The efficacy of subcutaneous slow-release melatonin implants in the prevention of canine flank alopecia recurrence is uncertain: A double-blind, randomized, placebo-controlled study.

BACKGROUND: Canine flank alopecia (CFA) is characterized by seasonally recurring noninflammatory, occasionally hyperpigmented alopecia predominantly in the thoracolumbar area. Previous studies suggest that reduced production of endogenous melatonin may play a role in the pathogenesis of this condition, and placebo-controlled studies on the efficacy of preventative melatonin treatment are lacking. OBJECTIVE: To evaluate the efficacy of subcutaneous slow-release melatonin implants in the prevention of CFA recurrence. ANIMALS: Twenty-one client-owned dogs with a history of CFA were included in the study. MATERIALS AND METHODS: At time (T)0, a general physical and dermatological examination was performed on each dog, blood was collected for serum biochemistry analysis and two skin biopsies were taken from alopecic areas on the nonsedated affected dogs after subcutaneous injection with 2% lidocaine. Dogs with normal blood work and histological results compatible with CFA were included in the study. Participating dogs were randomly assigned to receive either placebo or 18 mg melatonin subcutaneously in the interscapular area, approximately 2 months before expected CFA onset (T1). CFA recurrence was scored qualitatively as complete, ≤50% recurrence, or no recurrence at 5 and 7 months after the intervention (T2 and T3, respectively). RESULTS: At T3, in dogs treated with placebo (nine of 17), the percentages for complete recurrence, ≤50% recurrence and no recurrence were 44%, 0% and 56%, respectively. In dogs treated with melatonin (eight of 17), these percentages were 25%, 50% and 25%, respectively. There were no statistically significant differences in the scores between melatonin-treated dogs and placebo-treated dogs (p = 0.40). In three of eight melatonin-treated dogs, mild transient swelling was observed at the injection site. CONCLUSIONS: This study did not provide evidence that an 18 mg melatonin implant treatment, although well-tolerated, is efficacious in preventing recurrence of CFA in affected dogs.

Efficacy of subcutaneous allergen immunotherapy in atopic dogs: A retrospective study of 664 cases.

BACKGROUND: Canine atopic dermatitis (cAD) is an allergic skin disease affecting approximately 10% of dogs. allergen-specific immunotherapy (ASIT) is currently the only treatment option able to induce tolerance to the causative allergens. OBJECTIVE: To retrospectively establish the efficacy of ASIT in atopic dogs. ANIMALS: Client-owned (n  = 664) dogs with cAD presented between 2008 and 2018 to two dermatology referral clinics. MATERIALS AND METHODS: Clinical records of atopic dogs were reviewed to obtain information including the results of the intradermal skin test and/or allergen-specific immunoglobulin (Ig)E serological results, the allergens included in the ASIT, concurrent symptomatic medications, and ASIT efficacy after at least 9 months. RESULTS: Excellent (ASIT alone controlled clinical signs), good (≥50% reduction of clinical signs) and poor (<50% improvement) responses were seen in 31.5%, 28.5% and 40.1% of the dogs, respectively. No significant differences in efficacy were associated with breed, sex, age at initiation of ASIT, type of allergens in ASIT, and between clinics. Dogs re-examined regularly responded significantly better to ASIT than dogs that did not (>50% improvement in 69.3% and 55.4% of the dogs, respectively). Dogs treated with ASIT and concomitant systemic glucocorticoids showed a significantly poorer response (success rate of >50% improvement of 38.5%). CONCLUSIONS AND CLINICAL IMPORTANCE: In 59.9% of atopic dogs, subcutaneous ASIT can improve clinical signs by ≥50%. The beneficial effect of ASIT is higher if dogs are re-examined regularly and if systemic long-term corticosteroids are avoided, at least during the first 9 months of ASIT.

