. Correlations were investigated for each study separately as follows: CADESI and TEWL of individual regions, total CADESI and the log-transformed sum of TEWL of measured regions; total CADESI and TEWL of key regions measured. No significant correlations were found in the two studies using the closed chamber. The only significant correlation was found for concave pinna using an open-chamber device.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Relationship of body weight to ciclosporin dose in canine atopic dermatitis

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L. B. COHEN, S. ZABEL, F. OLEA-POPELKA, S. RAO and R. A. W. ROSYCHUK

Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Department of Clinical Sciences, Fort Collins, CO, USA

Ciclosporin A is a commonly prescribed and effective therapy for canine atopic dermatitis. Eighty-five cases of canine atopic dermatitis treated between 2000 and 2010 were evaluated retrospectively to determine whether canine body weight was related to the ultimate effective dose of ciclosporin (Atopica; Novartis Animal Health, Greensboro, NC, USA) required over a 6 month treatment time period. Patients were placed into groups based on weight, with dogs in group A weighing less than 15 kg and dogs in group B weighing more than 15 kg. Descriptive analysis and multivariable linear regression were performed to compare groups while adjusting for time. Compared with dogs in group A, dogs in group B required a mean of 4.40 mg/kg/day ciclosporin, 0.53 mg/kg/day less (P < 0.0001) across all time points. There was a mean 0.04 mg/kg/day decrease (P < 0.0001) in ciclosporin received per kilogram increase in body weight. Concurrent steroid dose, medication score and pruritus score did not differ between the groups. This finding suggests that differential ciclosporin dosing may be warranted based on body weight.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Type 1 and type 4 selective antihistamines do not prevent canine acute experimental atopic skin lesions

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T. OLIVRY*, J. STAHL†, K. SANDER‡, L. J. PETERSEN§, J. PAPS*, H. STARK‡, M. KIETZMANN† and W. BÄUMER*†

* Department of Clinical Sciences, College of Veterinary Medicine, and Center for Comparative Medicine and Translational Research, NC State University, Raleigh, NC, USADepartment of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Hannover, GermanyInstitute of Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Frankfurt, Germany § Laboratory of Experimental Physiology and Inflammation, Department of Clinical Physiology, Viborg Hospital, Viborg, Denmark

Type 1 histamine receptor (H1R) selective antagonists are commonly used for treatment of atopic skin lesions in dogs. The recently discovered type 4 histamine receptors (H4R), expressed on leukocytes, appear to be important in modulating allergic inflammatory responses in mice and humans. Our objectives were to determine whether H1R or H4R selective antagonists would prevent epicutaneous allergen-induced skin lesions in hypersensitive dogs. In these blinded, placebo-controlled cross-over experiments, six Maltese-beagle atopic (MBA) dogs sensitized to house dust mites (HDMs) were treated topically with 1% solutions of the H4R specific antagonists JNJ7777120 or JNJ28307474 or orally with JNJ28307474 at 15 mg/kg before and immediately after allergen challenge. Pretreatment with 0.015% triamcinolone acetonide solution (Genesis; Virbac Animal Health, Fort Worth, TX, USA) served as a positive control. In a second trial, the H1R specific antagonists hydroxyzine pamoate (three times 2 mg/kg twice daily; generic; PCCA, Houston, TX, USA) and cetirizine (two times 0.5 mg/kg once daily; Zyrtec; GSK, Research Triangle Park, NC, USA) were administered orally 1 h before challenge. Skin lesions were graded 24 h after epicutaneous HDM application. Dermal microdialysis confirmed the early release of histamine after epicutaneous HDM application in three of these mite-sensitized MBA dogs. Neither H1R nor H4R selective antihistamines prevented skin lesion development 24 h after epicutaneous HDM challenge compared with placebo; in contrast, triamcinolone application prevented all dogs from developing lesions. In conclusion, the preventive administration of H1R or H4R selective antagonists had no impact on the development of acute skin lesions in this experimental canine atopic dermatitis model.

Source of funding: Janssen Animal Health, Johnson & Johnson and German Research Foundation.

Conflict of interest: No conflicts of interest have been declared.

Evaluation of the clinical and skin repair effects of dimethicone topical therapy in dogs with naturally occurring atopic dermatitis

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C. PELLICORO*, R. MARSELLA† and K. AHRENS†

* College of Veterinary Medicine, University of Bari, ItalyDepartment of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

In human atopic dermatitis (AD), skin barrier dysfunction is proposed to increase allergen penetration and allergic sensitization. Skin barrier impairments have also been described in canine AD. In humans, skin protectants have beneficial effects on both the skin barrier and the clinical signs. Minimal information exists in veterinary medicine on this form of therapy. Thus, this study aimed to investigate the effect of a skin protectant solution (dimethicone 2% as active ingredient and cyclomethicone as vehicle) on clinical signs and skin barrier function in canine AD. Eighteen privately owned dogs with AD were enrolled and randomly divided into two groups. One group received topical dimethicone and the other one received the vehicle on selected areas (pinnae, groin and axillae) daily for 4 weeks. Owners and investigators were blinded regarding the allocation to groups. The clinical efficacy was evaluated by Canine Atopic Extent and Severity Index Score (CADESI), and skin barrier was assessed by measuring transepidermal water loss (TEWL) with a closed-chamber device on days 0, 14 and 28. Eighteen dogs were enrolled, and 10 completed the study (50% dropout rate in the vehicle group and 40% in the dimethicone group). For CADESI, ANOVA showed a main effect of time (P < 0.005; day 0 > day 28), an effect of region (axillae < groin < pinnae), but no effect of group or group × time interaction. For TEWL, ANOVA showed only a main effect of region (axillae > pinnae > groin). It is concluded that dimethicone has no effects on clinical signs and TEWL in canine AD.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Retrospective study on owner assessment of long-term management of canine atopic dermatitis

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D. DELL* and C. GRIFFIN†

* Animal Dermatology Clinic, Tustin, CA, USAAnimal Dermatology Clinic, San Diego, CA, USA

Canine atopic dermatitis (CAD) is described as requiring life-long or long-term management. The objective of this study was to evaluate owner assessments of long-term clinical outcomes of CAD 5 and 10 years after presentation to a veterinary dermatologist. A 28-question survey was developed. The questions were divided into the following four sections: owner satisfaction/current therapy; medical therapy; diet; and topical therapy. The 10 year study group (GI) consisted of dogs with atopic dermatitis first examined between 1 January and 31 December 2000. The 5 year group (GII) consisted of dogs with atopic dermatitis first examined between 1 January and 31 December 2005. A total of 334 records in GI and 545 records in GII met the inclusion criteria of the study, and the clients were contacted via telephone. A total of 136 surveys were completed, 39 from GI and 97 from GII. Of these patients, 62.96% (85 of 135) were still receiving some form of medical therapy for atopic dermatitis. Current therapies included allergen-specific immunotherapy (19%), ciclosporin (16.8%), oral steroids (32.6%), trimeprazine tartrate with prednisolone tablets (6%), steroid injections (3%), antihistamines (29.5%), fatty acids (9%), antibiotics (22%), antifungal agents (17%), homeopathic products (5%) and others (40%). Twenty-four dogs were receiving no medical or dietary therapy and were considered by the owners either cured or having disease mild enough not to require medication. Seven of those 24 were from GI and 17 were from GII. Sixteen of those 24 had previously received allergen-specific immunotherapy.

Source of funding: This study was funded by Dechra Veterinary Products.

Conflict of interest: No conflicts of interest have been declared.

