Isotype determination of circulating autoantibodies in canine autoimmune subepidermal blistering dermatoses

Abstract The three most common canine autoimmune blistering skin diseases (AISBD), bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA) have recently been separated based on clinical, histological and immunological grounds. The objectives of this study were to determine the isotype profiles of circulating autoantibodies in these dermatoses. Serum was collected from 5 dogs with BP, 15 with MMP and 11 with EBA. All sera were tested using an indirect immunofluorescence method using salt-split canine gingiva as substrate. Anti-basement membrane IgG autoantibodies were detected in all patients. Among the IgG autoantibodies, IgG1 and IgG4 were encountered most frequently, while IgG2 and IgG3 were uncovered in some dogs. IgE autoantibodies were detected more often than IgA or IgM autoantibodies in any of the three entities. The predominance of IgG1, IgG4 and IgE autoantibody isotypes in dogs with AISBD is very similar to the situation found in humans with the homologous diseases.     

Detection of circulating autoantibodies using living keratinocyte staining on MCA-B1 method in dogs with pemphigus foliaceus

In this study, we compared the sensitivity and specificity of three immunofluorescence techniques used to detect circulating autoantibodies in dogs with pemphigus foliaceus (PF); living keratinocyte staining on a canine keratinocyte cell line, MCA-B1, indirect immunofluorescence (IIF) on canine lip and IIF on bovine esophagus. Sera from canine PF cases were positive in four out of 27 dogs (14.8%) using living keratinocyte staining on MCA-B1 cells method, and five (18.5%) and eight sera (29.6%) using IIF on canine lip and bovine esophagus methods, respectively. By contrast, none of the 31 sera from dogs with non-pemphigus dermatoses reacted with MCA-B1 cells, whereas two (6.5%) as well as five sera (16.1%) obtained from those dogs showed positive reactivity with IIF on canine lip and bovine esophagus, respectively. Our results suggest that, although it exhibits the least sensitivity, the positive reactivity obtained by living keratinocyte staining on MCA-B1 cells can support the diagnosis of canine PF.     

Aspects of the humoral immune response to Staphylococcus intermedius in dogs with superficial pyoderma,deep pyoderma and anal furunculosis

Bacterial infection (pyoderma) of the canine skin is largely caused by Staphylococcus intermedius and may be a superficial or deep infection. Pyoderma may be a primary, idiopathic disease or secondary to a range of other dermatological disorders. In this study, the serum concentrations of IgG, IgA, antistaphylococcal IgG and antistaphylococcal IgA were measured by ELISA in normal dogs (n = 22), dogs with idiopathic deep pyoderma (n = 22), atopic dermatitis and superficial pyoderma (n = 24), atopic dermatitis without pyoderma (n = 25), flea bite dermatitis with superficial pyoderma (n = 8), pustular demodicosis (n = 8) and German shepherd dogs with anal furunculosis (n = 28). The serum IgG was significantly increased in dogs with atopy and superficial pyoderma (p < 0.001), and lower than normal in dogs with idiopathic deep pyoderma (p < 0.015). The concentration of serum IgA was significantly lower than normal in dogs with atopy uncomplicated by pyoderma (p < 0.015). The concentration of antistaphylococcal IgG in all clinical sera was significantly elevated (p < 0.001) when compared to normal dogs but concentrations of antistaphylococcal IgA were no greater than in normal dogs. Western blotting analysis for determination of the specificity of serum IgG antistaphylococcal antibody revealed that there were nine major epitopes. Discriminant analysis demonstrated that particular combinations of these epitopes were recognised more frequently by sera from dogs in different clinical groups.     

Histologic features and clinical outcomes of melanomas of lip, haired skin, and nail bed locations of dogs.

Outcomes, signalments, and the relationship of histologic features with the outcome of melanomas located in lip, nail bed, and haired skin of dogs were reviewed. These melanomas were diagnosed as benign or malignant based on histologic features. Melanomas of the lip arose from mucous membrane in most cases. 32 dogs with lip melanomas that had histologic features of malignancy, 22 died because of the tumor within 1 year and 10 were tumor free for at least 1 year following removal. Of 10 dogs with melanomas with benign histologic features on the mucous membrane of the lip, 9 were tumor free for at least 1 year. Of 4 dogs with benign appearing tumors of the haired skin of the lip, 3 were tumor free for at least 1 year. Melanomas with histologic features of malignancy occurred in many locations of haired skin, and 11 of 24 dogs were tumor free for at least 1 year. All nail bed melanomas had histologic features of malignancy and all invaded the third phalanx, but 6 of 14 dogs were tumor free for at least 1 year after amputation of the digit. Among these dogs, the 1-year survival rates for tumors classified as malignant by histologic features were 69% for lip, 46% for haired skin, and 43% for nail bed. However, the clinical outcome of an individual malignant tumor could not be predicted accurately by any specific histologic features.     