Contexte - La dermatite atopique canine (DAC) est une maladie cutanée allergique qui touche environ 10 % des chiens. L'immunothérapie allergénique (ASIT) est actuellement la seule option thérapeutique capable d'induire une tolérance aux allergènes responsables. Objectif - Établir rétrospectivement l'efficacité de l'ASIT chez les chiens atopiques. Animaux - Chiens de clients (n = 664) atteints de DAC présentés entre 2008 et 2018 à deux cliniques de référé en dermatologie. Méthodes - Les dossiers cliniques des chiens atopiques ont été examinés pour obtenir des informations, notamment les résultats du test cutané intradermique et / ou les résultats sérologiques de l'immunoglobuline (Ig) E spécifique d'allergène, les allergènes inclus dans l'ASIT, les médicaments symptomatiques concomitants et l'efficacité de l'ASIT après au moins neuf mois. Résultats - Des réponses excellentes (signes cliniques contrôlés par l'ASIT seul), bonnes (réduction ≥ 50 % des signes cliniques) et médiocres (amélioration < 50 %) ont été observées chez 31,5 %, 28,5 % et 40,1 % des chiens, respectivement. Aucune différence significative d'efficacité n'a été associée à la race, au sexe, à l'âge au début de l'ASIT, au type d'allergènes dans l'ASIT et entre les cliniques. Les chiens réexaminés régulièrement ont répondu significativement mieux à l'ASIT que les chiens qui ne l'ont pas fait (amélioration > 50 % chez 69,3 % et 55,4 % des chiens, respectivement). Les chiens traités avec l'ASIT et des glucocorticoïdes systémiques concomitants ont montré une réponse significativement plus faible (taux de réussite > 50 % d'amélioration de 38,5 %). Conclusions et importance clinique - Chez 59,9 % des chiens atopiques, l'ASIT sous-cutanée peut améliorer les signes cliniques de ≥ 50 %. L'effet bénéfique de l'ASIT est plus élevé si les chiens sont réexaminés régulièrement et si les corticostéroïdes systémiques à long terme sont évités, au moins pendant les neuf premiers mois de l'ASIT.

Introducción- la dermatitis atópica canina (cAD) es una enfermedad alérgica de la piel que afecta aproximadamente al 10 % de los perros. La inmunoterapia con alérgenos (ASIT) es actualmente la única opción de tratamiento capaz de inducir tolerancia a los alérgenos causantes. Objetivo- establecer retrospectivamente la eficacia de ASIT en perros atópicos. Animales- perros de propietarios particulares (n=664) con cAD presentados entre 2008 y 2018 a dos clínicas de referencia de dermatología. Métodos- se revisaron las historias clínicas de los perros atópicos para obtener información, incluidos los resultados de la prueba cutánea intradérmica y/o los resultados serológicos de la inmunoglobulina (Ig)E específica para alérgenos, los alérgenos incluidos en el ASIT, los medicamentos sintomáticos concurrentes y la eficacia del ASIT después de al menos menos nueve meses. Resultados- se observaron respuestas excelentes (la ASIT sola controló los signos clínicos), buenas (≥50 % de reducción de los signos clínicos) y malas (<50 % de mejora) en el 31,5 %, 28,5 % y 40,1 % de los perros, respectivamente. No se asociaron diferencias significativas en la eficacia con la raza, el sexo, la edad al inicio de la ASIT, el tipo de alérgenos en la ASIT y entre las clínicas. Los perros reexaminados con regularidad respondieron significativamente mejor a la ASIT que los perros que no lo hicieron (>50 % de mejora en el 69,3 % y el 55,4 % de los perros, respectivamente). Los perros tratados con ASIT y glucocorticoides sistémicos concomitantes mostraron una respuesta significativamente peor (tasa de éxito de >50 % de mejora del 38,5 %). Conclusiones e importancia clínica - En el 59,9% de los perros atópicos, la ASIT subcutánea puede mejorar los signos clínicos en ≥50%. El efecto beneficioso de la ASIT es mayor si los perros se vuelven a examinar con regularidad y si se evitan los corticosteroides sistémicos a largo plazo, al menos durante los primeros nueve meses de la ASIT.

Contexto - A dermatite atópica canina (DAC) é uma doença alérgica que afeta aproximadamente 10% dos cães. A imunoterapia alérgeno-específica (ASIT) é atualmente a única opção terapêutica capaz de induzir tolerância aos alérgenos causadores. Objetivo - Estabelecer retrospectivamente a eficácia da ASIT em cães atópicos. Animais - Cães de proprietários (664) com DAC apresentados a duas clínicas de referência em dermatologia entre 2008 e 2018. Métodos - Os prontuários dos cães atópicos foram revisados para se obter informações incluindo os resultados dos testes intradérmicos e/ou sorológicos para anticorpos (IgE) alérgeno-específicos, os alérgenos incluídos na ASIT, medicações sintomáticas concomitantes, e a eficácia de ASIT após um mínimo de nove meses. Resultados - Resposta excelente (ASIT controlou os sinais clínicos sozinha), boa (redução ≥50% nos sinais clínicos) e pobre (<50% de melhora) foi observada em 31,5%, 28,5% e 40,1% dos cães, respectivamente. Não foram observadas diferenças significativas na eficácia em relação a raça, gênero, idade à iniciação da ASIT, tipo de alérgenos na ASIT e entre as clínicas. Os cães reavaliados periodicamente responderam significativamente melhor à ASIT que cães que não foram reavaliados com frequência (>50% de melhora em 69,3% e 55,4% dos cães, respectivamente). Cães tratados com ASIT e glicocorticoides sistêmicos concomitantemente demonstraram uma resposta significativamente mais pobre (a taxa de sucesso >50% ocorreu em 38,5%). Conclusões e importância clínica - Em 59,9% dos cães atópicos, ASIT subcutânea é capaz de melhorar os sinais clínicos em ≥50%. O efeito benéfico de ASIT foi maior se os cães fossem reavaliados regularmente e se corticoides sistêmicos a longo prazo fossem evitados, ao menos durante os primeiros meses de ASIT.

