Immunoperoxidase staining for the detection of autoantibodies in canine autoimmune skin disease; comparison to immunofluorescence results

Skin sections from 22 dogs with autoimmune skin disease were stained with anti-canine IgG, IgM and IgA using an immunobridge immunoperoxidase method. Eight cases of lupus erythematosus, three cases of pemphigus vulgaris, and 11 cases of pemphigus foliaceus were included. Results of previously performed, direct immunofluorescence tests for the detection of canine immunoglobulin on skin were available on 17/22 cases. The immunoperoxidase method yielded an overall positive result in 59% (5/8 lupus erythematosus, 2/3 pemphigus vulgaris and 6/11 pemphigus foliaceus) versus an overall positive result of 47% for direct immunofluorescence (3/5 lupus erythematosus, 2/2 pemphigus vulgaris and 2/10 pemphigus foliaceus). The immunobridge immunoperoxidase method compared favorably to direct immunofluorescence testing of canine skin for autoantibody in cases of lupus erythematosis and pemphigus vulgaris, and was superior in cases of pemphigus foliaceus. This method should prove useful as an aid in the diagnosis of canine autoimmune skin disease.     

Pemphigus in the dog: comparison of immunofluorescence and immunoperoxidase method to demonstrate intercellular immunoglobulins in the epidermis

In 3 dogs with pemphigus vulgaris and 4 dogs with pemphigus foliaceus, intercellular immunoglobulins were demonstrated in the epidermal stratum spinosum. The immunofluorescence technique on cold ethanol-fixed and paraffin-embedded tissue sections was compared with the immunoperoxidase method on formalin-fixed paraffin-embedded tissue sections. The results of both methods were identical. However, the advantage of the unlabeled antibody-enzyme method was that the same formalin-fixed tissue specimens could be used for conventional light microscopy, as well as for immunohistologic studies.     

Immunohistochemical analysis of the distribution of desmoglein 1 and 2 in the skin of dogs and cats

OBJECTIVE: To compare the distribution of desmoglein (Dsg) 1 and 2 in skin specimens obtained from dogs and cats to provide information about the possible role of the density of Dsg 1 and 2 in the localization of lesions attributable to pemphigus foliaceus in these 2 species. SAMPLE POPULATION: Skin biopsy specimens obtained from 4 dogs and 4 cats. PROCEDURE: Biopsy specimens were collected from the muzzle, bridge of the nose, ear, dorsum, abdomen, area adjacent to the teats, and footpads of each animal. Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded skin samples by use of a biotinylated mouse monoclonal anti-Dsg 1 and 2 antibody raised against bovine muzzle. Color development was performed by use of the streptavidin-biotin-peroxidase method with a chromogenic substrate. RESULTS: Immunohistochemical staining yielded a positive reaction in skin samples obtained from all anatomic sites. The intensity and distribution of staining were related to the number of layers of the stratum spinosum. No differences were detected between samples obtained from dogs and cats. CONCLUSIONS AND CLINICAL RELEVANCE: No differences in intensity of Dsg 1 and 2 antigen were observed in the stratum spinosum between skin samples obtained from dogs and cats. Analysis of this result suggests that factors other than the distribution of Dsg may be responsible for the differences in localization of primary clinical lesions in dogs and cats with pemphigus foliaceus.     

Pemphigus foliaceus: a report of two cases in the dog

The clinical and diagnostic features of two cases of pemphigus foliaceus in the dog are described. Dermatohistopathology and direct immunofluorescence examination of skin biopsies were diagnostic for pemphigus foliaceus. Both dogs required immunosuppressive dosages of prednisolone to achieve control of the condition.     

Pemphigus foliaceus in dogs: a review of 37 cases

Thirty-seven dogs with pemphigus foliaceus were seen over a span of 9 years in a veterinary medical teaching hospital. Four breeds of dogs (Bearded Collie, Akita, Newfoundland, Schipperke) were at significant elevated risk when compared with both the dermatology canine case population and the hospital canine population. The mean age of onset was 4.2 years. The dorsal part of the muzzle was the most common site of initial involvement in over 50% of the dogs, and lesions of the head were seen first in 81% of the dogs. Disease progression was gradual (greater than 3 months) in 73% of the dogs. Somewhat bilaterally symmetric scaling, crusting, and alopecia were seen in all of the dogs. Vesicles, pustules, and bullae were not seen commonly, but target lesions with peripheral collarettes were seen frequently. Most dogs had characteristic footpad lesions, with erythematous swelling at the pad margins, cracking, and villous hypertrophy. Generalized exfoliative dermatitis was seen in dogs with widespread disease. Pruritus was noted in less than one half of the dogs. Typical histopathologic findings included subcorneal and intragranular cell layer epidermal pustules, or intrafollicular pustules with prominent acantholysis. Direct immunofluorescence in an intercellular pattern was noted in 76% of the dogs tested and indirect immunofluorescence was noted in 75% of a much smaller sample. Thirty-nine percent of the dogs responded to corticosteroid therapy alone, and 50% and 55% responded, respectively, to prednisone and cytotoxic drugs, and to prednisone with aurothioglucose. Aurothioglucose was successful alone in 27% of the dogs. One-year survival was achieved in 53% of the dogs.     