Clinical and immunological heterogeneity of canine subepidermal blistering dermatoses with anti‐laminin‐332 (laminin‐5) auto‐antibodies

Laminin‐332 (laminin‐5) is a basement membrane heterotrimeric protein composed of alpha‐3, beta‐3 and gamma‐2 laminin chains. Laminin‐332 polypeptides are targeted by auto‐antibodies in human patients with mucous membrane (cicatricial) pemphigoid or, more rarely, subepidermal vesicular diseases that resemble epidermolysis bullosa acquisita (EBA) or bullous pemphigoid (BP). The objectives of this report were to characterize the clinical, histopathological and immunological characteristics of nine dogs with auto‐antibodies targeting laminin‐332. Immunological investigations consisted of direct immunofluorescence (IF), indirect IF with intact and salt‐split canine gingival, and salt‐split normal or laminin‐332‐deficient human skin, immunoblotting with purified human laminin‐332 and immunoblotting with recombinant NC1 domain of human collagen VII. All dogs exhibited varying degrees of skin blistering and ulceration associated with microscopic subepidermal vesiculation with or without inflammatory cells. Indirect IF established that circulating IgG auto‐antibodies bound the dermal side of salt‐split canine lip and human skin. In five dogs, IgG variably recognized the basement membrane of laminin‐332‐deficient human skin (three dogs negative, two dogs positive). In all nine dogs, IgG auto‐antibodies detected purified human laminin‐332 by immunoblotting. In two dogs, additional targeting of collagen VII‐NC1 was present. These observations establish laminin‐332 as a novel basement membrane antigen in dogs with autoimmune blistering diseases with variable clinical phenotypes. The names ‘acquired junctional epidermolysis bullosa’, ‘anti‐laminin‐332 mucous membrane pemphigoid (MMP)’ and ‘mixed auto‐immune subepidermal blistering dermatosis’ are proposed for dogs with clinical signs reminiscent of EBA, MMP or BP respectively.     

IgG autoantibodies directed against desmoglein 3 cause dissociation of keratinocytes in canine pemphigus vulgaris and paraneoplastic pemphigus

Pemphigus is a group of autoimmune blistering diseases of the skin and mucous membranes. In human patients with pemphigus vulgaris (PV) and paraneoplastic pemphigus (PNP), IgG autoantibodies against desmoglein (Dsg) 3 and Dsg1 play pathogenic roles in blister formation. In contrast, the target for IgG autoantibodies that induce keratinocyte dissociation has not been elucidated in canine pemphigus. The aim of the present study was to determine whether anti-Dsg IgG autoantibodies are present and disrupt the cell-cell adhesion of keratinocytes in canine PV and PNP. The extracellular domains of canine Dsg3 were recognized by IgG in 3/5 (60%) canine PV sera tested. IgG against the extracellular domains of canine Dsg1 was detected exclusively in two dogs that had PV with the mucocutaneous phenotype. In addition, anti-Dsg3 IgG was identified in canine PNP serum. Furthermore, incubation of normal human keratinocytes (NHK) with mucocutaneous canine PV serum and canine PNP serum resulted in dissociation of the NHK sheets, whereas the removal of anti-Dsg3 IgG from these canine sera blocked this dissociation. The present study indicates for the first time that circulating anti-Dsg3 IgG antibodies capable of dissociating keratinocytes are present in dogs with PV and PNP.     