Clindamycin resistance of skin derived Staphylococcus pseudintermedius is higher in dogs with a history of antimicrobial therapy.

BACKGROUND: In the Netherlands there is a lack of data regarding resistance of Staphylococcus pseudintermedius to the systemic antimicrobial drugs used for the treatment of superficial pyoderma. OBJECTIVES: To assess antimicrobial resistance, with emphasis on resistance to clindamycin and meticillin, in clinical isolates of S. pseudintermedius isolated from dogs with superficial pyoderma. Results were compared between dogs with and without a history of systemic antimicrobial therapy during the previous year. ANIMALS: A retrospective study of 237 referral cases presented to an academic teaching hospital between 2014 and 2019, with the clinical and microbiological diagnosis of superficial pyoderma. METHODS AND MATERIALS: All clinical isolates were identified primarily by MALDI-TOF mass spectrometry. Antimicrobial susceptibility was tested either by an agar diffusion method (2014-2016) or by broth microdilution. Antimicrobial history in the preceding year was obtained from medical records. RESULTS: Meticillin-resistant S. pseudintermedius (MRSP) was isolated from 8% of superficial pyoderma cases. Within the meticillin-susceptible S. pseudintermedius (MSSP) population, clindamycin resistance was significantly more common in isolates derived from dogs with histories of antimicrobial treatment (37.7%) compared to dogs with no histories of exposure (21.7%; P = 0.03). CONCLUSIONS: Given the high prevalence of clindamycin resistance in MSSP isolated from dogs with prior antimicrobial exposure, it is recommended that bacterial culture and susceptibility testing be pursued before prescribing systemic antimicrobials. Clindamycin should be regarded as the preferred treatment option if susceptibility is confirmed, due to its narrow spectrum and reduced selective pressure for MRSP.

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study.

Canine atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease that is often complicated by (secondary) bacterial and fungal (yeast) infections. High-throughput DNA sequencing was used to characterize the composition of the microbiome (bacteria and fungi) inhabiting specific sites of skin in healthy dogs and dogs with atopic dermatitis (AD) before and after topical antimicrobial treatment. Skin microbiome samples were collected from six healthy control dogs and three dogs spontaneously affected by AD by swabbing at (non-) predilection sites before, during and after treatment. Bacteria and fungi were profiled by Illumina sequencing of the 16S ribosomal RNA gene of bacteria (16S) and the internally transcribed spacer of the ribosomal gene cassette in fungi (ITS). The total cohort of dogs showed a high diversity of microbes on skin with a strong individual variability of both 16S and ITS profiles. The genera of Staphylococcus and Porphyromonas were dominantly present both on atopic and healthy skin and across all skin sites studied. In addition, bacterial and fungal alpha diversity were similar at the different skin sites. The topical antimicrobial treatment increased the diversity of bacterial and fungal compositions in course of time on both AD and healthy skin.

A canine keratinocyte cell line expresses antimicrobial peptide and cytokine genes upon stimulation with bacteria, microbial ligands and recombinant cytokines.

Keratinocytes (KC) are the main cellular components of the stratum corneum that constitutes a solid physical skin barrier representing the first line of defense against pathogens. Moreover, KC are potent producers of inflammatory mediators and antimicrobial peptides (AMP) when activated through their pattern recognition receptors. In atopic dermatitis (AD) the protective skin barrier may be compromised due to barrier disruption, secondary infection and accelerated secretion of inflammatory cytokines which may also affect AMP expression in the skin. In the present study, we addressed the responses of a canine KC cell line upon exposure to Staphylococcus pseudintermedius, typically found on canine atopic skin during secondary infections, and stimulation by individual AD-associated ligands and cytokines. All stimuli induced a significant increase in expression of the pro-inflammatory cytokine genes tumor necrosis factor (TNF)-α and interleukin (IL)-8, but with different kinetics. Limited effects were observed on AMP gene expression except for K9CATH which was significantly upregulated upon bacterial infection but with none of the individual AD-associated ligands. Interestingly, K9CATH possessed antimicrobial activity towards Staphylococcus pseudintermedius, indicating that K9CATH expression is a specific defense reaction towards bacterial infection and not part of a general pro-inflammatory profile of KC.

