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Pemphigus foliaceus (PF) is an autoimmune skin disease of dogs characterized by intraepidermal pustules containing neutrophils and dissociated keratinocytes that develop in association with circulating and tissue-bound IgG autoantibodies. A subset of IgG autoantibodies in canine PF target desmocollin-1 (DSC1), a component of intercellular adhesion complexes within the epidermis. Passive transfer of IgG autoantibodies from canine PF sera to mice was previously shown to induce skin disease in the absence of infiltrating neutrophils. In attempts to identify a mechanism responsible for neutrophil recruitment, past studies evaluated the prevalence of IgA autoantibodies in canine PF sera where they were found in <20% of affected dogs. We re-evaluated the prevalence of anti-DSC1 IgA in canine PF due to concerns regarding the sensitivity of previously used methods. We hypothesized that anti-DSC1 IgA are present in most dogs with PF but have been under-detected due to competition with concurrent anti-DSC1 IgG for binding to their mutual antigenic target. Despite removing approximately 80% of IgG from patient sera using affinity chromatography, we did not detect an increase in anti-DSC1 IgA by performing indirect immunofluorescence on canine DSC1-transfected HEK293T cells. Taken together, our results do not support a role for pathogenic IgA in canine PF., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tyler J.M. Jordan reports financial support was provided by Morris Animal Foundation. Tyler J.M. Jordan reports financial support was provided by National Institutes of Health. Zhi Liu reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Pugs commonly present with thoracolumbar myelopathy, also known as pug dog myelopathy (PDM), which is clinically characterised by progressive signs involving the pelvic limbs, no apparent signs of pain and, often, incontinence. In addition to meningeal fibrosis and focal spinal cord destruction, histopathology has confirmed lymphohistiocytic infiltrates in the central nervous system (CNS) in a considerable number of pugs with PDM. Lymphohistiocytic CNS inflammation also characterises necrotising meningoencephalitis (NME) in pugs. This study aimed to investigate the potential contribution of an immunological aetiology to the development of PDM., Methods: The concentrations of glial fibrillary acidic protein (GFAP) in serum and CSF and of anti-GFAP autoantibodies in CSF were measured with an ELISA. In addition, a commercial test was used for genetic characterisation of the dog leukocyte antigen class II haplotype, which is associated with NME susceptibility., Results: This study included 87 dogs: 52 PDM pugs, 14 control pugs, four NME pugs and 17 dogs of breeds other than pugs that were investigated for neurological disease (neuro controls). Anti-GFAP autoantibodies were present in 15 of 19 (79%) of the PDM pugs tested versus six of 16 (38%) of the neuro controls tested (p = 0.018). All 18 PDM pugs evaluated had detectable CSF GFAP. Serum GFAP was detected in two of three (67%) of the NME pugs and in two of 11 (18%) of the control pugs but not in any of the 40 tested PDM pugs. Male pugs heterozygous for the NME risk haplotype had an earlier onset of clinical signs (70 months) compared to male pugs without the risk haplotype (78 months) (p = 0.036)., Limitations: The study was limited by the lack of healthy dogs of breeds other than pugs and the small numbers of control pugs and pugs with NME., Conclusions: The high proportion of PDM pugs with anti-GFAP autoantibodies and high CSF GFAP concentrations provide support for a potential immunological contribution to the development of PDM., (© 2024 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Canine diabetes has been considered a potential model of human type 1 diabetes (T1D), however the detection of autoantibodies common in humans with T1D in affected dogs is inconsistent. The aim of this study was to compare autoantibody responses in diabetic and healthy control dogs using a novel nucleic acid programmable protein array (NAPPA) platform. We performed a cross-sectional study of autoantibody profiles of 30 diabetic and 30 healthy control dogs of various breeds. Seventeen hundred human proteins related to the pancreas or diabetes were displayed on NAPPA arrays and interrogated with canine sera. The median normalized intensity (MNI) for each protein was calculated, and results were compared between groups to identify candidate autoantibodies. At a specificity of 90%, six autoantibodies had sensitivity greater than 10% (range 13-20%) for distinguishing diabetic and control groups. A combination of three antibodies (anti-KANK2, anti-GLI1, anti-SUMO2) resulted in a sensitivity of 37% (95% confidence interval (CI) 0.17-0.67%) at 90% specificity and an area under the receiver operating characteristics curve of 0.66 (95% CI 0.52-0.80). While this study does not provide conclusive support for autoimmunity as an underlying cause of diabetes in dogs, future studies should consider the use of canine specific proteins in larger numbers of dogs of breeds at high risk for diabetes., (© 2022. The Author(s).)