Prolonged remission after immunosuppressive therapy in six dogs with pemphigus foliaceus

Limited information is available on the long-term outcome of treatment of pemphigus foliaceus in dogs. The purpose of this study is to report that a prolonged remission can occur after discontinuation of immunosuppressive regimens in some animals with this disease. Six dogs were diagnosed with pemphigus foliaceus based on suggestive clinical signs and histopathology. These patients were treated either with immunosuppressive doses of oral glucocorticoids or with a combination of oral glucocorticoids and azathioprine. After clinical signs underwent complete remission, which occurred 1.5-5 months after immunosuppression was initiated, the drugs were tapered progressively and eventually withdrawn. The total duration of immunosuppressive therapy varied between 3 and 22 months. Skin lesions of pemphigus foliaceus did not recur for 1.5-6 years after treatment was stopped. These observations suggest that, in some dogs with pemphigus foliaceus, immunosuppression can lead to long-term remission of skin lesions, and that discontinuation of treatment is not necessarily followed by a recurrence of clinical signs.     

Comparative analysis of canine dermatophytosis and superficial pemphigus for the prevalence of dermatophytes and acantholytic keratinocytes: a histopathological and clinical retrospective study

Acantholytic dermatophytosis is a rarely reported condition of dogs that clinically and histopathologically mimics superficial pemphigus (erythematosus, foliaceus). Histologically, periodic acid-Schiff (PAS) and Grocott's methenamine-silver (GMS) are often necessary to show the fungus. A retrospective histopathological study was conducted on 190 canine skin biopsy specimens: 95 each with the diagnosis of canine dermatophytosis or of superficial pemphigus. All specimens were stained with haematoxylin and eosin, PAS, and GMS. Dermatophytes were not seen in any superficial pemphigus cases. Acantholytic keratinocytes were noted in 14% of the dermatophytosis cases, none of which had clinical signs consistent with superficial pemphigus. Among cases with acantholytic keratinocytes, superficial pemphigus had significantly more acantholytic cells than dermatophytosis (P = 0.02). When comparing face and nonface cases, there was no difference in prevalence of acantholytic keratinocytes in dermatophytosis or number of acantholytic keratinocytes in superficial pemphigus. All dermatophyte cases were both GMS and PAS positive with neither stain being visually superior. No dermatophyte cases where acantholytic keratinocytes were noted had a history, clinical signs and histopathological features compatible with acantholytic dermatophytosis.     

Use of tetracycline and niacinamide for treatment of autoimmune skin disease in 31 dogs.

A combination of niacinamide and tetracycline was used to treat 31 dogs with various autoimmune skin diseases (discoid lupus erythematosus, pemphigus foliaceus, pemphigus erythematosus, and bullous pemphigoid). Of the 20 dogs with discoid lupus erythematosus, 70% had excellent or good response to treatment. Serious side effects were not noticed in any dog.     

P-52 A case of juvenile cellulitis concurrent with hindlimb paresis in an English cocker spaniel puppy

Pemphigus vegetans is a very rare cutaneous autoimmune blistering acantholytic disease of humans that combines features of both pemphigus foliaceus and mucosal lesions of pemphigus vulgaris. We report here the clinical, histopathological and immunological findings in a dog whose lesions resembled those of pemphigus vegetans of humans. A 4-year old, greater Swiss mountain dog was presented with verrucous papules and crusts on the axillae and inguinal region. Within 3 months, lesions progressed to involve the thorax and ear pinnae, and then became generalized. Ulcers were observed in the oral cavity, anus and prepuce. Microscopic examination of mucosal and cutaneous biopsy specimens revealed a mixed pattern of deep intraepidermal neutrophilic and eosinophilic pustules with isolated and clustered acantholytic keratinocytes, along with suprabasal epidermal clefts leaving rounded basal keratinocytes at the bottom of the vesicles. These dual changes were also observed within the hair follicle epithelium. Dermal inflammation was mixed and perivascular. Direct immunofluorescence revealed IgG deposited around epidermal keratinocytes. Indirect immunofluorescence performed on normal canine gingival substrate uncovered antikeratinocyte IgG autoantibodies with a serum titre of 1:2500. Immunoblotting confirmed that circulating IgG autoantibodies recognized the extracellular segment of canine desmoglein-1 and human desmoglein-3. Treatment with azathioprine and oral glucocorticoids resulted in long-lasting complete remission. In this dog, clinical signs, microscopic skin lesions and immunological findings were deemed analogous to those of human Neumann-type pemphigus vegetans.
Funding: Self-funded.     