Detection of antinuclear antibodies by the Inno‐Lia ANA update test in canine systemic rheumatic disease

Background: Certain systemic autoimmune diseases in dogs are characterized by high titers of circulating antinuclear antibodies (ANA), which can be demonstrated by indirect immunofluorescence (IIF). In an earlier study of IIF‐ANA–positive dogs, the Ouchterlony double immunodiffusion (DID) test was used to identify specific autoantibodies. The DID test has largely been replaced with line blot tests in human diagnostic settings. Objective: The objective of this study was to investigate whether the line blot assay Inno‐Lia ANA update test is a useful tool in demonstrating ANA specificities in canine patients with previously diagnosed IIF‐ANA–positive rheumatic disorders. Methods: Serum samples from 3 clinically healthy control dogs and 20 canine patients with clinical signs of systemic rheumatic disease and documented positive results for IIF‐ANA and DID tests were included in the study. The Inno‐Lia ANA update assay was performed with an anti‐canine detection antibody. Results: Six serum samples that had DID positivity with anti‐spliceosomal small nuclear ribonucleoproteins (snRNP) reactivity showed reactivity to multiple snRNP proteins in the Inno‐Lia test. Samples from 2 dogs that had other types of DID positivity also had clear SmB reactivity and 1 had weak reactivity to RNP‐70K. The other serum samples, including controls, were negative. Conclusions: Using the Inno‐Lia ANA update test, multiple snRNP specificities were demonstrated in some canine patients with autoimmune rheumatic disorders. Other canine autoantibodies may exist that are not detected by this test. Further studies are necessary to characterize the target antigen(s) of these remaining autoantibodies in canine sera.     

Investigations on the nature and pathogenicity of circulating antikeratinocyte antibodies in dogs with pemphigus foliaceus

In humans with pemphigus foliaceus (PF), pathogenic autoantibodies are principally of IgG4 subclass and they cause superficial vesiculation when injected into neonatal mice. The objectives of this study were to determine the isotypes of circulating antikeratinocyte antibodies in dogs with PF, to assess whether serum antikeratinocyte antibody titres decreased during successful treatment, and to study whether such antibodies were pathogenic in passive transfers. Using indirect immunofluorescence with neonatal mouse skin substrates, circulating antikeratinocyte IgG antibodies were detected in 36 of 44 dogs with PF (82%). Serum autoantibodies belonged predominantly to IgG4 (three of 44; 80%) and IgG1 (30 of 44; 68%) subclasses. Antikeratinocyte IgG antibodies were detected in 16 of 20 normal dogs (80%), and these antibodies were IgG1 (16 of 20, 80%) but rarely IgG4 (two of 20; 10%) isotypes. In four dogs, IgG4 antikeratinocyte antibody titres decreased concomitantly to lesions nearing or reaching complete remission. In contrast, IgG or IgG1 titres remained stable or increased when lesions abated. Antikeratinocyte antibodies targeted mainly intercellular autoantigen(s) in the stratum granulosum, while in fewer dogs, such antibodies bound to cytoplasmic basal antigen(s). Intradermal injections of PF or pemphigus vulgaris (PV) IgG into neonatal mice caused subgranular or suprabasal acantholytic vesiculation without granulocyte infiltration, respectively. Similar transfers of normal dog IgG did not cause vesiculation. These observations suggest that antikeratinocyte IgG4 antibodies could be relevant to disease pathogenesis. Importantly, canine PF or PV IgG appear to be pathogenic when transferred passively into mice, causing vesiculation at epidermal levels similar to those of the natural disease.     

Laminin-5 is targeted by autoantibodies in feline mucous membrane (cicatricial) pemphigoid

In human and canine patients with mucous membrane (cicatricial) pemphigoid (MMP), circulating autoantibodies have been shown to target multiple epidermal basement membrane antigenic epitopes. These autoantigens include collagen XVII in humans and dogs, as well as laminin-5, laminin-6 or integrin alpha-6/beta-4 in human beings. The purpose of this study was to determine if autoantibodies targeted laminin-5 in a cat exhibiting clinical and microscopic lesions resembling those of MMP in humans. In this patient, an indirect immunofluorescence (IF) assay revealed circulating IgG and IgA autoantibodies that bound to the basement membrane zone on the dermal side of salt-split gingiva (titer 1:1000 for IgG and 1:50 for IgA). Immunoblotting, performed with affinity-purified human laminin-5, demonstrated that the autoantibodies bound the alpha-3 chain of this heterotrimer. These observations identify laminin-5 as one of the antigens recognized by circulating autoantibodies in this feline homologue of MMP in humans and dogs.     