Background: Antiplatelet antibodies are detected in multiple diseases including primary immune thrombocytopenia (ITP). Dynamics of how these antibodies change over time in ITP is unknown in dogs., Hypothesis/objectives: Antiplatelet antibodies (APA) will be detected in thrombocytopenic dogs with multiple etiologies and dynamics of APA in dogs with ITP can be used to evaluate response to treatment and relapse. Determine APA at the time of diagnosis in thrombocytopenic dogs and serially in primary ITP dogs., Animals: Seventy-nine thrombocytopenic dogs and 28 primary ITP dogs., Methods: Direct flow cytometry was performed in thrombocytopenic dogs at initial evaluation and serially in suspected primary ITP dogs. In primary ITP dogs, a 2-tailed Fisher's exact test was performed comparing survival to discharge between dogs with and without melena and to relate response to treatment and relapse to changes in APA and platelet count (repeated measures analysis, Spearman correlation)., Results: Twenty percent (16/79) of thrombocytopenic non-ITP dogs with infectious, neoplastic, or other diseases and all primary ITP dogs were positive for APA. Melena at initial evaluation was associated with decreased survival to discharge (odds ratio 0.06; P = .01). Persistence of APA was not associated with response to treatment, but recurrence of antibodies was associated with relapse (odds ratio 205.0; P < .01). There was no difference in percentage of APA or platelet count at initial diagnosis between dogs that did or did not respond to treatment., Conclusions and Clinical Importance: Serial monitoring of APA in dogs with primary ITP appeared beneficial for determining relapse of disease., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Presumed autoimmune diseases affecting the central nervous system (CNS) of dogs are common. In people, antibodies against neuronal cell surface antigens that are associated with a wide variety of neurological syndromes have been identified. The presence of cerebrospinal fluid (CSF) autoantibodies that target neuronal cell surface proteins has not been reported in dogs with neurologic disorders., Objectives: Autoantibodies to neuronal cell surface antigens can be found in the CSF of dogs with inflammatory CNS disease. Our aim was to determine whether 6 neuronal cell surface autoantibodies were present in the CSF of dogs diagnosed with inflammatory and noninflammatory CNS disease., Animals: Client-owned dogs with CNS disease and complete diagnostic evaluation including magnetic resonance imaging and CSF analysis were included. One healthy dog was included as a negative control., Methods: Cerebrospinal fluid was tested for 6 antigenic targets with a commercially available indirect immunofluorescence assay test kit., Results: There were 32 dogs with neurological disease, 19 diagnosed with inflammatory disease (encephalitis and meningitis), 10 with noninflammatory disease (neoplasia, intervertebral disk disease, degenerative myelopathy, and epilepsy), 2 with no diagnosis, and 1 with neoplasia and meningoencephalitis. Anti-N-methyl-d-aspartate receptor 1 (NMDAR1) antibodies were detected in 3 dogs (3/32; 9.38%). All 3 dogs responded to treatment of meningoencephalomyelitis of unknown etiology (MUE)., Conclusions and Clinical Importance: Further evaluation of the prevalence and clinical relevance of CSF and serum antibodies to neuronal cell surface antigens is warranted. Defining antigenic targets associated with encephalitis in dogs might allow diagnostic categorization of MUE antemortem., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Autoimmunity associated with autoantibodies against the β1-adrenergic receptor (β1-AAB) is increasingly accepted as the driver of human dilated cardiomyopathy (DCM). Unfortunately, there is a lack of animal models to extend the knowledge about β1-AAB autoimmunity in DCM and to develop appropriate treatment strategies., Objectives: To introduce an animal model, we investigated the β1-AAB associated autoimmunity in Doberman Pinscher (DP) with dilated cardiomyopathy, which has similarities to human DCM., Materials and Methods: Eighty-seven DP with cardiomyopathy in terms of pathological ECG and echocardiography (DoCM) and 31 dogs (at enrollment) without DoCM (controls) were analyzed for serum activity of β1-AAB with a bioassay that records the chronotropic response of spontaneously beating cultured neonatal rat cardiomyocytes to the DP's IgG. To locate the receptor binding site of β1-AAB and the autoantibody's sensitivity to inhibition, competing experiments with related blockers were performed with the bioassay. In controls that developed DoCM during follow-up, β1-AAB were analyzed during progress., Results: Fifty-nine (67.8%) DoCM dogs and 19 (61.3%) controls were β1-AAB positive. Of the controls that developed DoCM, 8 were β1-AAB positive (p = 0.044 vs. dogs remaining in the control group); their β1-AAB activity increased with the cardiomyopathy progress (p<0.02). To supplement DoCM group with the 9 animals which developed cardiomyopathy in the follow up, a more pronounced β1-AAB positivity became visible in the DoCM group (p = 0.066). Total and cardiac mortality were higher in β1-AAB positive DP (p = 0.002; p = 0037). The dogs' β1-AAB recognized a specific epitope on the second extracellular receptor and were sensitive to inhibition by drugs already successfully tested to inhibit the corresponding human autoantibody., Conclusions: Doberman Pinschers presented β1-AAB associated autoimmunity, similar as in the pathogenesis of human DCM. Consequently, DP could compensate the lack of animal models for the investigation of β1-AAB autoimmunity in human DCM., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: The authors declare that the study was not funded by any public, commercial or private funds. Gerd Wallukat, Niels-Peter Becker, Katrin Wenzel, Johannes Müller and Ingolf Schimke as employees and shareholder (GW, JM, IS) of Berlin Cures GmbH declare that this does not alter our adherence to PLOS ONE policies on sharing data and materials. Berlin Cures GmbH only provided support in the form of salaries and research materials but did not have any additional role in the study design, data collection, analysis and statistical evaluation, decision to publish or preparation of the manuscript. Anna Fritscher and Gerhard Wess declare that they have no conflict of interest. Berlin Cures GmbH, a spin-off company, founded in September 2014 for the commercial exploitation of patents held by Charité – Universitätsmedizin Berlin and Max-Delbrück-Center Berlin, Germany.