P‐50 Clinical,histological and immunological characteristics of Neumann‐type pemphigus vegetans in a dog

Pemphigus vegetans is a very rare cutaneous autoimmune blistering acantholytic disease of humans that combines features of both pemphigus foliaceus and mucosal lesions of pemphigus vulgaris. We report here the clinical, histopathological and immunological findings in a dog whose lesions resembled those of pemphigus vegetans of humans. A 4‐year old, greater Swiss mountain dog was presented with verrucous papules and crusts on the axillae and inguinal region. Within 3 months, lesions progressed to involve the thorax and ear pinnae, and then became generalized. Ulcers were observed in the oral cavity, anus and prepuce. Microscopic examination of mucosal and cutaneous biopsy specimens revealed a mixed pattern of deep intraepidermal neutrophilic and eosinophilic pustules with isolated and clustered acantholytic keratinocytes, along with suprabasal epidermal clefts leaving rounded basal keratinocytes at the bottom of the vesicles. These dual changes were also observed within the hair follicle epithelium. Dermal inflammation was mixed and perivascular. Direct immunofluorescence revealed IgG deposited around epidermal keratinocytes. Indirect immunofluorescence performed on normal canine gingival substrate uncovered antikeratinocyte IgG autoantibodies with a serum titre of 1:2500. Immunoblotting confirmed that circulating IgG autoantibodies recognized the extracellular segment of canine desmoglein‐1 and human desmoglein‐3. Treatment with azathioprine and oral glucocorticoids resulted in long‐lasting complete remission. In this dog, clinical signs, microscopic skin lesions and immunological findings were deemed analogous to those of human Neumann‐type pemphigus vegetans. Funding: Self‐funded.     

Localization of immunoglobulins and complement by the peroxidase antiperoxidase method in autoimmune and non-autoimmune canine dermatopathies

Formalin-fixed, paraffin embedded skin biopsy specimens from 44 dogs with various dermatopathies were examined for the presence of immunoglobulins (IgG, IgM, IgA) and complement (C3) by the peroxidase antiperoxidase method (PAP). Final diagnoses of the skin diseases were determined by clinical findings, fungal and bacterial cultures, skin scrapings, serum endocrinologic tests, and histologic features of cutaneous biopsies. The dermatopathies included examples of pyoderma (folliculitis/furunculosis), demodecosis, sarcoptic mange, dermatophytosis, endocrine dermatopathy, and autoimmune skin disease (AISD). In the latter category, 7 cases of pemphigus foliaceus, 1 pemphigus vulgaris, 4 discoid lupus erythematosus (DLE) and 4 examples of indeterminate AISD were examined. Immunoglobulins, C3, or both, were localized in tissue sections from AISD (15/16), pyoderma (7/11), demodecosis (4/8) sarcoptic mange (3/3), and dermatomycosis (2/3). Endocrine dermatopathy biopsies were consistently negative for Ig and C3 (0/3). The pattern of localization was most often intercellular (31/44 positive biopsies) with basement membrane immunoreactivity in 4 cases of DLE, and 1 case of indeterminate AISD. There was no consistent correlation between histologic features of hematoxylin and eosin-stained biopsies and the presence of Ig and C3. Clinical outcome was similar in both Ig and C3 positive and negative cases of non-AISD dermatitis. The high percentage (95%) of positive results in AISD biopsies indicates the usefulness and sensitivity of the PAP method for the localization of Ig and C3. Because of the high percentage of Ig localization in pyoderma (73%), biopsy specimens with extensive inflammatory skin disease are not suitable for diagnosis of AISD. The PAP method is useful in the diagnosis of AISD in biopsy specimens with minimal inflammation. Caution is warranted in the interpretation of immunoreactivity independent of clinical and histologic information.     