Altered lipid properties of the stratum corneum in Canine Atopic Dermatitis.

Skin barrier disruption plays a role in the pathogenesis of atopic dermatitis (AD) in humans. However, little is known about skin barrier (dys-) function in Canine Atopic Dermatitis. The properties of lipids located in the outermost layer of the skin, the stratum corneum (SC) are considered to be important for the barrier. In the present study the lipid composition and lipid organization of the SC of AD dogs and control dogs were examined. The lipid composition of lesional AD skin as compared to control skin, showed a reduced free fatty acid level and a decreased ratio of ceramide[NS] C44/C34, in which C44 and C34 are the total numbers of carbon atoms of the sphingosine (S) and non-hydroxy (N) acyl chains. As a consequence of the observed changes in lipid composition in AD lesional skin the lamellar organization of lipids altered and a shift from orthorhombic to hexagonal lipid packing was monitored. Simultaneously an increased conformational disordering occurred. These changes are expected to compromise the integrity of the skin barrier. The C44/C34 chain length ratio of ceramide[NS] also showed a decreasing nonlinear relationship with the AD severity score (CADESI). Taken together, canine atopic skin showed alterations in SC lipid properties, similar to the changes observed in atopic dermatitis in humans, that correlated with a disruption of the skin barrier. Hence lipids play an important role in the pathogenesis of Canine Atopic Dermatitis.

Disseminated Candidiasis in a Young, Previously Healthy, Dog and Review of Literature.

BACKGROUND: The reports on disseminated candidiasis in dogs so far describe at least one predisposing factor. This case report, however, highlights candidiasis in a dog without any known predisposition. PATIENT: A 1.5-year-old intact female Hovawart dog was presented with subcutaneous nodules and polyuria/polydipsia. An excisional biopsy revealed a chronic pyogranulomatous and necrotizing inflammation with mycotic structures. The patient became febrile and lethargic, and developed lameness. METHODS: A physical examination, blood tests, urinalysis, thoracic radiographs, abdominal ultrasonography of the abdomen, fine-needle aspiration biopsies, and a culture of a subcutaneous nodule aspirate were obtained. Selected sections of multiple organs were collected for routine histology postmortem. The isolate and a subcutaneous mass were subjected to molecular identification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. RESULTS: Clinical, laboratory, and radiological findings were consistent with a granulomatous chronic systemic inflammation. Cytology and histology showed a pyogranulomatous and necrotizing inflammation with myriads of intra- and extra-cellular yeasts and extracellular hyphae. Culture yielded numerous yeast colonies, which appeared Candida albicans-like, but showed a negative serum test and a low identification in API 20 C AUX. Nucleic acid sequences showed homology with the C. albicans-type strain CBS 562. Multilocus sequence typing (MLST) resulted in a new type with designation DST121. The identification of the isolates was confirmed by MALDI-TOF-MS analysis. CONCLUSION AND CLINICAL IMPORTANCE: Future MLST typing and investigation of virulence can provide further evidence whether this MLST-type is associated with clinical cases of disseminated candidiasis without an apparent predisposing condition.

Lesional skin in atopic dogs shows a mixed Type-1 and Type-2 immune responsiveness.

Canine atopic dermatitis (AD) is a chronic inflammatory and pruritic skin disease which shares several characteristics with its human counterpart. In chronic patch test lesions of human with AD mainly a Th1-type cellular response is found. Besides, non-lesional AD skin is already skewed for inflammation and therefore different from healthy skin. The goal of this study was to characterize local immune responsiveness in chronic canine AD lesions as compared to that in non-lesional AD skin by defining T cell subset relevant cytokine- and transcription factor expression profiles. The gene expression of the Th1 cytokines IL-12p35, IL-12p40 and IFN-γ and their related transcription factors STAT4, SOCS5 and T-bet, the Th2 cytokines IL-4 and IL-13 and transcription factors STAT6, SOCS3 and GATA-3 and the regulatory cytokines IL-10 and TGF-ß and the transcription factor FOXP3 was evaluated in healthy control and atopic dogs. In non-lesional (NLS) and chronic lesional skin (LS) of atopic dogs and control skin (CS) from healthy dogs mRNA expression of cytokines and transcription factors were measured by quantitative real-time PCR. Significantly different values were found for the following factors: IL-12p40 mRNA was lower in LS when compared to NLS. Expression of STAT4 was higher in LS compared to CS and NLS. More IL-13 and SOCS3 were found in LS and NLS when compared to CS and also in LS compared to NLS. GATA-3 was lower in LS compared to NLS. IL-10 expression was higher in both LS and NLS compared to CS and more IL-10 was present in LS compared to NLS. These findings indicate that both Th1- and Th2-type as well as T regulatory cells are present in NLS and LS in canine atopic skin.