Autoantibodies directed against the P450 side chain cleavage enzyme (P450scc) have been recently described in dogs affected with hypoadrenocorticism, consistent with an immune-mediated pathogenesis of this endocrinopathy. In human autoimmune Addison's disease, autoantibodies may have a predictive value, being detectable before clinical signs developing, and have been shown to persist for a period of time after diagnosis. Furthermore, an autoantibody positive status post-diagnosis has been associated with successful remission of Addison's disease following B-cell depletion, suggesting active immunopathology in these cases. The current study was designed to investigate changes in serum P450scc autoantibody status over time in dogs diagnosed with spontaneous hypoadrenocorticism. P450scc autoantibodies were measured using a species-specific radioimmunoprecipitation assay in an initial cohort of 213 dogs, indicating a prevalence of 24%. Thirty two of these dogs had repeat samples (n = 80 in total) available for analysis. Five dogs were consistently P450scc autoantibody positive in all samples, for up to 425 days following first sampling. Three dogs were initially autoantibody positive, then became seronegative at later time points. One dog, a 1 year old female entire standard poodle, was initially negative for P450scc autoantibodies, but seroconverted 18 months after diagnosis. The remaining 23 dogs with multiple samples available were consistently P450scc autoantibody negative. Persistence was not associated with sex (p = .673). This study demonstrates persistence of P450scc autoantibodies in a subset of dogs affected with hypoadrenocorticism and seroconversion over one year post-diagnosis. P450scc autoantibody reactivity in human autoimmune Addison's disease has been associated with sex, with females having a higher prevalence, possibly due to P450scc expression in the ovary acting as an additional source of antigenic stimulation. However, there was no sex difference in autoantibody persistence in the dogs affected with hypoadrenocorticism. Autontibody persistence in dogs with hypoadrenocorticism might represent persistent pathology, due to residual antigenic stimulation and autoimmune inflammation in the adrenal gland., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Immune system involvement is suggested as an underlying cause for Doberman hepatitis (DH) based on female predisposition, lymphocyte infiltration, abnormal hepatocyte expression of major histocompatibility complex class II antigens, and homozygosity for dog leukocyte antigen DRB1*00601., Objective: To measure serum antinuclear antibodies (ANA) and serum antihistone antibodies (AHA) in Dobermans with hepatitis. To determine whether increased serum ANA or serum AHA could be used to support the diagnosis of Doberman hepatitis (DH)., Animals: Privately owned 25 subclinically and 13 clinically affected DH Dobermans and 17 healthy control Dobermans., Methods: Case-control study. Indirect immunofluorescence (IIF) microscopy and line blot tests were employed for the ANA pilot studies and an enzyme-linked immunosorbent assay (ELISA) assay for detection of IgG AHA., Results: Indirect immunofluorescence revealed ANA-positive cases, and line blot showed AHA reactivity. In ELISA, importantly increased concentrations of AHA were found in 92% (23/25) of dogs in the subclinical stage and 84.6% (11 of 13) of dogs in the clinical stage of DH compared with no control dogs (0/17) (P < 0.0005). The mean AHA absorbance values of the blood samples obtained from the 25 subclinical DH dogs (1.36 ± 0.60, mean ± SD) and the 13 clinically affected dogs (1.46 ± 0.49) were significantly higher than in 17 control dogs (0.51 ± 0.18; P < 0.0001)., Conclusions and Clinical Importance: As the presence of AHA indicates autoimmune activity, our results favor an autoimmune background as one cause for DH. Antihistone antibody could represent a novel means for screening Dobermans with increased serum alanine transaminase concentrations and suspicion of DH., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Objective: To determine if concentrations of free thyroxine (FT4) measured by semi-automated chemiluminescent immunoassay (CLIA) correspond to FT4 determined by equilibrium dialysis (ED) in hypothyroid dogs positive for thyroglobulin antibody (TGA)., Animals: Thirteen TGA-positive dogs classified as hypothyroid based on subnormal FT4 concentrations by ED., Methods: Qualitative assessment of canine TGA was performed using an enzyme-linked immunosorbent assay. Serum total thyroxine and total triiodothyronine concentrations were measured by radioimmunoassay. Serum FT4 concentration was determined by ED, and also by semi-automated CLIA for human FT4 (FT4h) and veterinary FT4 (FT4v). Canine thyroid stimulating hormone concentration was measured by semi-automated CLIA., Results: Each dog's comprehensive thyroid profile supported a diagnosis of hypothyroidism. For detection of hypothyroidism, sensitivities of CLIA for FT4h and FT4v were 62% (95% CI, 32-85%) and 75% (95% CI, 36-96%), respectively, compared to FT4 by ED. Five of 13 (38%) dogs had FT4h and 2 of 8 (25%) dogs had FT4v concentrations by CLIA that were increased or within the reference range. Percentage of false-negative test results for FT4 by CLIA compared to ED was significantly (P < .0001 for FT4h and P < .001for FT4v) higher than the hypothesized false-negative rate of 0%., Conclusions and Clinical Importance: Caution should be exercised in screening dogs for hypothyroidism using FT4 measured by CLIA alone. Some (25-38%) TGA-positive hypothyroid dogs had FT4 concentrations determined by CLIA that did not support a diagnosis of hypothyroidism., (Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Objective: To detect antibodies for lens βH-crystallins in the serum from the American Cocker Spaniel (ACS) presenting with and without cataracts and with and without uveitis., Animal Studied: Seventy-three American Cocker Spaniels and six normal Beagles., Procedures: Sera were collected from 73 ACSs, including those with normal lenses and those with cataracts, or uveitis. Fractionated, normal Beagle lens βH-crystallins were separated by one- or two-dimensional electrophoresis. The separated lens βH-crystallins were used on immunoblots as sentinel substrates against which the ACS sera were tested for the presence of antibodies against βH-crystallins., Results: Sera from approximately two-thirds of study animals contained antibodies to some βH-crystallin polypeptides, but reactivity varied among patients. Contrary to some hypotheses, serum antibodies to groups of βH-crystallins did not relate to the stages of cataract. However, detailed analysis by two-dimensional immunoblotting and mass spectrometry showed that three spots originating from βA1-crystallin were detected only in sera from cataract patients., Conclusion: Serum antibodies to βA1-crystallin may be associated with the development of cataract., (© 2013 The Authors Veterinary Ophthalmology published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Ophthalmologists.)