Frequency of urinary tract infection in dogs treated with oral ciclosporin: a retrospective study of 87 cases

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A. PETERSON*, S. TORRES* and A. RENDAHL†

* Department of Veterinary Clinical Sciences andSchool of Statistics, University of Minnesota, St Paul, MN, USA

The main goal of this study was to evaluate the frequency of urinary tract infection (UTI) in dogs receiving ciclosporin therapy. Patients were enrolled if they met the following inclusion criteria: (i) at least one routine urine culture performed by cystocentesis after at least 5 months of ciclosporin therapy; (ii) known inflammatory skin condition; (iii) no antibiotic therapy at time of urine collection; and (iv) no concurrent diseases known to predispose to urinary tract infections. Control urine cultures were performed from 59 dogs with inflammatory skin conditions (almost 80% of these allergic dogs) that had not received glucocorticoids or ciclosporin for 6 months or antibiotics for 3 months. The first urine culture from all ciclosporin-treated dogs was compared with control samples. Compared with two of 59 (3%) positive samples in control dogs, 13 of 87 (15%) were positive in treated dogs. This difference was significant (Fisher's exact test, P = 0.027). The ciclosporin-treated cases were then divided into dogs concurrently receiving glucocorticoids (CsG) and dogs only receiving ciclosporin (Cs). Four of 16 (25%) first cultures were positive in CsG-treated dogs, and nine of 71 (13%) cultures were positive in Cs-treated dogs. Differences between these groups and the control group were statistically significant (Fisher's exact test, P = 0.019); after Bonferroni correction, the pairwise difference between CsG and control almost reached significance (P = 0.0503).Bacteriuria showed a sensitivity and specificity for predicting UTI of 64.1 and 98.1%, respectively. Routine urinalysis and urine culture are recommended with long-term ciclosporin therapy.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Patch testing in the evaluation of adverse food reactions in the dog

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SIMONE SCHÄFER*, JENNIFER BEXLEY† and RALF S. MUELLER*

* Center for Clinical Veterinary Medicine, Ludwig Maximilian University, Munich, GermanyAvacta Animal Health, York, UK

The only reliable method to rule out adverse food reactions is an elimination diet with ingredients previously not fed. The aim of this study was the evaluation of patch testing and food-specific IgE and IgG in the diagnostic work-up of canine adverse food reactions. In 11 control dogs and 26 dogs with atopic dermatitis, a patch test was conducted on the lateral chest 48 h after clipping. Finn chambers® were filled with various meat sources and fish (each raw and cooked) and three carbohydrate sources. Vaseline served as negative control. Evaluation of the reactions occurred after 24, 48 and 72 h; after 48 h the antigens were removed. Dogs with atopic dermatitis underwent an elimination diet for at least 6 weeks, with subsequent sequential rechallenge. Reactions with patch testing and increased IgE and IgG concentrations were interpreted as true positive when a rechallenge with the respective protein led to a deterioration and subsequent feeding of the elimination diet again led to improvement of clinical signs.

All but one of the dogs with atopic dermatitis showed at least one positive reaction on the patch test. Two dogs in the control group also showed one positive reaction each. Sensitivity of the patch testing was 96.7%, specificity 88.9%. Negative predictibility was 99.3%. Measurement of specific IgE (IgG) revealed a specificity of 91.6% (70.4%) and a negative predictive value of 81.1% (81.5%). Patch testing and food antigen-specific antibodies may be useful in selecting the food sources for the elimination diet due to their high negative predictibility.

Source of funding: This study is self-funded.

Conflict of interest: Jennifer Bexley is employed by the laboratory running the IgE and IgG assays, but was not informed of the results of the sequential rechallenges prior to running the assays. Hermal (Reinbeck, Germany) provided the Finn Chambers.

ELISA testing for soy antigens in dry dog foods used in dietary elimination trials

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C. WILLIS-MAHN*, D. RADITIC*†, K. TATER* and R. REMILLARD*

* MSPCA Angell Animal Medical Center, Boston, MA, USAUniversity of Tennessee, Knoxville, TN, USA

Elimination diet trials are used to determine food allergies and intolerances. Contamination of the diet with a known food antigen, such as soy, nullifies the results of the trial, whereas unintentional contamination confounds the results. The objective of this study was to determine whether: (1) four over-the-counter (OTC) dry dog foods carrying a ‘made with no soy’ claim and (2) eight veterinary therapeutic dry dog foods designed for food elimination trials were suitable for a canine soybean elimination trial. A 100 g sample of each diet plus a soy positive control diet were submitted for ELISA testing to an outside independent food laboratory. The ELISA test is quantitative for soy flour protein concentrations between 2.5 and 25 p.p.m. The positive control diet contained >25 p.p.m. soy protein antigens. Three of the four OTC ‘no soy’ claiming diets were positive for soy antigen; two contained >25 p.p.m. Four veterinary therapeutic diets had less than the lower detectable limit of soy protein. Two hydrolysed soy diets were positive (>2.5 p.p.m.). One veterinary therapeutic diet contained >25 p.p.m. soy but soybean oil was a listed ingredient, and one diet contained 4.6 p.p.m. with no soy ingredients listed. From these results we conclude that OTC dog food diets that claim to contain ‘no soy’ may contain high concentrations of soy protein antigen and therefore should not be considered for soy elimination trials. The veterinary therapeutic diet chosen for a soy elimination diet trial needs to be carefully selected based on these results.

Source of funding: Study funded by an ACVD and AAVD Research Award.

Conflict of interest: No conflicts of interest have been declared.

Cutaneous adverse reaction to several oral thyroid supplements in a dog with hypothyroidism and confirmed cutaneous adverse reaction to food

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A. PETERSEN

College of Veterinary Medicine, Department of Small Animal Clinical Sciences, Michigan State University, East Lansing, MI, USA

A 4-year-old female spayed Chinese crested dog was evaluated for nonseasonal, steroid-responsive pruritus affecting the face, legs, feet and ears and histopathological allergic dermatitis. A home-cooked food trial with venison and potatoes resulted in bloody diarrhoea. The pruritus had not responded to two subcutaneous ivermectin injections 2 weeks apart or to a year-long restricted diet trial with Z/D ultra (Hill's Science Diet, Topeka, KS, USA). A second home-cooked food trial with pinto beans and sweet potatoes resulted in complete resolution of the pruritus over a 4 week period. A year later, while still on the home-cooked diet, the dog was represented with lethargy and weakness. A thyroid profile revealed low thyroid hormone and elevated thyroid-stimulating hormone concentration. Thyroid supplementation with Thyrozine tablets (Phoenix Pharmaceutical, St. Joseph, MO, USA) resulted in resolution of weakness and lethargy, but pruritus flared within 24 h. Clinical signs were similar to the previous cutaneous adverse food reaction. Pruritus resolved again when the supplement was discontinued, but again flared with two other thyroid supplements, Soloxine tablets (Virbac AH, Fort Worth, TX, USA) and a compounded formulation with gelatin capsules. Finally, a liquid compounded formulation did result in resolution of lethargy without flare of the pruritus. The dog's diet was changed to a suitable commercial diet. In conclusion, this is a report of a dog with a cutaneous adverse reaction to several thyroid supplements with signs identical to the dog's cutaneous adverse food reaction. Thus, thyroid supplements can produce signs of cutaneous adverse food reaction.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and carriage sites of dogs after treatment of their meticillin-resistant or meticillin-sensitive staphylococcal pyoderma

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K. M. BECK*, S. E. WAISGLASS*, H. L. N. DICK† and J. S. WEESE‡

* Veterinary Emergency Clinic and Referral Centre, Toronto, ON, CanadaIDEXX Laboratories, Markham, ON, CanadaDepartment of Pathobiology, Ontario Veterinary College, Guelph, ON, Canada

Meticillin-resistant staphylococci are critical pathogens in veterinary dermatology, yet longitudinal studies of the impact of routine antimicrobial therapy on emergence or resolution of resistance are lacking. Skin, nasal and rectal swabs were collected from dogs with clinical and cytological evidence of pyoderma at time of referral and after clinical resolution. Initially, skin cultures yielded 39% (64 of 165) MRSP, 1.8% (three of 165) meticillin-resistant Staphylococcus aureus (MRSA) and 1.8% (three of 165) meticillin-resistant Staphylococcus schleiferi ssp. coagulans (MRSScoag). MRSP was isolated from nose or rectum (carriage sites) of 31% (51 of 165), MRSA from 6.7% (11 of 165) and MRSScoag from 3.0% (five of 165). Of 85 follow-up cases, skin cultures revealed 33% (28 of 85) MRSP and 1.2% (one of 85) each for MRSA and MRSScoag. MRSP was isolated from carriage sites in 32% (27 of 85), while MRSA was isolated in 1.2% (one of 85) and MRSScoag in 2.4% (two of 85). Of dogs initially diagnosed with MRSP pyoderma, MRSP was isolated at follow-up from skin in 37% (11 of 30) and carriage sites in 40% (12 of 30). The prevalence of MRSP carriage was significantly lower than at initial presentation (72%, P = 0.006). Of dogs that did not have MRSP pyoderma initially, MRSP was isolated from the skin of 31% (17 of 55) and MRSP from carriage sites increased from 5.0 to 27% (15 of 55, P = 0.0002). There was no difference in the follow-up skin or carriage site prevalence of MRSP in dogs that initially had MRSP pyoderma versus others (P = 0.32 and 0.63). Persistence of MRSP on skin and carriage sites is common after resolution of MRSP pyoderma. Acquisition of MRSP during treatment appears to be common.

