Abnormal keratinocyte differentiation in the nasal planum of Labrador Retrievers with hereditary nasal parakeratosis (HNPK).

Hereditary nasal parakeratosis (HNPK) is an inherited disorder described in Labrador Retrievers and Greyhounds. It has been associated with breed-specific variants in the SUV39H2 gene encoding a histone 3 methyltransferase involved in epigenetic silencing. Formalin-fixed biopsies of the nasal planum of Labrador Retrievers were screened by immunofluorescence microscopy for the presence and distribution of epidermal proliferation and differentiation markers. Gene expression of these markers was further analysed using RNA sequencing (RNA-seq) and ultrastructural epidermal differences were investigated by electron microscopy. Differentiation of the nasal planum in the basal and suprabasal epidermal layers of HNPK-affected dogs (n = 6) was similar compared to control dogs (n = 6). In the upper epidermal layers, clear modifications were noticed. Loricrin protein was absent in HNPK-affected nasal planum sections in contrast to sections of the same location of control dogs. However, loricrin was present in the epidermis of paw pads and abdominal skin from HNPK dogs and healthy control dogs. The patterns of keratins K1, K10 and K14, were not markedly altered in the nasal planum of HNPK-affected dogs while the expression of the terminal differentiation marker involucrin appeared less regular. Based on RNA-seq, LOR and IVL expression levels were significantly decreased, while KRT1, KRT10 and KRT14 levels were up-regulated (log2fold-changes of 2.67, 3.19 and 1.71, respectively) in HNPK-affected nasal planum (n = 3) compared to control dogs (n = 3). Electron microscopical analysis revealed structural alterations in keratinocytes and stratum corneum, and disrupted keratinocyte adhesions and distended intercellular spaces in lesional samples (n = 3) compared to a sample of a healthy control dog (n = 1). Our findings demonstrate aberrant keratinocyte terminal differentiation of the nasal planum of HNPK-affected Labrador Retrievers and provide insights into biological consequences of this inactive SUV39H2 gene variant.

FAM83G/Fam83g genetic variants affect canine and murine hair formation.

FAM83G/Fam83g genetic variants have been described in dogs, mice and recently also in humans. They are associated with palmoplantar keratoderma and altered hair or coat phenotype, reported as wooly phenotype in mice. FAM83G/Fam83g is an unexplored effector of temporally and spatially coordinated Wnt and BMP signalling which are key pathways in pre- and postnatal hair follicle morphogenesis and differentiation. The aim of this study was to unravel phenotypic consequences of FAM83G/Fam83g variants on hair coat formation in dogs and mice. Our results show differences in hair types and hair shaft structures in both species. Additionally, mice exhibit deregulated hair cycle progression which timely correlates with defective Wnt signalling (Axin2) and Bmp2/4 expression. These results affirm the involvement of FAM83G in hair morphogenesis, hair follicle differentiation and cycling.

Macrococcus canis and M. caseolyticus in dogs: occurrence, genetic diversity and antibiotic resistance.

BACKGROUND: The discovery of a new Macrococcus canis species isolated from skin and infection sites of dogs led us to question if Macrococcus spp. are common in dogs and are resistant to antibiotics. HYPOTHESIS/OBJECTIVES: To evaluate the occurrence of Macrococcus spp. in dogs, determine antibiotic resistance profiles and genetic relationships. ANIMALS: One hundred and sixty two dogs (mainly West Highland white terriers and Newfoundland dogs) were screened for the presence of Macrococcus, including six dogs with Macrococcus infections. METHODS: Samples were taken from skin, ear canal and oral mucosa using swabs. Macrococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, 16S rRNA sequencing and nuc-PCR. Minimal inhibitory concentrations of 19 antibiotics were determined using broth microdilution. Resistance mechanisms were identified by microarray and sequencing of the fluoroquinolone-determining region of gyrA and grlA. Sequence type (ST) was determined by multilocus sequence typing. RESULTS: Out of the 162 dogs, six harboured M. caseolyticus (n = 6) and 13 harboured M. canis (n = 16). Six isolates of M. canis and one of M. caseolyticus were obtained from infection sites. The 22 M. canis strains belonged to 20 different STs and the seven M. caseolyticus strains to three STs. Resistance to antibiotics was mostly associated with the detection of known genes, with mecB-mediated meticillin resistance being the most frequent. CONCLUSION AND CLINICAL IMPORTANCE: This study gives some insights into the occurrence and genetic characteristics of antibiotic-resistant Macrococcus from dogs. Presence of M. canis in infection sites and resistance to antibiotics emphasized that more attention should be paid to this novel bacteria species.