The pathogenesis of necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalomyelitis (GME) is still uncertain, although they are considered immune-mediated diseases. The purpose of the present study is to generate a rodent model(s) of these diseases. Rats were injected with rat cerebral or cerebellar homogenate. Rats injected with cerebral homogenate (Cbr) exhibited vacuolar or malacic changes mainly in the cerebral cortex. CD3-positive T cells and Iba-1-positive and CD163-negative microglia infiltrated and activated around the lesions. IgG deposited in the glial fibrillary acid protein (GFAP)-positive glia limitans from the early phase, and CD3-positive T cells attached to GFAP-positive astrocytes. Autoantibodies against GFAP were detected in the sera. These pathological features of Cbr rats were consistent with those of canine NME. In contrast, rats injected with cerebral homogenate (Cbe) exhibited demyelinating lesions with inflammatory reactions in the cerebellum, brainstem, and spinal cord. The presence of demyelination and autoantibodies against myelin proteins in Cbe rats was similar to murine experimental autoimmune encephalitis and differed from NME, NLE, and GME. All the present findings indicate that autoantibodies together with microglia and T cells may play a major role in the pathogenesis of idiopathic canine meningoencephalomyelitis., (© The Author(s) 2014.)

Background: Pemphigus foliaceus (PF) is the most common IgG-mediated autoimmune skin disease in dogs. Studies of human PF have revealed the presence of other antigen-specific autoantibody isotypes, thereby uncovering new avenues of investigation of the disease pathomechanism., Hypothesis/objectives: The aim was to obtain information about the autoantibody isotype response in canine PF., Methods: Sera from 34 dogs with PF were tested for the presence of antikeratinocyte, anti-desmocollin-1 and anti-desmoglein-1 IgA, IgE and IgM using indirect immunofluorescence., Results: Using our indirect immunofluorescence technique, IgA, IgE and IgM autoreactivities were detected in six, one and zero of 34 sera from PF-affected dogs, respectively. Two of the six IgA-positive sera contained antikeratinocyte and anti-desmocollin-1 IgA, while the four remaining sera tested positive either for antikeratinocyte IgA (two of six) or for anti-desmocollin-1 IgA (two of six). A single serum contained anti-desmocollin-1 IgE. None of the six sera from healthy dogs contained detectable IgA, IgE or IgM autoantibodies., Conclusions and Clinical Importance: Our findings suggest that sera from dogs with PF rarely contain IgA or IgE autoantibodies at levels detectable by indirect immunofluorescence, while IgM autoreactivity appears not to be a feature of this disease. Considering these findings, it appears that canine PF is aetiologically and immunologically similar to that of the classic human PF, in which the IgG autoantibody response is also the predominant type., (© 2014 ESVD and ACVD.)

Objective: To investigate whether differences existed between clinically normal dogs and dogs with goniodysgenesis-related glaucoma (GDRG) in serum autoantibodies against optic nerve antigens., Animals: 16 dogs with GDRG, 17 healthy dogs with unremarkable pectinate ligament and iridocorneal angle morphology, and 13 euthanized dogs with no major ocular abnormalities or underlying diseases., Procedures: Western blotting was performed with optic nerve extracts from the euthanized dogs as an antigen source and serum from clinically normal dogs and dogs with GDRG as a primary antibody (autoantibody) source. Blots were evaluated for presence and density of bands., Results: Multiple bands were identified on western blots from all dogs with GDRG and all clinically normal dogs, with a high degree of variability among individual dogs. Dogs with GDRG were significantly more likely than healthy dogs to have bands present at 38, 40, and 68 kDa. Dogs with GDRG had significant increases in autoreactivity at 40 and 53 kDa and a significant decrease in autoreactivity at 48 kDa., Conclusions and Clinical Relevance: Significant differences in serum autoantibodies against optic nerve antigens were found in dogs with versus without GDRG. Although it remains unclear whether these differences were part of the pathogenesis of disease or were sequelae to glaucomatous changes, these findings provide support for the hypothesis that immune-mediated mechanisms play a role in the development or progression of GDRG. However, the high degree of variability among individual dogs and the considerable overlap between groups suggest that the clinical usefulness of this technique for distinguishing dogs with GDRG from clinically normal dogs is likely limited.

Objective: To determine the prevalence of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) in dogs with confirmed or suspected immune-mediated hemolytic anemia (IMHA) or dogs infected with various vector-borne pathogens, including Rickettsia rickettsii, Bartonella henselae, Bartonella vinsonii subsp berkhoffii, Ehrlichia canis, Borrelia burgdorferi, and Leishmania infantum., Animals: 55 dogs with confirmed or suspected IMHA, 140 dogs seroreactive for vector-borne pathogens, and 62 healthy dogs and dogs seronegative for vector-borne pathogens., Procedures: Samples were allocated to subgroups on the basis of the health status of the dogs and the degree of seroreactivity against various vector-borne pathogens. Serum samples were tested retrospectively via indirect immunofluorescence assay to determine pANCA status., Results: 26 of 55 (47%) dogs with confirmed or suspected IMHA and 67 of 140 (48%) dogs seroreactive for vector-borne pathogens had positive results when tested for pANCA. Serum samples with the highest antibody concentrations against L infantum antigen had the highest proportion (28/43 [65%]) that were positive for pANCA. One of 20 (5%) dogs seronegative for tick-borne pathogens and 8 of 22 (36%) dogs seronegative for L infantum had positive results for pANCA. One of 20 (5%) healthy dogs had serum antibodies against pANCA., Conclusions and Clinical Relevance: pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.