Source of funding: This study was supported by the Ontario Veterinary College Pet Trust Fund.

Conflict of interest: No conflicts of interest have been declared.

Evaluation of a potential role for pet animals to serve as reservoirs for meticillin-resistant Staphylococcus aureus (MRSA) within households

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D. O. MORRIS*, E. LAUTENBACH†, T. ZAOUTIS‡ and S. C. RANKIN§

* Departments of Clinical Studies and § Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USADepartment of Healthcare, Epidemiology and Infection Control, School of Medicine, Philadelphia, PA, USADepartment of Pediatric Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, PA, USA

Colonization by meticillin-resistant Staphylococcus aureus (MRSA) may persist for months to years in people. Horizontally, transmissiion occurs. The scientific literature suggests that domestic pets may also participate in cross-transmission of MRSA within households. The objectives of this study were to evaluate the prevalence of and risk factors for cross-transmission of MRSA between people with active infections and their household pets. From 66 households in which an MRSA-infected patient resided, we screened 47 dogs and 52 cats using a swab protocol. Isolates from pets and humans were genotyped using two techniques, and compared for concordance. Human subjects completed a 22-question survey of demographic and epidemiological data relevant to staphylococcal transmission. Of 66 participating households, nine (13.5%) harboured at least one MRSA-positive pet, but in only six (9%) of these households were the human- and animal-source strains genetically concordant. The MRSA strain isolated from the majority of human infections was USA 300, while the most common strain isolated from pets was USA 100. Significant risk factors for pet carriage included human exposure to amoxicillin–clavulanic acid within the month prior to diagnosis [odds ratio = 16.6 (95% confidence interval 2.6–107.9), P = 0.003], and human infection by strain USA 100 [odds ratio = 11.4 (95% confidence interval 1.7–76.9), P = 0.013]. Yet, for each day of delay in sampling the pet after the person's MRSA diagnosis, the odds of isolating MRSA from the pet decreased by 13%. It may be concluded that the source of MRSA to carrier pets is not always the infected person within the household.

Source of funding: This study was supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, no. R21-AI-073328.

Conflict of interest: No conflicts of interest have been declared.

Treatment outcome of dogs with meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius pyoderma

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J. BRYAN, L. FRANK, L. BURGETTE and C. CAIN

Department of Small Animal Clinical Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, TN, USA

Staphylococcus pseudintermedius is the most common causative organism associated with canine pyoderma. Meticillin-resistant strains are increasingly more prevalent, and management of these infections is often challenging. The aim of this study was to investigate the treatment outcome, as defined by clinical resolution and treatment duration, and adverse effects of medication in dogs with pyoderma caused by meticillin-susceptible S. pseudintermedius (MSSP) and meticillin-resistant S. pseudintermedius (MRSP) between January 2008 and April 2010. Medical records were reviewed for antimicrobial therapy, treatment outcome and adverse effects. A total of 219 cases were included [125 (57.1%) MSSP, 94 (42.9%) MRSP]. In MSSP infections, cefalexin (20.4–39.0 mg/kg twice daily) and cefpodoxime (6.0–13.2 mg/kg four times per day) were the most commonly prescribed antimicrobials, accounting for 55 (44.0%) and 44 (35.2%) cases, respectively. In MRSP infections, chloramphenicol (30.5–61.9 mg/kg three times per day) and doxycycline (4.6–11.1 mg/kg twice daily) were most commonly prescribed, accounting for 53 (56.4%) and 13 (13.8%) cases, respectively. Adverse effects were reported in 41 (18.7%) cases (seven MSSP, 34 MRSP). The most common adverse effects were gastrointestinal, prompting antibiotic discontinuation in 22 of 41 cases (three MSSP, 19 MRSP). Chloramphenicol was associated with the highest incidence of adverse reactions (29 of 53 dogs). Of 162 cases with follow-up, 46 of 86 (53.5%) MSSP infections and 28 of 76 (36.8%) MRSP infections achieved complete clinical resolution at the first recheck examination after 3–4 weeks. All but two cases of MSSP and seven cases of MRSP improved or resolved on subsequent visits. This study demonstrates that resistant infections may require additional time to resolve and that chloramphenicol is associated with frequent adverse effects.

Source of funding: This study was funded by Center of Excellence, University of Tennessee College of Veterinary Medicine, Knoxville, TN, USA.

Conflict of interest: No conflicts of interest have been declared.

Clinical characterization of Staphylococcus schleiferi infections in dogs and identification of risk factors for acquisition of oxacillin-resistant strains

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C. L. CAIN*, D. O. MORRIS† and S. C. RANKIN‡

* Department of Small Animal Clinical Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, TN, USADepartments of Clinical Studies andPathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

Staphylococcus schleiferi is an emerging veterinary pathogen reported to have two subspecies based on coagulase status, although recent work has supported a single species with variable coagulase production. The objective of this retrospective case series was to define clinical differences in canine infections caused by coagulase-positive and coagulase-negative S. schleiferi and to identify risk factors for the isolation of oxacillin (meticillin)-resistant strains. Two-hundred and twenty-five S. schleiferi isolates from dogs were identified by a commercial microbiology system and antibiotic susceptibility data recorded. Medical records of dogs were reviewed regarding isolates, patient demographics, antimicrobial use and primary disease. Multiple logistic regression was performed to identify risk factors for infection by coagulase status and for oxacillin resistance. Forty-nine per cent of dogs were allergic, and most isolates were from the ears (45%) or skin (42%). Isolate coagulase status was not significantly associated with any patient-level factors. Fifty-seven per cent of isolates were oxacillin resistant. Coagulase-negative isolates were more likely to be oxacillin resistant than coagulase-positive isolates [odds ratio, 1.8 (95% confidence interval, 1.1–3.0); P < 0.04]. Use of penicillin-based or first-generation cephalosporin drugs [odds ratio, 3.0 (95% confidence interval, 1.8–5.9); P < 0.001] and third-generation cephalosporins [odds ratio, 3.7 (95% confidence interval, 1.1–12.3); P < 0.04] within 30 days were risk factors for oxacillin resistance. Both coagulase-negative and coagulase-positive S. schleiferi are potential pathogens. Staphylococcus schleiferi was commonly oxacillin resistant, and recent penicillin or cephalosporin use were risk factors for oxacillin resistance.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

In vitro efficacy and safety of topical biocides and essential oils

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B. VALENTINE*, S. WEESE†, A. YU* and J. LAMARRE‡

* Departments of Clinical Studies andPathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, CanadaDepartment of Biomedical Sciences, University of Guelph, Guelph, ON, Canada

The emergence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) has complicated treatment of canine pyoderma. Multidrug-resistant infections may require alternative approaches, such as the use of topical therapy, but there is minimal information about in vitro susceptibility and safety of biocides and essential oils. The objective of this study was to determine susceptibility of canine meticillin-susceptible S. pseudintermedius (MSSP) and MRSP to various biocides and to assess the cytotoxic effects of a subset of these compounds on canine keratinocytes. The minimal inhibitory concentration (MIC) of chlorhexidine digluconate, benzalkonium chloride, triclosan, accelerated hydrogen peroxide (AHP), geranium oil, tea tree oil and grapefruit seed extract (GSE) was assessed for 25 MRSP and 25 MSSP isolates by agar dilution. Triclosan demonstrated excellent activity against all bacterial isolates (MIC50 ≤ 0.5 μg/mL), while GSE had no effect at the highest concentration tested (3.84 μg/mL). Other biocides had MIC50 values ranging from 2 to 32 μg/mL, while MIC50 values for other oils ranged from 0.6 to 0.48%, with user concentration typically 100%. There was no difference between MRSP and MSSP for any biocide or antimicrobial (all > 0.17). Progenitor canine keratinocytes were cultured and subsequently subjected to treatment with AHP, triclosan, geranium oil and GSE. Cellular cytotoxicity was assessed using a crystal violet assay for estimation of cell viability. All compounds exhibited cytopathic effects at high concentrations in vitro, including concentrations well beyond commercial concentrations for the essential oils. These safety and efficacy data will allow for development of evidence-based recommendations for topical therapy for canine pyoderma.

Source of funding: This study was funded by the Ontario Veterinary College Pet Trust Fund.

Conflict of interest: No conflicts of interest have been declared.