Pemphigus foliaceus of the footpads in three dogs

Severe hyperkeratinization and villous hypertrophy of the footpads were seen in 3 middle-aged dogs. Peeling, fissuring, swelling, and ulcerations were noted on the margins of severely affected pads. Pain was evident in palpation and ambulation. Lesions were compatible with the traditional diagnosis of "hard pad disease". Histopathologic findings were diagnostic for canine pemphigus foliaceus in all 3 dogs, and direct immunofluorescence in an intercellular pattern was seen in both dogs that were tested. All 3 dogs responded to immunosuppressive dosages of corticosteroids.     

Clinical and histopathological features of pemphigus foliaceus with and without eosinophilic infiltrates: a retrospective evaluation of 40 dogs

The importance of cellular infiltrates in tissues has been investigated as a diagnostic tool, mechanism of pathogenesis, and prognostic indicator in certain human diseases. Eosinophils, in particular, have a distinct role in the development of cutaneous lesions in human autoimmune diseases. Identification of an eosinophilic infiltrate can aid the diagnosis of immunobullous disease in the early stages of the disease process. In canine pemphigus foliaceus, eosinophils are present to a variable degree within lesional tissue. This study retrospectively evaluated 40 dogs with pemphigus foliaceus, and examined clinical and histologic features and final outcomes in cases with and without eosinophilic infiltrates. Twenty‐five of 40 dogs (63%) had an eosinophilic infiltrate in either the pustules/crust, follicular infundibulum or dermis. There was no statistically significant difference in clinical distribution or appearance of dermatological lesions, response to treatment, or disease outcome in dogs with or without an eosinophilic infiltrate. However, dogs with concurrent disease were significantly more likely to have an eosinophilic infiltrate (P = 0.01). Dogs with adverse effects associated with immunosuppressive therapy were significantly more likely to have an eosinophilic infiltrate (P = 0.05). Fifteen of 40 dogs (38%) had a history of allergic disease and a significantly higher proportion of these dogs had an eosinophilic infiltrate (P = 0.04). An eosinophilic infiltrate was found in more than half of the dogs in this study. These findings justify further studies to investigate the role of eosinophils in the pathogenesis, therapy and prognosis in dogs with pemphigus foliaceus.     

The duration and quality of positive direct immunofluorescence in skin biopsies using michel's fixative on a case of equine pemphigus foliaceus

A special buffered ammonium sulfate fixative (Michel's fixative) designed for use as a liquid media capable of preserving in vivo tissue fixed immunoglobulins and complement was evaluated. The preservative ability of this fixative was studied by using skin biopsies from a confirmed case of spontaneously occurring equine pemphigus foliaceus. Once obtained, 16 skin biopsies were placed in Michel's fixative. Eight samples were stored at ambient temperature (21°C) and 8 samples were stored in a conventional refrigerator at 4°C. Over an 8 month time period, direct immunofluorescence was performed on these skin biopsies looking for the abnormal presence of IgG, IgM (when done) and C³ deposition within the intercellular spaces of the epidermis. The results of this study indicated that Michel's fixative was capable of preserving in vivo tissue fixed immunoglobulin and complement for 8 months. This was demonstrated by positive direct immunofluoresence spaces of the epidermis in all of the skin biopsies examined. In addition, it was shown that refrigerated and non-refrigerated skin biopsies in Michel's fixative both yielded a diagnostic quality of direct immunofluorescence. This will allow the mailing of skin biopsies without requiring refrigeration and specimens mailed in the winter should demonstrate reliable results on fluorescent antibody testing when performed.     

Metaflumizone-amitraz (Promeris)-associated pustular acantholytic dermatitis in 22 dogs: evidence suggests contact drug-triggered pemphigus foliaceus

Promeris Duo (PD) is a novel topical flea and tick preventative for dogs, which is also licensed for treatment of canine demodicosis. In this article, we present 22 dogs that all developed pemphigus foliaceus (PF)-like cutaneous drug reactions at the site of PD application. In eight dogs, the lesions were restricted to the application site (localized group). Signs of systemic illness were reported in three dogs, and four required immunosuppressive treatment. Direct immunofluorescence for IgG was positive in four dogs, although circulating antikeratinocyte IgG could not be detected in any tested sera. Complete remission was achieved in all dogs, with one patient still remaining on treatment. Fourteen dogs developed skin lesions at the application site as well as other noncontiguous areas (distant group). Systemic signs were reported in 11 dogs, and immunosuppression was required in 10 cases. Direct and indirect immunofluorescence tests were positive for antikeratinocyte autoantibodies in 10 of 13 and six of 10 patients with distant disease, respectively. Complete remission was achieved in 10 of 13 dogs with distant disease; one-third are still on treatment. Histological changes were similar to canine PF. Desmosomal architectural changes, assessed by desmoglein-1 immunostaining, were also similar to those of dogs with spontaneous autoimmune PF. Apoptosis did not appear to contribute to lesion formation, in either autoimmune or PD-associated PF. In conclusion, PD has the potential of triggering a variant of PF that resembles spontaneously occurring autoimmune PF at clinical, morphological, immunological and treatment outcome levels.     