Objective: To estimate the prevalence of perinuclear antineutrophilic cytoplasmic autoantibodies (pANCA) in the serum of healthy Soft Coated Wheaten Terriers (SCWTs) in the United Kingdom and to identify potential risk factors and heritability patterns associated with a positive result for pANCA., Animals: 188 SCWTs (age range, 18 months to 14.3 years)., Procedures: Blood samples were obtained from SCWTs in various locations in England. Serum was tested for pANCA by use of an immunofluorescence assay, and total protein and albumin concentrations were determined. Pedigrees were evaluated to identify close relatives that had protein-losing enteropathy (PLE) or protein-losing nephropathy (PLN)., Results: 39 of 188 (20.7%) dogs, including young dogs, had positive results for pANCA. Dogs had significantly higher odds of having positive results for pANCA if they had at least 1 littermate that had PLE or PLN (odds ratio, 12.1) or if they had at least 1 full sibling from another litter known to be affected with PLE or PLN (odds ratio, 4.0). CONCLUSIONS AND CLINICAL RELEVANCE; This study revealed a high prevalence of pANCA in the serum of a representative sample of healthy SCWTs in the United Kingdom and a significant association between positive results for pANCA and a diagnosis of PLE or PLN in a sibling.

Objective: To determine whether dogs with spontaneously-occurring diabetes mellitus demonstrate serological reactivity to proinsulin., Sample Population: Serum samples were collected from 15 newly-diagnosed diabetic, 15 insulin-treated diabetic and 15 non-diabetic control dogs., Procedures: Canine proinsulin was cloned into a prokaryotic expression vector to generate recombinant poly-histidine-tagged protein in Escherichia coli. A Western blotting assay was developed for detection of proinsulin autoantibodies in canine sera., Results: Reactivity to canine proinsulin was detected in 3 of 15 control dogs, 8 of 15 newly-diagnosed diabetic dogs and 6 of 15 insulin-treated diabetic patients. Of these reactors, only 1 control dog, 1 newly-diagnosed diabetic dog and 3 insulin-treated diabetic dogs recognised porcine insulin by ELISA, suggesting that the remaining proinsulin reactors might have been recognising proinsulin-specific epitopes., Conclusions and Clinical Relevance: This study suggests that proinsulin autoantibodies are present in a proportion of diabetic dogs. Further work is required to refine the assay and clarify the significance of these autoantibodies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Autoantibodies against thyroxin (T4AA) and triiodothyronine (T3AA) are present in dogs with autoimmune thyroiditis and have been reported to interfere with immunoassays. The objectives of this study were to determine the frequency of autoantibodies and to determine whether interference occurs by T4AA, using a non-immunological method (high performance liquid chromatography, HPLC) for thyroxin (T4) measurement. Based on clinical symptoms, T4 and thyroid stimulating hormone (TSH) concentration, 1,339 dogs were divided into six groups: Group 1: hypothyroid (n = 149); Group 2: subclinical thyroiditis (n = 110); Group 3: suspicious for non thyroidal illness (n = 691); Group 4: biochemical euthyroid (n = 138); Group 5: hypothyroid dogs under substitution therapy (n = 141); Group 6: healthy dogs (n = 110). The incidence of T4AA and T3AA, determined using radiometric assay, was low (0.5% and 3.8%) and higher in hypothyroid dogs compared to dogs suspicious for hypothyroidism (Group 2-4) (p < 0.05). T4AA was not detected in dogs with normal T4 and elevated TSH. T4 concentrations of T4AA positive samples determined using HPLC were comparable to results obtained by chemiluminescence immunoassay. These findings indicate that the probability of interference of T4AA leading to falsely elevated T4 concentration in the T4 assay seems to be low.

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., (© 2010 The Authors. Journal compilation © 2010 ESVD and ACVD.)

Survivin overexpression has been reported in relation to tumor malignancy, suggesting that it is an unfavorable prognostic marker, and antibody responses to this protein have been confirmed in human cancer patients. In this study, we investigated antibody responses to survivin in canine cancer cases, and examined the prevalence of such responses by enzyme-linked immunosorbent assay (ELISA) using recombinant canine survivin protein as the antigen. The cut-off value for positivity in the anti-survivin ELISA was 0.35, as determined using the mean absorbance +2 S.D. of samples from healthy dogs. Sera from 16 of 59 (27.1%) cancer and 3 of 25 (12%) non-cancer disease dogs were positive on ELISA. The highest positivity rates (>50%) among the cancer cases were seen in dogs with mammary tumor, squamous cell carcinoma and melanoma.

Vaccination against pathogenic microorganisms is one of the major achievements of modern medicine, but due to an increasing number of reports of adverse reactions the vaccination procedure has induced also considerable debate. It is well known that certain infections are involved in triggering the production of autoantibodies, which could lead to autoimmune adverse reactions in genetically predisposed subjects. Based on these findings it was assumed that vaccinations might induce similar autoimmune reactions. At present there is no clear-cut evidence that vaccinations are associated with overt autoimmune diseases but it has been demonstrated that in genetically predisposed persons vaccination can trigger the production of autoantibodies and autoimmune adverse reactions. The first studies investigating the production of autoantibodies following vaccination were done in dogs and mice. Several studies investigated the production of autoantibodies following vaccination in patients with autoimmune diseases, but there are only limited data on the autoimmune responses after vaccinations in apparently healthy humans. This review summarizes current evidence on the vaccination-induced autoantibodies in apparently healthy subjects including studies in animals and humans.

Background: Dogs with immune-mediated hemolytic anemia (IMHA) and certain inflammatory diseases are at high risk of developing thromboembolic disease. The presence of anti-endothelial cell autoantibodies (AECA) has been associated with an increased risk of thromboembolism in humans., Hypothesis: AECA will be detected more often in dogs at risk of thromboembolism than in healthy control animals or dogs with diseases not associated with a higher risk of thromboembolism., Animals: Ninety-one sick dogs and 22 healthy control dogs., Methods: Retrospective case-controlled study. Serum was screened for the presence of AECA. Dogs were identified for the study based on the risk of thromboembolism as determined by clinical impression and the underlying disease process. Flow cytometry and normal canine endothelial cells were used to screen serum samples from sick and healthy control dogs for the presence of AECA. In addition, serum from dogs with confirmed thromboemboli was also screened for the presence of AECA by immunohistochemistry., Results: AECA were detected in 2/91 sick dogs, both with infectious diseases, but were not found in healthy dogs. Anti-endothelial antibodies were not detected in 21 dogs with IMHA and 20 dogs with systemic inflammatory response syndrome, sepsis, or both., Conclusions: We conclude that AECA are rarely detectable in dogs considered at high risk of thromboembolism. These findings suggest that AECA may not play an important role in the pathogenesis of thromboembolism in dogs with IMHA and other inflammatory diseases.