Contact time of 2% chlorhexidine acetate for canine superficial pyoderma

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N. MURAYAMA*†, Y. TERADA*, M. OKUAKI*, K. HARADA†, Y. KATAOKA†, T. TAKAHASHI† and M. NAGATA*

* ASC Dermatology service, Tokyo, JapanNippon Veterinary and Life Science University, Tokyo, Japan

Two per cent chlorhexidine acetate (2CA) is a topical agent used for canine pyoderma. The aim of this study was to evaluate appropriate contact time of 2CA (Nolvasan Surgical Scrub; Fort Dodge Animal Health, IA, USA) for canine pyoderma. Ten dogs with superficial pyoderma without underlying disorders using standard diagnostic procedures were allocated. Staphylococcus pseudintermedius, S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans were identified in seven, two and one cases, respectively, and five isolates were mecA positive Staphylococcus spp. Two different contact times (group 1, 1 min; group 2, 10 min) were investigated in symmetrical lesions at different sites every 2 days four times with a dosage of 0.5 ml (one coin size of the product per two hands). Sixty and 70% of dogs subjectively evaluated by owners and investigators showed excellent to good improvement in group 1 and 2, respectively. Erythema, papules, crusts/scales and alopecia were objectively scored in four grades. Erythema and crusts/scale, and erythema, papules and crusts/scale significantly improved in group 1 and 2, respectively (Wilcoxon signed rank test), and no significant differences were found for each lesion type (Mann–Whitney U-test). No adverse reactions were observed during this study. An in vitro study compared the rate of killing at 1:5 and 1:25 dilutions of 2CA. All isolates were eliminated in <30 s at both dilutions. It was indicated that a short contact time of 2CA seems to be sufficient 2CA therapy for canine superficial pyoderma.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Evaluation of the tympanic membrane presence and integrity, and bulla in dogs with otitis by performing MRI pre- and post-ear flushing

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R. CERUNDOLO, G. B. CHERUBINI, A. CAINE, R. A. S. WHITE and S. BRAVO

Dick White Referrals, Veterinary Specialist Centre, Six Mile Bottom, Suffolk CB8 0UH, UK

Evaluating the tympanic membrane (TM) presence/integrity is often difficult in dogs with chronic otitis, with stenotic or hairy ear canals. The presence of a normal TM influences the prognosis and therapy of otitis.

A prospective study was performed in 30 dogs (60 ears) with recurrent/chronic bilateral otitis where the TM was not visualized using a hand-held otoscope. All dogs underwent MRI pre- (MRI-Pre-F) and post-ear flushing (MRI-Post-F). T2, T1 and T1 + gadolinium images were obtained in the transverse plane on a 0.4 T Hitachi Aperto MRI scanner.

Video otoscopy performed after the MRI-Pre-F showed that the TM was present in 12 ears, and in all but one case the MRI-post-F did not show any fluid accumulation in the bulla, suggesting an intact TM. The TM was not visualized in the other 48 ears, and the absence of fluid accumulation in the bulla in the MRI-post-F suggested that the TM was intact in 35 of 48 ears. In the other 13 of 48 ears, there was a small/large amount of fluid, suggesting partial/complete rupture of the TM, which was already present in the MRI-pre-F in eight of 13 ears. In three dogs (three ears) the bulla was filled with debris that, despite repeated lavage–aspiration cycles, was still present in the MRI-post-F.

Evaluation of the ear canal and bullae with MRI when the TM cannot be visualized is a useful tool to guide ear flushing. MRI-post-F also provides information on the integrity of the TM and indicates presence of otitis media, which can be used to guide treatment (surgical or medical) and prognosis.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Bacterial contamination of commercial ear cleaners following routine use

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S. BARTLETT*, W. ROSENKRANTZ† and S. SANCHEZ‡

* Animal Dermatology Clinic, Marietta, GA, USAAnimal Dermatology Clinic, Tustin, CA, USADepartment of Infectious Diseases and Athens Veterinary Diagnostic Laboratory, University of Georgia, Athens, GA, USA

Ear cleaning solutions are designed for multiple uses, raising the possibility of bacterial contamination leading to recurrent or persistent infectious otitis. The purpose of this study was to investigate the prevalence of bacterial contamination of commercial ear cleaners following home use in dogs and to describe the characteristics associated with contamination. Used ear cleaner bottles and information regarding their use were obtained from canine owners visiting veterinary dermatologists. Bottle applicator tips and the solution contents were cultured for aerobic bacteria. Bacterial contamination was present on 10% of the bottle tips and in 2% of the solutions. Isolated bacteria included Staphylococcus pseudintermedius, Bacillus spp, coagulase-negative Staphylococcus spp., Micrococcus spp. and Burkholderia cepacia. The contamination rate was significantly higher on the applicator tips than in the solutions (P = 0.0076). The applicator tip contamination rate was significantly higher in expired samples (17%) than in-date samples (4%; = 0.0277). The bottle sizes were significantly greater in the samples with contaminated applicator tips compared with noncontaminated tips (P = 0.0455). The contamination rate was significantly higher when Tris–EDTA was an ingredient. Cleanliness of the bottle, contact with the ear canal and infection status of the ear at time of culture had no bearing on the contamination rate. In summary, with routine home use of commercial ear cleaners, pathogenic bacterial contamination is of minor concern. This concern may increase when expired products or larger bottles of ear cleaner are used and when Tris–EDTA is an ingredient.

Source of funding: This study was supported by a grant from the American College of Veterinary Dermatology.

Conflict of interest: No conflicts of interest have been declared.

Epithelial migration on the canine tympanic membrane

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N. E. TABACCA*, L. K. COLE*, A. HILLIER* and P. J. RAJALA-SCHULTZ†

* Departments of Veterinary Clinical Sciences andVeterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Epithelial migration (EM) is a process that serves as a self-cleaning and repair mechanism for the ear canal and tympanic membrane (TM) and has been evaluated in humans and several other species, but not dogs. The objective of this study was to determine the rate and pattern of EM on the TM in clinically normal dogs. Eighteen research dogs (eight beagles and 10 fox hounds) were anaesthetized and, using an operating microscope, three drops of waterproof India ink were placed on the TM, two on the caudal half of the pars tensa (PT1 and PT2) and one on the centre of the pars flaccida (PF). Images were recorded with a video otoscope and digital capture system. Each dog was evaluated and images recorded every 6–8 days for a maximum of four evaluations (until the ink drops migrated off the TM). Morphometric pattern analysis and EM rate calculation were performed with ImagePro Analyzer® version 6.3. Descriptive statistics for EM rate (means ± SD) were calculated in GraphPad Prism®version 5.02 for all ink drop locations on the TM (PT1, PT2 and PF) at each time point. The mean overall EM rate was 96.4 ± 43.1 μm/day for the PT and 225.4 ± 128.1 μm/day for the PF. All drops moved outward, the majority in a radial direction, from the original location to the periphery of the TM. The ink drop placement method evaluated in this study can be used to determine the EM rate of a normal canine TM.

Source of funding: This study was supported by The Ohio State University, College of Veterinary Medicine, Canine Intramural Grants and VCA Antech, Inc.

Conflict of interest: No conflicts of interest have been declared.

Diagnosis of primary secretory otitis media in the cavalier King Charles spaniel

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L. K. COLE*, V. F. SAMII*, S. WAGNER* and P. J. RAJALA-SCHULTZ†

* Departments of Veterinary Clinical Sciences andVeterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

Primary secretory otitis media (PSOM) is a disease reported in the cavalier King Charles spaniel (CKCS). In a previous study, diagnosis of PSOM was made based only on visualization of a bulging tympanic membrane and mucus in the middle ear postmyringotomy. No additional tests were used to evaluate for PSOM, and CKCS with early disease may have been missed. The purpose of this study was to compare three diagnostic tests (tympanometry, pneumotoscopy and tympanic bulla ultrasonography) using computed tomography (CT) as the gold standard for the diagnosis of PSOM in CKCS. Sixty CKCS (31 females and 29 males) with clinical signs suggestive of PSOM (e.g. hearing loss, neck scratching, pruritic ears) were enrolled in the study. Mean age at presentation was 54 months (range 5–151 months). Forty-three (72%) CKCS had PSOM (30 bilateral, 13 unilateral). A large, bulging pars flaccida was identified in only those CKCS with PSOM (specificity of 100%); however, only 21 of 73 ears with PSOM had a large, bulging pars flaccida (sensitivity of 29%). Sensitivities and specificities for tympanometry, pneumotoscopy, and tympanic bulla ultrasonography were (84 and 47%), (75 and 79%) and (67 and 47%), respectively. Based on the results of this study, a large, bulging pars flaccida indicates the presence of PSOM, while a flat pars flaccida may be present in CKCS that have PSOM as well as those that do not. In CKCS with a flat pars flaccida, unfortunately, none of the above diagnostic tests can be recommended in place of CT scan for the diagnosis of PSOM.