Identification of rickettsiae in cutaneous biopsy specimens from dogs with experimental Rocky Mountain spotted fever

Direct immunofluorescence reaction for Rickettsia rickettsii was performed on formalin-fixed, paraffin-embedded cutaneous biopsy specimens collected from dogs with experimental Rocky Mountain spotted fever (RMSF). A technique of trypsin digestion of deparaffinized, rehydrated sections was successful in demonstrating discrete, immunofluorescent organisms in endothelia and adjacent vessel walls in the dermis. R rickettsii was identified only in grossly evident dermal lesions (macular rash or oral vesicles) and was not apparent in randomly collected biopsy specimens from clinically normal inguinal skin. These results suggest that clinical application of this technique for diagnosis of RMSF may be limited in dogs without cutaneous lesions.     

A review of autoimmune skin diseases in domestic animals: I - superficial pemphigus

In humans, the pemphigus denomination encompasses a group of autoimmune blistering skin diseases with intraepidermal separation resulting from cell-cell detachment by acantholysis. Entities are classified based on the level of blistering in the epidermis, and both superficial (pemphigus foliaceus, IgA pemphigus) and deep (pemphigus vulgaris, pemphigus vegetans and paraneoplastic pemphigus) variants are recognized. In domestic animals, subsets of pemphigus have been recognized since the mid-1970s, and the disease classification resembles that used for human patients. This article reviews up-to-date knowledge on the epidemiology, clinical signs, histopathology, immunopathology and treatment outcome of superficial pemphigus in domestic animals. Detailed information on canine, feline, equine and caprine pemphigus foliaceus, canine and feline pemphigus erythematosus and canine panepidermal pustular pemphigus is provided.     

Identification of Rickettsiae in Cutaneous i Biopsy Specimens From Dogs With Experimental Rocky Mountain Spotted Fever

Direct immunofluorescence reaction for Rickettsia rickettsii was performed on formalin-fixed, paraffin-embedded cutaneous biopsy specimens collected from dogs with experimental Rocky Mountain spotted fever (RMSF). A technique of trypsin digestion of deparaffinized, rehydrated sections was successful in demonstrating discrete, immunofluorescent organisms in endothelia and adjacent vessel walls in the dermis. R rickettsii was identified only in grossly evident dermal lesions (macular rash or oral vesicles) and was not apparent in randomly collected biopsy specimens from clinically normal inguinal skin. These results suggest that clinical application of this technique for diagnosis of RMSF may be limited in dogs without cutaneous lesions. (Journal of Veterinary Internal Medicine 1989; 3:8–11)     

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

FC-20 Efficacy of griseofulvin for juvenile cellulitis in dogs

Medical records of 97 dogs with pemphigus foliaceus were evaluated. The average age of onset was 6.3 years (range 0.5–16 years). Crusts were the most common lesions in 79 dogs; pustules were observed in 36 dogs. No gender predisposition was identified. The trunk was the most commonly involved area (51 dogs), followed by the inner pinnae (46), dorsal muzzle (37), footpads (32), periocular area (26), outer pinnae (23) and planum nasale (23). Facial involvement only was noted in 15 dogs. Of the 48 dogs in which cytology was recorded, concurrent infections were identified in 32 dogs, acantholytic cells were seen in 37 dogs, numerous neutrophils in 35 dogs, and numerous eosinophils in eight cases. Final control of the disease was achieved with: glucocorticoids (24 dogs); azathioprine (9); chlorambucil (1); aurothioglucose (1); a combination of glucocorticoids and azathioprine (31); glucocorticoids and aurothioglucose (2); tetracycline/doxycycline and niacinamide (8); prednisolone, tetracycline and niacinamide (1); fatty acid supplementation (2); and tacrolimus (1). One dog was completely tapered off drugs and stayed in remission. Average time to improvement was 6 weeks, and average time to remission was 9.3 months. Forty-three dogs were followed for <12 months, and 12 of these were euthanized: eight for other diseases and four due to a lack of response or adverse effects of treatment. In 54 dogs, the follow up was >12 months; four of these dogs were euthanized (one due to an unrelated cause, one due to neoplastic disease and two related to pemphigus foliaceus).
Funding: Self-funded.