Chronological clinical and pathological documentation of porcine ulcerative dermatitis.

BACKGROUND: Porcine ulcerative dermatitis syndrome (PUDS) is a rare disease of breeding sows with an unknown pathogenesis. OBJECTIVE: To describe the evolution of clinical and histopathological lesions over the course of the disease and to elucidate the pathogenesis. ANIMAL: A 24-month-old, pluriparous, large white sow presented during gestation with ulcerations around the teats compatible with PUDS. METHODS AND RESULTS: Clinical and histopathological lesions were monitored over the course of the disease (i.e. during and after the subsequent pregnancy). A clear gestation-dependent flare of the lesions was observed with partial resolution occurring postpartum. The histological pattern presented as a lymphocytic interface dermatitis. CONCLUSIONS AND CLINICAL IMPORTANCE: The findings in this case report link gestation with the development of clinical signs and histological changes. Multiparity appears to enhance severity and may finally result in a self-perpetuating disease. Therefore, it seems advisable to cull breeding sows after they have developed PUDS.

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria.

BACKGROUND: Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. HYPOTHESIS/OBJECTIVE: The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. ANIMALS: Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. METHODS: The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B ells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T-helper 2 cytokines (interleukins IL-4, IL-5 and IL-13), a T-helper 1 cytokine (interferon-γ), IL-4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT-PCR. Results - In subepidermal lesional skin of RU-affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79-positive (P ≤ 0.01), MAC387-positive (P ≤ 0.01) and tryptase-positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU-affected horses contained more eosinophils (P ≤ 0.05) and tryptase-positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL-4 (P ≤ 0.01), IL-13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL-4 receptor α (P ≤ 0.05) was increased in lesional skin of RU-affected horses compared with control horses. Expression of IL-4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. CONCLUSIONS AND CLINICAL IMPORTANCE: Analysis of cytokine expression and inflammatory infiltrate suggests that T-helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry.

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.

Unusual systemic signs in a dog with sterile neutrophilic-macrophagic lymphadenitis and nodular panniculitis.

A 7 mo old female English springer spaniel was presented with diarrhea, vomiting, apathy, and hyperthermia. Further examinations revealed generalized lymphadenomegaly consistent with sterile neutrophilic-macrophagic lymphadenitis and pulmonary involvement. Subcutaneous nodules developed one day after presentation. Histology was consistent with sterile idiopathic nodular panniculitis and vasculitis. No infectious organism was isolated. The dog responded to prednisolone, but relapsed during medication tapering. Cyclosporine had to be added to control the disease. No further relapse had occurred 98 wk after the first presentation. This is an unusual presentation of a systemic sterile neutrophilic-macrophagic lymphadenitis with nodular panniculitis and vasculitis associated with gastrointestinal and pulmonary signs.

Combined moxidectin and environmental therapy do not eliminate Chorioptes bovis infestation in heavily feathered horses.

Chorioptes bovis infestation is a common cause of pastern dermatitis in the horse, with a predilection in draft horses and other horses with thick hair 'feathers' on the distal limbs. The treatment of this superficial mite is challenging; treatment failure and relapse are common. Furthermore, C. bovis infestation may affect the progression of chronic pastern dermatitis (also known as chronic proliferative pastern dermatitis, chronic progressive lymphoedema and dermatitis verrucosa) in draft horses, manifesting with oedema, lichenification and excessive skin folds that can progress to verruciform lesions. An effective cure for C. bovis infestation would therefore be of great clinical value. In a prospective, double-blind, placebo-controlled study, the efficacy of oral moxidectin (0.4 mg/kg body weight) given twice with a 3 week interval in combination with environmental treatment with 4-chloro-3-methylphenol and propoxur was tested in 19 heavily feathered horses with clinical pastern dermatitis and C. bovis infestation. Follow-up examinations over a period of 180 days revealed significantly more skin crusting in the placebo group than in the treatment group. However, no other differences in clinical signs or the numbers of mites detected were found between the two groups. The results of this study suggest that moxidectin in combination with environmental insecticide treatment as used in this study is ineffective in the treatment of C. bovis in feathered horses.