Source of funding: This study was supported by the American Cavalier King Charles Spaniel Club Charitable Trust Inc. and The Ohio State University, College of Veterinary Medicine, Canine Intramural Grant (Paladin Fund).

Conflict of interest: No conflicts of interest have been declared.

Primary secretory otitis media (PSOM) in dogs with suspected Chiari-like malformation: 120 cases (2007–2010)

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C. A. LOUGHIN, D. J. MARINO and S. GOVIER

The Canine Chiari Institute at Long Island Veterinary Specialists, Plainview, NY, USA

Primary secretory otitis media (PSOM) is a disease that commonly affects cavalier King Charles spaniel dogs (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, head tilt, nystagmus and facial/cervical pruritus resembling those of Chiari-like malformation (CLM) and syringomyelia. Traditionally, a diagnosis of PSOM is made by otoscopic examination, computed tomography (CT) or magnetic resonance imaging (MRI); however, the sensitivity and specificity of CT and MRI has not been reported. The purpose of this study was to describe the prevalence of PSOM in dogs with CLM, to describe clinical signs and to compare the sensitivity and specificity of CT with MRI used in the diagnosis of PSOM. The MRI and CT images from 310 dogs with CLM were evaluated for PSOM and compared with surgical findings (myringotomy). The MRI results were identical to those of surgical findings; 38.7% of dogs with CLM had PSOM; 32% of dogs were asymptomatic. Clinical signs included pruritus (31%), hyperpathia (28%) and hearing loss (2.5%). There was a significant difference between the prevalence of PSOM in CKCS (46%) and non-CKCS (13%); P < 0.0001. Compared with MRI, CT had a 62 and 100% sensitivity and specificity, respectively, for the right bulla and a 72.5 and 97.9% sensitivity and specificity, respectively, for the left. Based on these findings, PSOM is a common finding in dogs with CLM. The CKCS breed was found to be overrepresented. Magnetic resonance imaging is superior to CT for the diagnosis of PSOM in dogs.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Absorption of transdermal ciclosporin versus orally administered ciclosporin in six healthy cats

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R. MILLER*, A. SCHICK*, D. BOOTH† and T. LEWIS*

* Dermatology for Animals, Gilbert, AZ, USAClinical Pharmacology Laboratory, College of Veterinary Medicine, Auburn University, Auburn, AL, USA

Ciclosporin is a commonly used oral medication in the treatment of feline dermatoses. Due to the difficulty of administering oral medications in cats, compounded transdermal cyclosporin is prescribed by veterinarians, although human studies show limited transdermal absorption. The objective of this pilot study was to compare blood ciclosporin concentrations (Atopica; Novartis Animal Health, Greensboro, NC, USA) after oral administration to blood ciclosporin concentrations after a compounded transdermal formulation. Ciclosporin was given orally at 5.1–7.4 mg/kg once daily for 7 days to six healthy cats. On day 7, ciclosporin was measured in whole blood samples collected 2 and 12 h postdosing. Ciclosporin was detected using a monoclonal-based immunoassay (limit of quantification 25 ng/ml). After a 2 week washout period, ciclosporin concentrations were measured, and 0.1 ml (5.1–7.4 mg/kg) of transdermal ciclosporin, formulated in pluronic lecithin organogel at 250 mg/ml, was applied to the nonhaired portion of the pinna once daily on each cat for 21 days. Ciclosporin was measured 2 and 12 h postdosing on day 7, 14 and 21. Median oral-dosed ciclosporin concentration (in nanograms per millilitre) at 2 h on day 7 was 2208 (range 1357–3419). Median transdermal-dosed ciclosporin concentration at 2 h on day 7 was 37 (range 25–290) and for day 21 was 58 (range 51–878). Concentrations were quantifiable for transdermal ciclosporin, but concentrations were therapeutic in only one of six cats. Based on the results of this study, transdermal ciclosporin should not be recommended in cats because of inconsistent absorption.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Selected feline infectious disease test results from client-owned cats before and after administration of ciclosporin

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S. ZABEL*, N. OKUNAKA†, K. E. LOFT†, M. BREWER‡ and M. R. LAPPIN*

* Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Department of Clinical Sciences, Fort Collins, CO, USAVCA South Shore Animal Hospital, South Weymore, MA, USAColorado State University, College of Veterinary Medicine and Biomedical Sciences, Center for Companion Animal Studies, Fort Collins, CO, USA

Ciclosporin A (CsA) is prescribed frequently to cats for the management of dermatological and other immune-mediated diseases. Activation of chronic infectious disease agents has occurred in some cats administered CsA, presumably because of immune suppression induced by the drug. The purpose of this study was to report the results of diagnostic tests for selected infectious disease agents before and after the administration of CsA (5–10 mg/kg/day). Serum, blood and faeces samples were collected from 20 cats before and approximately 60 days after CsA therapy for various diseases. Samples were assayed for Toxoplasma gondii IgM and IgG (TG), Bartonella (BA) spp. IgG, Bartonella spp. DNA (BA DNA), Mycoplasma haemofelis DNA (MH), ‘Candidatus M. haemominutum’ DNA (CMH), ‘Candidatus M. turicensis’ DNA (CMT) and faecal parasites using standard procedures. Concentrations of CsA were measured in the day 60 sera. Overall, evidence of an infectious disease agent was detected in nine of 20 (45%) on day 0 (1 TG, 4 BA, 1 BA DNA, 1 MH and 2 CMH) and all but one stayed positive on day 60 (4 BA, 1 BA DNA, 1 MH and 2 CMH). None of the cats had evidence of clinical disease attributable to the agents on day 60. Only one of three cats with CsA concentrations >900 μg/mL had been exposed to an infectious agent (Bartonella spp. IgG). While serology or PCR showed evidence of exposure to infectious disease agents in this study population, none of the cats developed clinical evidence of any of the infectious disease agents.

Source of funding: This study was funded by the Colorado State University College research fund.

Conflict of interest: No conflicts of interest have been declared.

Comparison of modified ciclosporin to chlorambucil in the management of feline pemphigus foliaceus

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K. E. IRWIN, K. M. BEALE and V. A. FADOK

Gulf Coast Veterinary Dermatology and Allergy, Houston, TX, USA

Ciclosporin was compared with chlorambucil in management of feline pemphigus foliaceus. Fifteen cats presented from 1999 to 2006 with pemphigus foliaceus confirmed by histopathology, that received ciclosporin (median dose 5.2 mg/kg/day, range 4.4–6.9) and/or chlorambucil (median dose 0.17 mg/kg/day, range 0.07–0.2) as part of their treatment and had adequate follow-up to assess treatment response were evaluated. Cats were placed within two treatment groups: ciclosporin ± glucocorticoid and chlorambucil ± glucocorticoid. Each group contained nine patients, with three represented in both. Time to disease remission, mean remission-inducing glucocorticoid dose, mean maintenance or final glucocorticoid dose, disease response and adverse effects were assessed. There was no significant difference in remission times or disease response between groups. To compare between glucocorticoid protocols, each patient's glucocorticoid dose was converted to its relative hydrocortisone equivalency and calculated as an average daily dose. Patients managed with ciclosporin required significantly less glucocorticoids both for remission induction and for maintenance/final therapy (Student's paired t-test, P = 0.027 and P < 0.0001, respectively). Six of eight patients maintained on ciclosporin and one of seven on chlorambucil were weaned off all systemic glucocorticoids. Ciclosporin-associated adverse effects included hypertrichosis, self-limiting diarrhoea and intermittent soft stools. One patient's ciclosporin was discontinued after developing a disseminated Mycobacterium avium infection. Chlorambucil-associated adverse effects included leukopenia and anorexia with weight loss. Ciclosporin was tapered to alternate day dosing in five of eight patients, while chlorambucil was tapered to alternate days in seven of seven patients. We conclude, relative to chlorambucil, ciclosporin provides comparable efficacy for management of feline pemphigus foliaceus while providing superior glucocorticoid-sparing effect.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Desmocollin-1 is a major autoantigen in canine pemphigus foliaceus