Canine necrotizing meningoencephalitis (NME) is characterized by autoantibodies against glial fibrillary acidic protein (GFAP) in cerebrospinal fluids (CSFs). To clarify the time-course changes in autoantibodies, serial examinations were conducted in three dogs with NME (two Pugs and a Pomeranian) that were treated by immunosuppressive therapy. The Pugs retained high autoantibody titers throughout the observation periods (146 and 813 days) and died with neurological signs. On the other hand, the Pomeranian switched from being positive for autoantibody to negative after day 580, and its NME seemed to be in clinical remission until death on day 1238. Therefore, the anti-GFAP autoantibodies can be detected over time in canine NME even during immunosuppressive therapies. However, the autoantibodies can also disappear within a certain period after onset.

Acquired canine myasthenia gravis is an autoimmune disease in which autoantibodies are directed against muscle postsynaptic nicotinic acetylcholine receptors. Three adult great dane littermates were evaluated over a four month time period for an acute onset of generalised neuromuscular signs. All three dogs had elevated serum acetylcholine receptor antibody titres, which were considered diagnostic for acquired myasthenia gravis. Identification of three littermates with acquired myasthenia gravis in a breed with a low relative risk of developing the disease suggests a familial and possibly a genetic predisposition to myasthenia gravis in this family of dogs.

Diabetes mellitus in dogs shares many characteristics with the human type 1 disease and virtually all diabetic dogs require insulin therapy to control hyperglycaemia. Insulin deficiency is suspected to result from immune-mediated destruction of pancreatic beta cells in some cases. Human patients suffering from Type 1A (immune-mediated) diabetes or latent autoimmune diabetes of the adult (LADA) demonstrate circulating autoantibodies against the 65kDa isoform of glutamic acid decarboxylase (GAD65) and/or insulinoma antigen-2 (IA-2). The aims of the current study were to develop radio-immunoassays to detect serum antibodies against recombinant canine GAD65 and IA-2 and to identify diabetic dogs showing serological evidence of autoreactivity to these pancreatic beta cell antigens. Canine GAD65 and the 3' end of IA-2 (coding for amino acids 771-979 of the intracellular domain) were amplified by PCR from cDNA prepared from canine insulinoma tissue and cloned into the pCRII vector. The canine sequences were later confirmed by identifying GAD2 and PTPRN genes from the dog genome assembly. Recombinant (35)S-methionine-radiolabelled canine GAD65 and IA-2 (771-979) proteins were used in radio-immunoprecipitation assays to screen sera from 30 newly diagnosed diabetic dogs and 30 control dogs. Four of 30 canine diabetic patients had significant GAD65 autoreactivity (p<0.01) compared to controls and 3 dogs were positive for autoantibodies to IA-2 (771-979). Two diabetic dogs showed dual autoantigen reactivity. These preliminary data indicate that serological reactivity to GAD65 and IA-2 is present in a proportion of diabetic dogs and suggests that, in some cases, canine diabetes is associated with an autoimmune response to these antigens.

Objective: To evaluate perinuclear anti-neutrophilic cytoplasmic autoantibody (pANCA) status in Soft Coated Wheaten Terriers (SCWTs) and SCWT-Beagle crossbred dogs and to correlate pANCA status of dogs with clinicopathologic variables of protein-losing enteropathy (PLE), protein-losing nephropathy (PLN), or both., Animals: 13 SCWTs and 8 SCWT-Beagle crossbred dogs in a research colony and a control group comprising 7 dogs with X-linked hereditary nephropathy and 12 healthy SCWTs > 9 years old., Procedures: Samples were obtained from dogs in the research colony every 6 months. At each sample-collection time point, serum concentrations of albumin, globulin, creatinine, and urea nitrogen; fecal concentration of alpha-proteinase inhibitor; and urinary protein-to-creatinine ratios were determined and correlated with pANCA status., Results: 20 of 21 dogs in the research colony had positive results for pANCAs at a minimum of 2 time points, and 18 of 21 dogs had definitive evidence of disease. None of the control dogs had positive results for pANCAs. A positive result for pANCAs was significantly associated with hypoalbuminemia, and pANCAs preceded the onset of hypoalbuminemia on an average of 2.4 years. Sensitivity and specificity for use of pANCAs to predict development of PLE or PLN were 0.95 (95% confidence interval, 0.72 to 1.00) and 0.8 (95% confidence interval, 0.51 to 0.95), respectively., Conclusions and Clinical Relevance: Most dogs in this study affected with PLE, PLN, or both had positive results for pANCAs before clinicopathologic evidence of disease was detected. Thus, pANCAs may be useful as an early noninvasive test of disease in SCWTs.