Missing C-terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis.

Filaggrin loss-of-function mutations resulting in C-terminal protein truncations are strong predisposing factors in human atopic dermatitis (AD). To assess the possibility of similar truncations in canine AD, an exclusion strategy was designed on 16 control and 18 AD dogs of various breeds. Comparative immunofluorescence microscopy was performed with an antibody raised against the canine filaggrin C-terminus and a commercial N-terminal antibody. Concurrent with human AD-like features such as generalized NFKB activation and hyperproliferation, four distinctive filaggrin expression patterns were identified in non-lesional skin. It was found that 10/18 AD dogs exhibited an identical pattern for both antibodies with comparable (category I, 3/18) or reduced (category II, 7/18) expression to that of controls. In contrast, 4/18 dogs displayed aberrant large vesicles revealed by the C-terminal but not the N-terminal antibody (category III), while 4/18 showed a control-like N-terminal expression but lacked the C-terminal protein (category IV). The missing C-terminal filaggrin in category IV strongly points towards loss-of function mutations in 4/18 (22%) of all AD dogs analysed.

Alopecia areata in Eringer cows.

Alopecia areata is a hair loss disorder in humans, dogs and horses with a suspected autoimmune aetiology targeting anagen hair follicles. Alopecia areata is only sporadically reported in cows. Recently, we observed several cases of suspected alopecia areata in Eringer cows. The aim of this study was to confirm the presumptive diagnosis of alopecia areata and to define the clinical phenotype and histopathological patterns, including characterization of the infiltrating inflammatory cells. Twenty Eringer cows with alopecia and 11 Eringer cows without skin problems were included in this study. Affected cows had either generalized or multifocal alopecia or hypotrichosis. The tail, forehead and distal extremities were usually spared. Punch biopsies were obtained from the centre and margin of alopecic lesions and normal haired skin. Histological examination revealed several alterations in anagen hair bulbs. These included peri- and intrabulbar lymphocytic infiltration, peribulbar fibrosis, degenerate matrix cells with clumped melanosomes and pigmentary incontinence. Mild lymphocytic infiltrative mural folliculitis was seen in the inferior segment and isthmus of the hair follicles. Hair shafts were often unpigmented and dysplastic. The large majority of infiltrating lymphocytes were CD3(+) T cells, whereas only occasional CD20(+) lymphocytes were present in the peribulbar infiltrate. Our findings confirm the diagnosis of T-cell-mediated alopecia areata in these cows. Alopecia areata appears to occur with increased frequency in the Eringer breed, but distinct predisposing factors could not be identified.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Intradermal injection of heat-killed Mycobacterium vaccae in dogs with atopic dermatitis: a multicentre pilot study.

Canine atopic dermatitis (cAD) is a common disease with a multifactorial aetiology associated with impaired immunoregulation. The immunopathogenesis has similarities to that of human atopic dermatitis. Clinical signs of allergic disease in humans and mice are reduced by administration of saprophytic mycobacteria that amplify regulatory cytokines and hence the effect of Mycobacterium vaccae on the clinical severity of cAD was investigated. Sixty-two dogs with cAD, selected according to strict criteria, were treated with a single intradermal injection and evaluated monthly for 3 months in a placebo-controlled double-blind clinical trial. Clinical severity was quantified using standardized scores and by owner assessment of pruritus. A single injection of a heat-killed suspension of M. vaccae was found to be well tolerated and effective in treating mild to moderate cases of cAD demonstrable for 3 months, but was insignificant in more severely affected dogs.