A Screening for the Occurrence of Autoreactive Antibodies in Sera of Pregnant and Non-pregnant Bitches

Sera of healthy pregnant (group I, n = 11) and non-pregnant (group II, n = 11) bitches were screened for autoantibodies (AAb). In both groups, blood samples were drawn every fifth day between days 5 and 55 after mating. Serum was analysed via indirect immunofluorescence (IIF) with the Canine ANA HEp-2 Screening Kit. In all animals, anticytoplasmic AAb were detected. Utilizing primate-heart substrate slides AAb against contractile proteins of the cytoplasm could be observed. The predominating fluorescence pattern in pregnant animals resembled above all desmin, which was proven via Western blot. The sera were then pre-incubated with tropomyosin, actin, vimentin, vinculin and keratin solutions, and assessed on HEp-2 slides and on human and canine fibroblasts as well. The latter substrate was used to verify whether the detected Ab were in fact AAb. Utilizing tropomyosin, revealed elimination of the cytoplasmic fluorescences on all three substrates. It is therefore assumed, that in sera of healthy dogs, AAb against contractile structure proteins of the cytoplasm are present regularly. The majority of pregnant bitches presented with higher end-point titres (EPT), than to be found in non-pregnant dogs. AAb against desmin played the key role in those patterns. In addition, sera were screened for thyroid specific AAb, namely thyroglobulin, thyroid peroxidase (TPO), T3 and T4, and for AAb against insulin by ELISA or Western blot (TPO). Only in two of the pregnant bitches a weak positive reaction (1:100) for T3-AAb was detected.     

Conflicting results of serological,PCR and microscopic methods clarify the various risk levels of canine babesiosis in Slovakia: A complex approach to Babesia canis diagnostics

We have performed a survey of Babesia canis prevalence within group of dogs living in Southern and Western Slovakia. Blood samples and sera from 217 dogs, including individuals suspected of having babesiosis, were examined by nested PCR-RFLP, light microscopy and indirect fluorescence antibody test (IFAT). The detection of B. canis DNA revealed the highest number of infected dogs in the region of Nové Zámky, with 23 B. canis-positive blood samples (35.4%, n = 65), followed by an area close to Komárno (both areas of Southern Slovakia), where 1 dog out of 52 collected (1.9%) had detectible B. canis DNA in the blood stream. The serological method revealed an opposing pattern, with only 3 dogs (4.8%, n = 63) sampled at Nové Zámky presenting IgG antibodies against B. canis, while in Komárno region such antibodies were detected in 15 dogs (28.8%, n = 52). This discrepancy may be because the majority of samples from Nové Zámky were dogs suspected of an acute phase of canine babesiosis, whereas dogs at Komárno were sampled during a vaccination campaign, and thus were without any clinical signs of the disease. The latter group contains evidently recovered carriers of IgG against B. canis. Hence, the combination of PCR-based and serological methods enabled us to discover both recently infected as well as recovered dogs, thus obtaining a more realistic view on the epidemiological situation. Remarkably, we did not find any positive samples in the vicinity of Stupava (district Malacky, Western Slovakia), either by PCR-RFLP, microscopy or IFAT (n = 100). Considering the numerous falsely diagnosed cases of canine babesiosis, we suggest that light microscopy as the simplest and most accessible diagnostic test. Southern Slovakia was confirmed as an area of high risk of canine babesiosis, whereas conclusions about B. canis spreading over Western Slovakia should be considered with wariness.     

Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus

The majority of human patients with pemphigus foliaceus (PF) have circulating IgG autoantibodies that target conformational epitopes on the desmosomal cadherin desmoglein-1 (dsg1). Limited studies using immunoblot techniques suggested that the principal autoantigen in dogs with PF might also be dsg1. It was the objective of this study to test this hypothesis. A comprehensive survey of canine PF sera was conducted using a novel screening strategy that detects conformational epitopes. This method consists of the ectopic expression of canine dsg1 at the surface of human 293T epithelial kidney cells and their live screening, i.e. prior to fixation. Out of seven control human PF sera that bound to canine epidermis, three (57%) contained IgG autoantibodies that recognized ectopically expressed canine dsg1 with a membrane and punctate pattern. Out of 83 canine PF sera only five (6%) contained IgG that recognized canine dsg1. Consistent with findings for human PF sera obtained in this study, autoantibody binding was conformation- and glycosylation-dependent as demonstrated by calcium chelation with EDTA and tunicamycin or wheat germ agglutinin treatment, respectively. In conclusion, these studies establish canine dsg1 as a minor autoantigen for canine PF. Antigenic epitopes appear to be conformation- and glycosylation-dependent.     