Human patients with atopic dermatitis (AD) commonly exhibit IgE reactivity to cutaneous self-antigens. The presence of serum IgE autoantibodies appears to correlate with disease severity, and it is suspected to reflect or contribute to tissue damage. The objective of this study was to determine whether IgE autoantibodies specific for cutaneous antigens could be detected in the serum of dogs with AD. Serum was collected from 19 dogs with untreated moderate to severe AD and four specific-pathogen free (SPF) dogs. Indirect immunofluorescence was performed using normal canine skin collected at four different locations (concave ear, nose, medial thigh and lateral thorax), while Western immunoblotting was done using normal canine ear pinna epidermal and dermal extracts and reducing conditions. In both methods, IgE was detected using a monoclonal antibody specific for heat stable epitopes of canine IgE. At 1:10 dilution, specific IgE autoantibodies against cutaneous autoantigens were not detected, with either method, in AD and SPF canine sera. Either IgE autoreactivity is not associated with moderate to severe AD in dogs, or the methods employed herein were not sensitive enough to permit IgE autoantibody detection.

There is increasing evidence that a large proportion of dilated cardiomyopathy (DCM) cases are mediated by autoimmune processes. Since DCM is a fatal disorder with rapid aggravation and is the leading cause of heart transplantation, further insights into disease pathogenesis are needed. Recent studies have separated the pathogenic capacity of autoantibodies and initial clinical trials removing such autoantibodies via immunoadsorption have been promising. In order to elucidate the full autoantibody repertoire involved in DCM, we applied an autoantibody screening test using ventricular and atrial proteomes as autoantigenic sources and subsequently tested the autoantibody-binding patterns of sera from dogs with spontaneous DCM. With this method, we detected five potentially DCM-related autoantigens which were identified by MS as being: myosin heavy chain cardiac muscle alpha isoform, alpha cardiac actin, mitochondrial aconitate hydratase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and brain glycogen phosphorylase (GPBB). The recovery of two known DCM autoantigens (myosin heavy chain and alpha cardiac actin) and the discovery of three novel autoantigens (mitochondrial aconitate hydratase, GADPH, and GPBB) underscore the efficacy of this experimental method and the significance of the spontaneous canine DCM model.

Inflammatory myopathies are a group of autoimmune diseases that affect muscles. In humans, the most common inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis. Autoantibodies may be found in humans with inflammatory myopathies, and these play an important role in diagnosis and disease classification. However, these Abs are typically not muscle specific. Spontaneously occurring canine inflammatory myopathies may be good parallel disorders and provide insights into human myositis. In dogs with inflammatory myopathy, muscle-specific autoantibodies have been found, especially in masticatory muscle myositis. We have identified the major Ag recognized by the autoantibodies in canine masticatory muscle myositis. This Ag is a novel member of the myosin binding protein-C family, which we call masticatory myosin binding protein-C (mMyBP-C). mMyBP-C is localized not only within the masticatory muscle fibers, but also at or near their cell surface, perhaps making it accessible as an immunogen. The gene for mMyBP-C also exists in humans, and mMyBP-C could potentially play a role in certain human inflammatory myopathies. Understanding the role of mMyBP-C in this canine inflammatory myopathy may advance our knowledge of mechanisms of autoimmune inflammatory muscle diseases, not only in dogs, but also in humans.

To establish clinical markers for canine necrotising meningoencephalitis (NME) and to elucidate its pathogenesis, glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibodies were measured in the cerebrospinal fluid (CSF) of 32 dogs with NME, 23 dogs with other inflammatory central nervous system (CNS) diseases, 27 dogs with miscellaneous CNS diseases and 25 healthy dogs, including five pugs. The dogs with NME had the highest levels of anti-GFAP autoantibodies. The diagnostic sensitivity and specificity of anti-GFAP autoantibodies for NME were 91 per cent and 73 per cent, respectively. Some of the dogs with NME and the healthy pugs, had high CSF concentrations of GFAP, suggesting a breed-specific fragility of astrocytes. The leakage of GFAP and the development of autoimmunity may be key to understanding the pathogenesis of NME.

A 4-year-old, male Labrador retriever, weighing 27 kg, presented with abrupt clinical signs including mental retardation, circling and head pressing. The dog never ingested water by choice. An adipsia of the dog was persisted and developed to hypernatremia with artifactual hyperchloremia. Serial endocrine results and image findings were suggestive of a hypothyroidism. The dog revealed the presence of antithyroid antibodies in the cerebrospinal fluid and serum. With the administration of levothyroxine sodium, his neurologic signs were alleviated within the first week of treatment and adipsia was also resolved.

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.

Objective: To evaluate anticollagen type I antibodies in synovial fluid of the affected stifle joint, the contralateral stifle joint, and the left shoulder joint of dogs with unilateral cranial cruciate ligament (CrCL) rupture during an extended period of 12 to 18 months., Animals: 13 client-owned dogs with CrCL rupture and 2 sham-operated dogs., Procedures: All dogs were examined and arthrocentesis of all 3 joints was performed every 6 months after surgery. Synovial fluid samples were tested for anticollagen type I antibodies by use of an ELISA., Results: Dogs with partial CrCL rupture had higher antibody titers than dogs with complete rupture. Six of 13 dogs ruptured the contralateral CrCL during the study, whereby higher antibody titers were found for the stifle joints than for the shoulder joint. Seronegative dogs or dogs with extremely low antibody titers and 2 dogs with high antibody titers did not sustain a CrCL rupture in the contralateral stifle joint., Conclusions and Clinical Relevance: In most dogs that had a CrCL rupture of the contralateral stifle joint, a distinct antibody titer gradient toward the stifle joints was detected, suggesting that there was a local inflammatory process in these joints. However, only a small number of sham-operated dogs were used to calculate the cutoff values used to determine the anticollagen type I antibody titers in these patients. Synovial fluid antibodies against collagen type I alone do not initiate CrCL rupture because not all dogs with high antibody titers sustained a CrCL rupture in the contralateral stifle joint.