Feline herpes dermatitis treated with interferon omega.

This case report describes the diagnosis, demonstration and treatment of feline herpes virus-induced facial dermatitis in a cat. The cat was successfully treated with interferon omega (IFN-omega).

Penetration of ASM 981 in canine skin: a comparative study.

ASM 981 has been developed for topical treatment of inflammatory skin diseases. It specifically inhibits the production and release of pro-inflammatory cytokines. We measured the skin penetration of ASM 981 in canine skin and compared penetration in living and frozen skin. To make penetration of ASM 981 visible in dog skin, tritium labelled ASM 981 was applied to a living dog and to defrosted skin of the same dog. Using qualitative autoradiography the radioactive molecules were detected in the lumen of the hair follicles until the infundibulum, around the superficial parts of the hair follicles and into a depth of the dermis of 200 to 500 microm. Activity could not be found in deeper parts of the hair follicles, the dermis or in the sebaceous glands. Penetration of ASM 981 is low in canine skin and is only equally spread in the upper third of the dermis 24 hours after application. Penetration in frozen skin takes even longer than in living canine skin but shows the same distribution.

Immunoglobulin E-bearing cells and mast cells in skin biopsies of horses with urticaria.

The pathogenesis of equine urticaria is not well understood. In man, urticaria has been associated with immunological and nonimmunological mechanisms leading to the release of various mediators by mast cells. Skin biopsies of 32 horses with a history of urticaria were stained with toluidine blue, a double-labelling method for chymase and tryptase, and immunohistochemistry for immunoglobulin (Ig)E. These horses were compared with horses with pemphigus foliaceus, insect bite hypersensitivity and control horses with healthy skin. Neither formalin fixation time nor biopsy site influenced the staining methods. No chymase-positive cells were found. In all groups of horses, cells staining with toluidine blue and for tryptase and IgE were found in the epidermis and hair follicle papilla and significantly more positively staining cells were observed in the subepidermal dermis compared with the deep dermis. Horses with urticaria had significantly more IgE-bearing cells in the subepidermal dermis than control horses. However, horses with urticaria had significantly fewer toluidine-blue-stained mast cells in both subepidermal and deep dermis compared with the insect bite hypersensitivity and pemphigus foliaceus groups. This study suggests that IgE-mediated reactions play a role in the pathogenesis of urticaria.

Evaluation of the usefulness of sensitization to aeroallergens as a model for canine atopic dermatitis in genetically predisposed Beagles.

OBJECTIVE: To evaluate a model for atopic dermatitis (AD) and to measure the effect of sensitization in Beagles genetically predisposed to produce high serum concentrations of allergen specific IgE. ANIMALS: 22 laboratory Beagles. PROCEDURE: Seventeen dogs were sensitized from birth to 3 allergens (recombinant birch pollen, Dermatophagoides pteronyssinus, and D farinae). Five nonsensitized dogs from the same litters served as controls. Clinical scoring, regular intradermal testing, measurement of serum concentrations of allergen-specific IgE, and collection of biopsy specimens of skin at 23, 32, and 43 weeks of age were performed. Serial tissue sections were stained for identification of IgE+ cells, mast cells and their subtypes, T-cells, Langerhans cells, and major histocompatibility complex class-II+ cells. At the age of 15 months, dogs were continuously exposed to 2 microg of mite allergen/g of dust. RESULTS: Sensitized dogs had positive intradermal test reactions and significantly higher serum concentrations of allergen specific IgE, compared with nonsensitized dogs. In sensitized and nonsensitized dogs, a significantly higher number of mast cells was found at predilection sites, compared with the control biopsy site. The number of mast cells at predilection sites increased with age. Sensitization significantly increased the number of epidermal Langerhans cells by 23 weeks of age. The number of epidermal Langerhans cells significantly increased in nonsensitized dogs by 32 weeks of age. Clinical scoring only revealed mild transient erythema in some dogs. CONCLUSIONS: increases in concentrations of serum allergen-specific IgE and exposure to allergens is not sufficient to induce clinical signs of AD in genetically predisposed dogs.