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P. BIZIKOVA*, G. A. DEAN†‡, T. HASHIMOTO§ and T. OLIVRY*‡

* Departments of Clinical Sciences andMolecular Biomedical Sciences andCenter for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA § School of Medicine, Kurume University, Japan

A recent immunomapping study proposed desmocollin-1 (DSC1) as a relevant candidate autoantigen in canine pemphigus foliaceus (cPF). To investigate this hypothesis, we cloned and transfected 293T human epithelial kidney cells to produce canine DSC1. We selected 52 canine PF sera with positive indirect immunofluorescence (IIF) testing on canine epithelia; 43 sera (83%) exhibited the classical IIF pattern (footpad positive, buccal mucosa negative) and nine (17%) had atypical IIF results (footpad and buccal mucosa positive). Sera from 29 normal dogs served as negative controls. All sera were tested by IIF on fixed DSC1-transfected 293T cells, while canine desmoglein-1 (DSG1) and untransfected cells served as negative controls. All DSC1-negative sera were retested in the same manner using live cells. None of the normal control canine sera recognized any of the transfected cells. Of 43 cPF sera exhibiting the classical staining pattern, 36 (84%) contained IgG recognizing the DSC1-expressing cells but none of the control cells. Eight of nine cPF sera with atypical IIF pattern (89%) contained IgG recognizing canine DSC1-producing cells. The live-cell staining revealed a superior sensitivity to that using fixed cells. In three dogs with PF and classical IIF patterns, serial serum samples were collected during treatment, and their titres of anti-DSC1 IgG decreased in parallel with the reduction in disease activity. In all, 44 of 52 IIF-positive cPF sera (85%) contained IgG autoantibodies that recognized DSC1, thereby suggesting that it is a major target autoantigen in this common autoimmune skin disease.

Source of funding: This project was funded by the American College of Veterinary Dermatology. P. Bizikova is the current recipient of the American Academy of Veterinary Dermatology PhD fellowship.

Conflict of interest: No conflicts of interest have been declared.

Successful treatment of a novel variant of canine chronic cutaneous lupus erythematosus with oral hydroxychloroquine, topical tacrolimus and prednicarbate

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U. OBERKIRCHNER*, K. LINDER† and T. OLIVRY*

* Departments of Clinical Sciences andPopulation Health and Pathobiology, North Carolina State University, Raleigh, NC, USA

We report herein a new variant of canine cutaneous erythematosus (CLE) successfully treated with the antimalarial immunomodulator hydroxychloroquine (HCQ). A 9-year-old Chinese crested dog was presented with annular and polycyclic hyperpigmented and scaly lesions with central erosions, hypopigmentation and/or scarring on the trunk, neck and lateral extremities. Associated systemic signs were not seen. In this dog, the clinical diagnosis of CLE was supported by the demonstration of lymphocyte-rich interface dermatitis with epidermal atrophy and dermo-epidermal deposition of immunoglobulins and activated complement. As for human CLE, treatment was initiated with HCQ at 5 mg/kg once daily (Gallipot, St Paul, MN, USA) along with 2 weeks of 0.1% tacrolimus ointment (Protopic; Astellas Pharma, Deerfield, IL, USA) and restriction of sun exposure. Over the following year, the complete remission was maintained with only three relapses; two occurred during treatment induction and the third arose when the frequency of HCQ administration was reduced to every other day. Disease flares were controlled with 5–10 days of tacrolimus ointment alternating with generic 0.1% prednicarbate cream (Prasco Laboratories, Mason, OH, USA). Altogether, adverse drug events were not seen with this treatment regimen. In summary, this dog's disease mirrored clinically, histologically and immunologically the generalized discoid variant of chronic CLE of humans. The apparent benefit of HCQ and its low cost warrant future investigations of its use for treatment of canine CLE variants.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Optimization of a Staphylococcus aureus adhesion assay for equine corneocytes

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G. GRIFFETH*, N. CALLORI†, S. RANKIN‡ and D. MORRIS*

* Department of Clinical Studies,Class of 2008 andDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

Meticillin-resistant Staphylococcus aureus (MRSA) causes serious skin and soft-tissue infections of humans and animals. Multiple strains of MRSA have been characterized, and one particular strain, USA 500, causes infections predominantly of horses and the people who work with them. We hypothesize that this MRSA strain's avidity for equine tissue is due to host-adapted adhesion factors. This study was designed to optimize a Staphylococcus–equine corneocyte adhesion assay, which could then be used to compare the relative adhesion of MRSA strains, including USA 500. Corneocytes were collected from the perineal skin of 10 horses onto adhesive discs (D-Squame®; CuDerm, Dallas, TX, USA). The discs were incubated with a S. aureus field strain at three concentrations (107, 108 and 109 CFU/mL) each for three incubation periods (45, 90 and 180 min). After standardized rinsing and staining, areas containing confluent corneocytes were examined at ×1000 magnification and photographed. The percentage of surface area occupied by adherent bacteria was analysed using image processing and analysis software (ImageJ 1.44c; NIH, Bethesda, MD, USA). Significant colour space image processing was required to distinguish bacteria from the ubiquitous melanin granules present in equine corneocytes. Subjective and objective methods were used to determine optimal conditions for specific adherence without introducing confounding factors. Using 108 CFU/mL incubated for 45 min before rinsing produced binding in significant numbers with less nonspecific interbacterial adhesion, and will be used as the initial protocol for our multistrain comparative studies.

Source of funding: This project was partially funded by the NIH/Merial Summer Research Program.

Conflict of interest: No conflicts of interest have been declared.

Dermatohistopathological changes in hirsute horses with pituitary pars intermedia dysfunction (PPID)

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M. INNERÅ*, A. PETERSEN*, D. DESJARDINS†, B. STEFICEK†, E. ROSSER JR* and H. SCHOTT II‡

* Departments of Small Animal Clinical Sciences andLarge Animal Clinical Sciences, Veterinary Medical Center andDiagnostic Center for Population and Animal Health, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA

This study evaluated hair follicle morphology in PPID-affected, hirsute horses. Eight-millimetre punch skin biopsies were collected from the neck and rump region of 17 PPID-affected horses and six age-matched control horses. Their PPID status was established on the basis of hirsutism and supportive overnight dexamethasone supression test results. Biopsies were obtained between 1 November and 31 January, placed in formalin and sectioned at the level of, or slightly below, entry of the sebaceous duct in a plane parallel to the skin surface. Sections were stained with haematoxylin and eosin, and hair cycle growth stage (anagen or telogen) was determined based on morphometric characteristics previously established for human scalp hair follicles. These included degree of tricholemmal keratinization, inner root sheath histology and volumetric involution of the outer root sheath. The percentage of hair follicles in anagen and telogen in a 50 mm2 area was counted for each biopsy. A split-plot analysis of variance demonstrated that neck skin of PPID-affected horses had a greater percentage of anagen (90%) and a lower percentage of telogen (10%) follicles than control horses (anagen 26%, telogen 74%). In rump skin, PPID-affected horses also had a higher percentage of anagen hair follicles (97%) and a lower percentage of telogen (3%) follicles compared with control horses (59% anagen, 41% telogen). In conclusion, our findings provide support that hirsutism in PPID-affected horses is characterized by persistence of hair follicles in anagen.

Source of funding: This study was funded by an ACVD Resident's Research Award.

Conflict of interest: No conflicts of interest have been declared.