Characterisation of T cell phenotypes, cytokines and transcription factors in the skin of dogs with cutaneous adverse food reactions.

The immunopathogenesis of cutaneous adverse food reactions (CAFRs) in dogs is unknown. Since the clinical manifestations in the skin are like those found in canine atopic dermatitis (AD), this study investigated the similarity in T cell phenotypes and gene expression of cytokines and transcription factors in CAFRs. In addition, the influence of an elimination diet on these parameters was tested. In the skin of canine CAFRs, a predominant presence of CD8(+) T cells and increased expression of the IL-4, IL-13, Foxp3 and SOCS-3 genes were observed. IFN-γ gene expression was increased in lesional compared to non-lesional skin. The predominance of CD8(+) T cells indicates that the immunopathogenesis of CAFRs is different from that of canine AD. The elimination diet relieved clinical signs, but did not influence T cell phenotypes or expression of the cytokine and transcription factor genes in the skin of dogs with CAFRs, indicating a continuously pre-activated immune status in dogs sensitised to food constituents.

Enzymes involved in the conversion of arachidonic acid to eicosanoids in the skin of atopic dogs.

Canine atopic dermatitis (AD), a chronic inflammatory skin disease, shares characteristics with its human counterpart. To get insight into the role of enzymes involved in production of prostaglandin E(2) (PGE2) and leukotriene B(4) (LTB(4)), potent inflammatory mediators originating from membrane-derived arachidonic acid (AA), expression of genes encoding these enzymes and receptors was quantified by qPCR in non-lesional and lesional skin from atopic dogs and in healthy skin. Significantly higher mRNA expression of the key enzymes 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP), leukotriene A(4) hydrolase (LTA(4)H) and prostaglandin E synthase 1 (mPGES-1) and their receptors (PGE receptors 2 and 3) were observed. Being responsible for elevated levels of metabolites of the 3-series prostaglandins and the 5-series leukotrienes these enzymes may be interesting targets for therapy that should result in amelioration of clinical signs in canine atopic dermatitis.

A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis.

Quantitative real time PCR (Q-PCR) is the method of choice to study mRNA expression levels. Since Q-PCR is very sensitive, normalization of the data with stably expressed reference genes if of utmost importance. The stability of reference genes depends on the tissue and the species of interest. Therefore, evaluation of the stability of reference genes must be performed for each new tissue and species under study. The stability of B2M, GAPDH, HPRT, SRPR, hnRNPH, GUSB, RPL8, RPS5, and RPS19 was analyzed with the GeNorm software in snap frozen canine skin biopsies. Healthy dogs (n=7) and dogs with confirmed atopic dermatitis (n=28) were included. Lesional and non-lesional skin was analyzed. The study indicated that the most appropriate reference genes in canine skin are the ribosomal gene products RPL8, RPS5 and RPS19 besides GUSB and HPRT. As little as three reference genes will reveal highly reliable Q-PCR calculations.

Two canine CD1a proteins are differentially expressed in skin.

Lipid antigens are presented to T cells by the CD1 family of proteins. In this study, we characterize the complete dog (Canis familiaris) CD1 locus, which is located on chromosome 38. The canine locus contains eight CD1A genes (canCD1A), of which five are pseudogenes, one canCD1B, one canCD1C, one canCD1D, and one canCD1E gene. In vivo expression of canine CD1 proteins was shown for canCD1a6, canCD1a8, and canCD1b, using a panel of anti-CD1 monoclonal antibodies (mAbs). CanCD1a6 and canCD1a8 are recognized by two distinct mAbs. Furthermore, we show differential transcription of the three canCD1A genes in canine tissues. In canine skin, the transcription level of canCD1A8 was higher than that of canCD1A6, and no transcription of canCD1A2 was detected. Based on protein modeling and protein sequence alignment, we predict that both canine CD1a proteins can bind different glycolipids in their groove. Besides differences in ectodomain structure, we observed the unique presence of three types of cytoplasmic tails encoded by canCD1A genes. cDNA sequencing and expressed sequence tag sequences confirmed the existence of a short, human CD1a-like cytoplasmic tail of four amino acids, of an intermediate length form of 15 amino acids, and of a long form of 31 amino acids.