Immunomapping of desmosomal and nondesmosomal adhesion molecules in healthy canine footpad,haired skin and buccal mucosal epithelia: comparison with canine pemphigus foliaceus serum immunoglobulin G staining patterns

Pemphigus foliaceus (PF) is the most common canine autoimmune skin disease. In contrast to human PF (hPF), desmoglein‐1 is a minor autoantigen in the canine disease. The major autoantigen(s) of canine PF (cPF) remain(s) unknown, which limits the ability to perform mechanistic studies of lesion formation and the development of novel diagnostic and therapeutic strategies for this disease. The immunofluorescence patterns of selected desmosomal (desmoglein‐1, desmoglein‐3, desmocollin‐1, desmocollin‐3, desmoplakin‐1/2, plakoglobin and plakophilin‐1) and nondesmosomal adhesion proteins (E‐cadherin, claudin‐1, zona occludens‐1 and occludin) in healthy canine footpad, haired skin and buccal mucosal epithelia were determined using hPF and pemphigus vulgaris sera and specific antibodies. The immunostaining patterns were then compared with that of indirect immunofluorescence staining with 66 cPF sera. Most cPF sera (58 of 66; 88%) exhibited positive staining along keratinocyte margins in the stratum spinosum and stratum granulosum of canine footpad. One serum contained autoantibodies binding solely to stratum granulosum keratinocytes. Concurrent intercellular fluorescence in the stratum basale was limited to seven of 66 cPF sera (11%). Only 12 of 66 cPF sera (18%) also exhibited positive IF staining of the buccal mucosa. This study confirms the immunological heterogeneity of cPF immunoglobulin G autoantibodies. Moreover, the major indirect immunofluorescence staining pattern and the inability of most cPF sera to label the buccal mucosa closely matched that of desmocollin‐1. These observations warrant further investigation of desmocollin‐1 as a potential major cPF autoantigen.     

Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of specific immunoglobulin G (IgG) against canine distemper virus (CDV) antigens. Sucrose gradient separation of viral and cellular proteins was required to produce coating antigens for the ELISA. The specificity of the ELISA was demonstrated by blocking CDV-positive canine sera with CDV-specific antisera produced in goats and rabbits and adsorption of positive sera with CDV antigens. A comparison of the ELISA with the serum-neutralization technique for the detection of CDV antibodies was conducted. Anti-CDV IgG was detected in conventional dogs as early as 6 days after inoculation with a commercial vaccine to CDV. Paired sera from the immunized dogs were evaluated by both techniques and a statistically (P less than 0.01) significant agreement between the ELISA and the serum-neutralization technique was shown (r = 0.6121, n = 75).     

Autoantibodies against the processed ectodomain of collagen XVII (BPAG2, BP180) define a canine homologue of linear IgA disease of humans

Linear IgA disease (LAD) is an acquired autoimmune subepidermal blistering dermatosis that affects human children and adults. In contrast to bullous pemphigoid, in which autoantibodies recognize transmembrane type XVII collagen (BP180, BPAG2), LAD is associated with skin-fixed and circulating IgA autoantibodies that target LAD-1, the processed extracellular form of type XVII collagen. An immunologic homologue of LAD in humans was identified in two dogs according to the following criteria: 1) erosive, ulcerative, and crusted lesions seen on the face, in the oral cavity, and on the extremities, 2) dermoepidermal clefting present in the basement membrane lamina lucida without inflammation or with mild neutrophilic infiltration, 3) basement membrane-fixed IgG and/or IgA antibodies, and 4) circulating IgA and IgG autoantibodies that target the 120-kd soluble protein LAD-1. The present study establishes unequivocally the existence of a naturally occurring canine model of LAD of humans.     

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.     

Evaluation of a standard immunofluorescence assay and a new flow cytometric method for the detection of autoreactive antibodies in dogs with tumours

The major purpose of the presented study was to develop and to evaluate a flow cytometry-based assay (IIFC) for the determination of autoreactive antibodies in sera from canine cancer patients. A blinded study demonstrated the poor reproducibility of the standard, slide-based and microscopically evaluated indirect immunofluorescence test (IIF), especially with sera displaying a cytoplasmic reactivity. In the IIFC, the intra assay coefficient of variance ranged between 5% and 11%, the inter assay variance between 8% and 25%. The IIFC resulted in significantly less positive results among canine cancer patients (16%) than the IIF (40%). The latter results were due to low titered sera indicating that the standard assay may lead to a high proportion of false positive results. The limitation of the IIFC is that no conclusions can be made about the sub cellular localization of the fluorescence. However, this cytometry-based assay makes a more objective and standardized detection of canine autoreactive antibodies possible.     