Equine atopic skin disease and response to allergen-specific immunotherapy (ASIT): a retrospective study at the University of California-Davis (1991–2008)

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C. T. STEPNIK*, C. A. OUTERBRIDGE*, S. D. WHITE* and P. H. KASS†

* Departments of Medicine and Epidemiology andPopulation Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA

The purpose of the study was to better define the clinical presentation of equine atopic skin disease and evaluate the response to treatment with ASIT based on intradermal testing and/or serum testing. Fifty-four horses (35 males, 19 females; variety of breeds) were included in the study. There was no statistical significance in sex distribution. The Hackney breed (two of 54) may be predisposed to atopic skin disease (χ2 test, P = 0.0001). Presenting clinical signs (CS) included urticaria (28), pruritus (8) or both (18). Forty-one of 54 horses received ASIT; 32 of these were evaluated for response to ASIT via telephone survey. Eighty-four per cent (27 of 32) of owners reported that ASIT reduced their horse's CS [urticaria (9), pruritus (4), both (14)]. There was no statistical significance between type of allergy test performed and reported success of ASIT (exact χ2 test, P = 0.53). Ninety-three per cent (30 of 32) of owners reported use of antipruritic medications prior to starting ASIT; 60% (18 of 30) reported discontinuing those medications after starting ASIT. The only adverse effect reported was swelling at the injection site, seen in 16% (five of 32). Seventy-five per cent (24 of 32) reported discontinuing ASIT (mean 2.2 years, range 6 months to 8 years); 15 due to resolution of CS, six due to persistence of CS, two because the horse was sold, and one due to cost. Thirteen owners reported no recurrence of CS after discontinuing ASIT; five horses had recurrence within a median of 2 years of discontinuing ASIT (range 1–12 years). Allergen-specific immunotherapy is a safe and effective way to manage equine atopic skin disease.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

A semiquantitative method to evaluate cutaneous cytology

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S. BUDACH and R. S. MUELLER

Center for Clinical Veterinary Medicine, Ludwig Maximilian University, Munich, Germany

Cytology provides important information relevant for the diagnosis and treatment of skin disease. The aim of this study was to evaluate the intra- and interobserver variability of a practical semiquantitative method to assess cytology specimens. Participants were divided into experienced and inexperienced cytologists. All participants were asked to twice evaluate 10 cytology slides under the microscope and 18 photographs of cutaneous cytology. The two evaluations had to beat least 6 h apart. Cocci, rods, yeast, neutrophils, eosinophils and macrophages each were assigned to one of five classes from 0 to ++++. Sixty veterinarians participated in the study: 29 experienced and 31 unexperienced cytologists. The mean intraobserver reproducibility for the photographs was 87.4% in the experienced and 92.1% in the unexperienced group. With the cytology slides, the mean reproducibility was 90.5% for the experienced and 82.6% for the inexperienced group. The interobserver reproducibility for the photographs was 91.3% (91.5%) for the experienced (inexperienced) group and for the cytology slides 84.2% (82.2%). The reproducibility for cytology slides for the experienced (inexperienced) group was 70.8% (71%) for cocci, 91.3% (92%) for rods, 87.2% (79.5%) for yeasts, 78.7% (76.3%) for neutrophils, 93.7% (94.5%) for eosinophils and 82.1% (78%) for macrophages. Agreement for the photographs was even higher. These results indicate that the above method is suitable for the evaluation of cutaneous cytology in the diagnosis and treatment monitoring of skin infections.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Spontaneous remission in canine generalized demodicosis – predisposing factors

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R. BRUZINSKAITE-SCHMIDHALTER and C. S. NETT-METTLER

Tierärztliche Spezialistenklinik, Hünenberg, Switzerland

Canine localized (LD) and generalized demodicosis (GD) are common parasitic skin diseases in veterinary practice. Miticidal therapy is used to treat GD, whereas LD is often self-curing. The aim of this study was to investigate spontaneous remission in GD and predisposing factors for such a remission. Records of 78 dogs with demodicosis were retrospectively analysed. Demodicosis was defined as GD when more than five focal areas were affected or an entire body region and/or one or more paws were involved. Dogs with GD (n = 34) either underwent systemic avermectin therapy (24 of 34) or were left untreated (10 of 34) based on various factors, including clinical impression of disease severity, persistence of disease and owner consent. Only local and/or systemic antimicrobial therapy as needed to treat concurrent bacterial infection was allowed in both groups. Dogs with GD treated with systemic avermectin therapy went into remission within 5 months on average compared with an average of 2 months in the untreated group. Twenty of 24 cases received systemic avermectin therapy at the time of diagnosis. In four of 24, spontaneous remission was not observed within 1–2 months after the initial visit, and miticidal therapy was subsequently initiated. Most cases with spontaneous remission showed young age of onset and small numbers of juvenile demodex stages. In summary, a subgroup of GD (29% of all GD cases) clinically behaves like LD. Prospective studies are needed to investigate these observations further.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Sebaceous gland dysplasia in two dogs

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J. PETERS-KENNEDY* and D. W. SCOTT†

* Departments of Biomedical Sciences andClinical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, NY, USA

Normal sebaceous glands are an integral component of healthy skin and have many important functions, including the excretion of sebum and normal hair growth. Sebaceous gland dysplasia is a rare condition of the dog and cat. Although little is known about this condition, it is thought that a genetic defect causes abnormal sebaceous gland development. Clinically, this condition occurs in juvenile dogs and cats and is characterized by adherent scale, hair casts, poor coat quality and progressive hypotrichosis. Successful treatment has not been described. We describe two adult dogs with a keratinization defect characterized by progressive scaling, hair casts, dull, dry, brittle hair coat and hypotrichosis beginning at several months of age. Histologically, all sebaceous glands were small to absent and composed of a mixture of mature sebocytes and basal cells. The basal cell population was often haphazardly arranged and irregularly clustered. Epidermal and follicular infundibular orthokeratotic hyperkeratosis were prominent features. Both dogs showed marked clinical improvement in the scaling, hair casts and hair coat quality with topical therapy consisting of antiseborrhoeic shampoo, moisturizing conditioner and propylene glycol and oral therapy including ω-6/ω-3 fatty acids and vitamin A. There was minimal improvement in the hypotrichosis. Although rare, sebaceous gland dysplasia should be considered in cases where a primary keratinization defect is suspected. As sebaceous gland dysplasia is a structural defect of the skin, long-term maintenance treatment is aimed at ameliorating the clinical signs rather than curing the disease.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Brooke–Spiegler-like syndrome: multiple trichoepitheliomas in a cat

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J. PIEPER*, M. SONG* and R. LIRTZMAN†

* Animal Dermatology PC, Phoenix, AZ, USAAnimal Specialty Group of Scottsdale, Scottsdale, AZ, USA

A 10-year-old neutered male, domestic short-haired cat presented with a 7 year history of multiple firm, raised nodules on the head, neck, dorsum and tail, ranging from 0.2 to 3 cm in diameter. Five masses were removed, and the three masses submitted for histopathology revealed trichoepithelioma with secondary bacterial infection (dorsal aspect of the head), epidermal inclusion cyst (ventral abdomen) and squamous carcinoma in situ (neck). Surgical excision has been repeated. Over 25 nodules have been removed to date; additional histopathology results are pending. The cat is otherwise healthy and has also been treated with imiquimod. Brooke–Spiegler syndrome is a rare disease in humans with a predisposition to develop cutaneous adnexal neoplasms such as trichoepitheliomas, cylindromas and spiradenomas. Brooke–Spiegler syndrome in humans has been identified as an autosomal dominant disorder caused by mutations of the cylindromatosis gene (CYLD), which is located on chromosome 16q12–13. Several studies have revealed that CYLD functions as a tumour-suppressor gene.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Terbinafine pharmacokinetics after single-dose oral administration in the dog

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M. R. SAKAI*, E. R. MAY*, S. A. CARLSON†, P. IMERMAN‡, C. FELZ*, T. A. DAY† and J. O. NOXON*

* Departments of Veterinary Clinical Sciences andBiomedical Sciences andVeterinary Diagnostic Laboratory, Iowa State University, Ames, IA, USA

Terbinafine is an allylamine antifungal that is prescribed for the treatment of mycoses in humans and has recently gained attention for use in veterinary patients. The purpose of this study was to evaluate the pharmacokinetic properties of terbinafine in dogs after a single oral dose. Ten healthy adult dogs were enrolled in the study. A single dose of terbinafine (∼30 mg/kg) was administered orally, and blood samples were periodically collected over a 24 h period during which dogs were monitored for adverse effects. No significant adverse effects were noted. An HPLC assay was developed and used to determine plasma terbinafine levels. Pharmacokinetic analysis was performed using PK Solutions® computer software. Area under the curve (AUC) from time 0 to 24 h (15.4 μg h/mL, range 5–27), maximal plasma concentration (Cmax; 3.5 μg/mL, range 3–4.9) and time to Cmax (tmax; 3.6 h, range 2–6) values were empirically deemed adequate for an oral drug. Statistical analysis indicated no difference between subjects in terms of AUC, Cmax or tmax values (P < 0.05). The time above minimal inhibitory concentration (t > MIC) as well as AUC/MIC was calculated for important invasive fungal pathogens and dermatophytes. The t > MIC was 17–18 h for Blastomyces dermatitidis, Histoplasma capsulatum and dermatophytes (Microsporum spp.and Trichophyton mentagrophytes), while the MIC for Sporothrix schenckii was exceeded for 9.5 h. The AUC/MIC values ranged from 9 to 13 μg h/mL for these fungi. Our results provide evidence supporting the use of terbinafine as an oral therapeutic agent for treating systemic and deep mycoses in the canine.