Detection of autoantibodies against thyroid peroxidase in serum samples of hypothyroid dogs

OBJECTIVE: To establish a sensitive test for the detection of autoantibodies against thyroid peroxidase (TPO) in canine serum samples. SAMPLE POPULATION: 365 serum samples from dogs with hypothyroidism as determined on the basis of serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid-stimulating hormone, of which 195 (53%) had positive results for at least 1 of 3 thyroid autoantibodies (against thyroglobulin [Tg], T4, or T3) and serum samples from 28 healthy dogs (control samples). PROCEDURE: TPO was purified from canine thyroid glands by extraction with detergents, ultracentrifugation, and precipitation with ammonium sulfate. Screening for anti-TPO autoantibodies in canine sera was performed by use of an immunoblot assay. Thyroid extract containing TPO was separated electrophoretically, blotted, and probed with canine sera. Alkaline phosphatase-conjugated rabbit anti-dog IgG was used for detection of bound antibodies. RESULTS: TPO bands were observed at 110, 100, and 40 kd. Anti-TPO autoantibodies against the 40-kd fragment were detected in 33 (17%) sera of dogs with positive results for anti-Tg, anti-T4, or anti-T3 autoantibodies but not in sera of hypothyroid dogs without these autoantibodies or in sera of healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The immunoblot assay was a sensitive and specific method for the detection of autoantibodies because it also provided information about the antigen. Anti-TPO autoantibodies were clearly detected in a fraction of hypothyroid dogs. The value of anti-TPO autoantibodies for use in early diagnosis of animals with thyroid gland diseases should be evaluated in additional studies.     

Diagnosing new autoimmune blistering skin diseases of dogs and cats

Autoimmune blistering skin diseases have been recognized for decades in humans and dogs. In the dog, most of these diseases unfortunately were grouped under the generic denomination of bullous pemphigoid without any confirmation that the autoantibodies targeted bullous pemphigoid antigens. In recent years, advanced diagnostic methods have permitted the recognition of new autoimmune blistering skin diseases in humans and companion-animal species. At this time, the diagnosis of these entities is made by combining clinical signs and results of histopathology. Immunologic methods serve to establish the presence of skin-fixed and circulating autoantibodies that target various epidermal or basement membrane antigens. In this article, salient features of the most common canine and feline subepidermal blistering dermatoses (mucous membrane pemphigold, bullous pemphigold, epidermolysis bullosa acquisita) and new variants of cutaneous lupus (type I bullous systemic lupus erythematosus and vesicular cutaneous lupus erythematosus) are presented.     

Determination of autoantibodies to thyroglobulin,thyroxine and triiodothyronine in canine serum

Enzyme immunoassays (EIAs) for the determination of autoanti-bodies (AA) to thyroid antigens in canine serum were developed. Streptavidin (SA) was immobilized as capture molecule on microtitreplates (MTP). Thyroglobulin (Tg) purified from canine thyroids and the thyroid hormones thyroxine and triiodothyronine (T3 and T4) were conjugated to biotin labelling reagents and attached to the MTP over the SA-biotin bridge. Bound AA were detected with anti-dog-immunoglobulin G (IgG) labelled with horseradish peroxidase. Serum samples from dogs which were allotted to four groups were analysed: A (n = 31), biochemical evidence of hypothyroidism; B (n = 76), clinical signs of hypothyroidism; C (n = 47), euthyroid with non-thyroidal disease; D (n = 186), clinically healthy. The validity of the assays was tested with two different methods. After thiophilic absorption chromatography of positive sera, a positive reaction in the EIA was only detected in those fractions which coeluted with the canine IgG standard. Furthermore, the positive reaction was blocked by the addition of the corresponding antigen. In 55% of the hypothyroid dogs AA to Tg and/or T3 and T4, respectively, were found (up to a titre of 1 : 1600). In group B 34% of the dogs were diagnosed positive, but the titre was lower (up to 1 : 400). In the groups C and D the number of dogs with AA and their titre was significantly lower. Two different methods for distinguishing positive and negative test results were compared in order to increase the specificity of the tests without decreasing the sensitivity. The EIAs are precise and based on high agreement with previous reported assays able to discriminate dogs with thyroiditis from healthy ones. These assays represent a good alternative to the isotope assays generally used for the analysis of AA to T4 and T3.