Source of funding: This study was funded by the Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA and research start-up funds provided by Iowa State University, Ames, IA, USA.

Conflict of interest: No conflicts of interest have been declared.

Cutaneous T-cell lymphoma in a dog exhibiting vasotropism and epitheliotropism

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G. A. CAMPBELL*, T. SHAPIRO-DUNLAP†, T. A. BROWN‡ and E. C. HODGIN§

* Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, OK, USACollierville Animal Clinic, Collierville, TN, USAMedVet Veterinary Specialty Hospital, Memphis, TN, USA § Pathodgin Veterinary Dermatopathology Service, Baton Rouge, LA, USA

A 12-year-old spayed female shar-pei dog was presented with diffuse erythema, crusting, pigmentary changes and friable skin following a meloxicam regimen. Yeast, bacterial rods and cocci were observed on skin scrapes. The patient was treated with antibiotics, antifungals, selamectin, corticosteroids, thyroxine and medicated baths, with no improvement over 9 weeks, and skin biopsy was performed. Initial diagnosis was severe periadnexal to mural granulomatous folliculitis, superficial perivascular to diffuse lymphohistiocytic dermatitis and ulceration. This was initially interpreted as follicle-centred erythema multiforme and possible drug reaction secondary to the meloxicam. The patient was referred to a veterinary dermatologist and rebiopsied. Severe follicular atrophy with less inflammation and apparent interface folliculitis was present. A trichogram revealed >90% of hairs in telogen. The dog continued to develop progressive skin lesions and oral lesions, with severe weight loss over 3 months. A liver mass was palpated, and the patient was humanely euthanized based on the poor condition and prognosis. The liver mass was compatible with haematoma. Examination of skin and oral cavity biopsy specimens revealed extensive infiltration of cutaneous vasculature by medium-to-large pale lymphocytes that were CD3 positive. There were CD3-positive cells infiltrating follicular epithelium, epidermis and oral mucosal epithelium. Examination of original sections revealed significant numbers of CD3-positive cells in the periadnexal and mural follicular infiltrates. This case represents both vasotropism and epitheliotropism in a cutaneous T lymphocyte malignancy. On initial presentation, no convincing vasotropism was seen. However, the vasculotropic neoplastic cells were probably affecting periadnexal vasculature.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Topical imiquimod in the treatment of two cutaneous melanocytomas in a dog

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K. COYNER* and D. LOEFFLER†

* Dermatology Clinic for Animals, Las Vegas, NV, USASouthwest Dermpath, Gilbert, AZ, USA

Imiquimod is a topical biological response modifier, which has been shown to be useful in the treatment of viral and nonviral skin tumours in humans and animals. Lentigo maligna (melanoma in situ) is common in older humans, and can be successfully treated with imiquimod. Melanocytomas are common skin tumours in dogs, and although surgical removal is the treatment of choice, tumour location and patient morbidity can make excision difficult. This case report describes the use of imiquimod (applied 3 days/week for 3–4 weeks) to treat two melanocytomas in a dog. Initial inflammation and crusting was followed by a decrease in both vertical tumour size and pigmentation. Dermatohistopathology demonstrated an interface lymphoid infiltrate obscuring the dermo-epidermal junction, a lymphocytic epidermal infiltrate, occasional apoptotic cells, loss of epidermal melanocytic cells, and a moderate dermal superficial lichenoid and perifollicular lymphocytic infiltrate admixed with plasma cells, neutrophils and large melanin-containing cells. Immunohistochemistry revealed a mixed T cell (CD4, CD8ab, TCRab) and B cell (CD79a) lymphocytic infiltrate with melanin-laden macrophages (CD18). Numerous active dermal dendritic cells were present (CD18, CD1, CD11c, CD4). Some dendritic-shaped, pigmented cells lacked expression of leukocyte markers and were interpreted as melanocytes, but it was not clear whether the cells were residual neoplastic melanocytes or activated dermal melanocytes. To the best of the authors’ knowledge, this is the first report of the use of imiquimod to treat canine melanocytomas, and the first description of post-treatment histopathology and immunohistochemistry after imiquimod use in companion animals.

Source of funding: This study is self-funded.

Conflict of interest: No conflicts of interest have been declared.

Posters

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Assessment and comparison of reference intervals for mast cells in peripheral lymph nodes of normal dogs and dogs with allergic skin disease

C. L. BAUER*, P. M. RAKICH†, K. S. LATIMER‡, A. WHITE§ and P. HENSEL†

* Southeast Veterinary Dermatology & Ear Clinic, Greenville, SC, USAUniversity of Georgia, College of Veterinary Medicine, Athens, GA, USACovance Laboratories, Inc., Vienna, VA, USA § Auburn University, College of Veterinary Medicine, Auburn, AL, USA

Clinical staging of dogs with mast cell tumours (MCTs) involves cytological examination of regional lymph nodes (LNs) for evidence of metastasis. Mast cells (MCs) also play an important role in allergic skin conditions, which have been suspected to heighten the occurrence of MCTs. Lymph nodes may normally contain MC, but reference values for MC counts in LNs of normal dogs or dogs with allergic skin diseases are not established. The aim of this study was to determine cytological reference intervals for MCs in fine-needle aspirates of LNs of normal dogs and compare them with those of dogs with allergic skin disease. Reference intervals were determined using aspirates of the prescapular and popliteal LNs from 30 healthy dogs and compared with MC counts from 20 allergic dogs. The following three methods were used to assess the MC counts: ‘fields’ (number of MCs counted in 20 fields); ‘cells’ (number of MCs/500 lymphoid cells); and ‘total’ (total number of MCs/entire smear). Mast cells were found in LN aspirates from five of 30 healthy dogs and from 15 of 20 allergic dogs. Reference intervals for MCs in normal dogs were as follows: ‘fields’ = 0–2 (mean 0.2); ‘cells’ = 0–1 (mean 0.07); and ‘total’ = 0–13 (mean 2.97). The intervals for the allergic dogs were as follows: ‘fields’ = 0–3 (mean 0.5); ‘cells’ = 0–1 (mean 0.2); and ‘total’ = 0–84 (mean 13.5). The numbers of MCs were significantly higher in the allergic dog group (P = 0.0005). These findings indicate that the interpretation of LN aspirates in clinical staging of MCTs may be complicated in dogs with MCTs and concurrent allergic skin disease.

Source of funding: This study was supported by the Companion Animal Clinical Research Award, University of Georgia.

Conflict of interest: No conflicts of interest have been declared.

Cell cultures of keratinocytes harvested from atopic Beagle dogs and investigation of filaggrin expression

R. MARSELLA, K. AHRENS

Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL, USA

Several studies have investigated cell cultures of keratinocytes in normal dogs, but no study has been done on cell cultures from canine atopic skin. Decreased filaggrin expression has been reported in human atopic dermatitis. No information exists on filaggrin expression in atopic canine keratinocytes in cell culture. Skin biopsies from atopic beagle dogs were taken from the shaved inguinal area. Samples were initially placed in Dispase overnight at 4°C. Epithelium was teased from dermis and placed in trypsin–EDTA. Neutralizing solution was added, and cells were resuspended and filtered through sterile 40 μm mesh and placed in culture flask containing serum-free keratinocyte growth media (KGM; Lonza-Clonetics). Cholera toxin and fetal bovine serum were added to encourage growth. Cells were plated into chamber slides and fed every 2 days. After 6 days, cells had adhered, flattened and spread in cobblestone clusters. By day 14, cells were confluent on glass chamber slides or were subcultured from the flask. Cells differentiated with CaCl2 for 5 days were fixed in cold 3.7% paraformaldehyde in phosphate-buffered saline, washed and stored in a refridgerator. Cells were stained with cytokeratin 10 monoclonal antibody to human and canine, with anti-canine cytokeratin polyclonal antibody and a polyclonal antibody against canine filaggrin peptide sequence. It was found that cell culture can be established in atopic keratinocytes (1 × 105 cells per 8 mm biopsy), and filaggrin was visualized using the antibody produced against the canine peptide. Before addition of CaCl2, filaggrin was detectable in perinuclear aggregates and after differentiation with addition of CaCl2, filaggrin organized in cytoplasmic filaments.

Source of funding: European Society of Veterinary Dermatology.

Conflict of interest: No conflicts of interest have been declared.