Cerebrospinal fluids (CSFs) from 9 Pug dogs with necrotizing meningoencephalitis (NME: Pug dog encephalitis) were examined to identify the antigens for anti-astrocyte autoantibodies. Each CSF exhibited a positive reaction to the cytoplasm of cultured canine astrocytes by an indirect fluorescent antibody test. In an immunoblotting analysis on normal canine brain proteins, eight of 9 CSFs showed a common band of 52 kDa, corresponding to glial fibrillary acidic protein (GFAP), and all of 9 CSFs reacted with purified bovine GFAP. From these results, GFAP is one of the common autoantigens in Pug dogs with NME. On the other hand, the reactivity of CSFs to chymotrypsin-digested bovine GFAP fragments were variable among dogs, indicating that the antibodies in the CSFs recognized different epitopes on GFAP.

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.

Inflammatory myopathies (IM) are relatively common in dogs with an increased incidence in the Boxer and Newfoundland breeds. Here, we show that a high proportion of affected Boxers and Newfoundlands have circulating autoantibodies against unknown sarcolemma antigens, that are muscle-specific but not species specific. We further show that the autoantigen can be extracted from muscle membranes with non-ionic detergent, and that such detergent extracts can be used in a sensitive ELISA for detection and quantitation of antibodies. The relatively high incidence of IM with autoantibodies in selected breeds of dogs indicates a genetic predisposition for a particular form of IM. In these breeds, this form of IM could be diagnosed and monitored with a simple serum assay.

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.

Objective: To determine the presence of serum antiretinal antibodies in sudden acquired retinal degeneration syndrome (SARDS) affected dogs and the size of the antigen to which these antibodies bind via the use of enzyme-linked immunosorbent assay (ELISA) and Western blot immunoassays., Animals Studied: Serum was collected from 13 dogs affected by SARDS and five dogs with normal ocular examinations., Procedures: All serum samples were subjected to ELISA with saline-soluble canine retinal tissue and Western blot analyses with SDS solubilized normal canine retinal tissue as the antigen. Antirecoverin (23 kDa) and antiheat shock cognate (65 kDa) antibodies were used as positive controls for both procedures. Affinity-purified goat antidog IgG and IgM labeled with horseradish peroxidase were used for all clinical samples and goat antirabbit IgG was used as the secondary antibody for the positive controls., Results: ELISA demonstrated antibody reaction with all samples. Western blot immunoassays identified multiple bands in all canine serum samples, as well as in negative controls. Approximate sizes of the bands were 25 and 50 kDa, corresponding to IgG light and heavy chains, respectively., Conclusion: No antiretinal autoantibodies were identified in the serum of dogs affected by SARDS as compared to normal canine patients.

Steroid-responsive meningitis-arteriitis (SRMA) is a disease of dogs familiar in small animal practice for decades. A combined evaluation of IgA in serum and cerebrospinal fluid (CSF) is an important diagnostic tool. It is suspected that immunpathological mechanisms are involved in the pathogenesis of SRMA because of the marked response to steroids. Excessive production of IgA seems to play a central role and might be caused by an immune reaction to self-antigens of the central nervous system (CNS). To test this hypothesis, we analyzed CSF samples from 55 dogs with SRMA using the western blot method. After blotting canine brain tissue, IgA, IgM and IgG of the CSF samples were tested for their binding to CNS antigens. We also evaluated CSF samples from 45 dogs with other brain diseases, including different encephalitides and intracranial tumors, and from healthy dogs as controls. Positive reactions (mostly IgA) were observed in the CSF samples from dogs with SRMA, different encephalitides and brain tumors (a total of 8% positive samples). The occurrence of autoantibodies against CNS structures was significantly higher in the control group "other brain diseases" than in the SRMA group (p = 0.0135). There was no significant difference in the number of positive samples between dogs with SRMA and the negative control group (healthy dogs, p = 0.1535). Despite the small number of positive samples, only dogs with abnormal findings in the CSF analysis also had autoantibodies in the CSF. There was no significant correlation between the occurrence of autoantibodies and levels of IgA, protein content and cell counts in the cerebrospinal fluid. However, there was a certain trend toward positive reactions in CSF samples with high protein content. The occurrence of autoantibodies in dogs with SRMA thus seems to be an epiphenomenona rather than the cause of the disease.

A seven-year-old, spayed female mongrel dog was diagnosed as pemphigus foliaceus (PF) by clinical, histopathological and immunopathological observations. Serum antibodies against the cell surface of keratinocytes in the dog were detected by indirect immunofluorescence (IIF) using cryosectioned bovine esophagus as well as living cultured-canine keratinocytes as the substrates. When we compared the titers of IIF on bovine esophagus with its disease activity, the IIF titers reflected the disease activity throughout the time course. Our findings will suggest that sequential titration of serum antibodies by IIF will be useful for monitoring the serological disease activity in canine PF.

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.

Thyroglobulin autoantibodies (TgAA) were detected in sera of hypothyroid (n=19), obese (n=28) and clinically healthy dogs (n=52) using a commercially available immunoassay kit. TgAA-positive results occurred in 10 of 19 hypothyroid, 1 of 28 obese and 1 of 52 clinically healthy dogs. The clinically healthy TgAA-positive dog had additional evidence of hypothyroidism supported by low total T(4), low free T(4) and high canine TSH. Among the breeds, Golden Retriever had the highest frequency of hypothyroid (9/19) and TgAA-positive hypothyroid dogs (6/10). This study was the first survey about the prevalence of canine TgAA in Japan and could be a useful reference for clinicians.

To examine the prevalence of autoantibody in canine cerebrospinal fluids (CSFs), CSFs were collected from 14 healthy controls and 88 clinical cases with various diseases in the central nervous system (CNS), and were analyzed by an indirect fluorescence antibody test on frozen sections of the cerebrum from normal Beagle dogs. An anti-astrocyte autoantibody was detected in 31 clinical cases with titers ranging from 1:1 to >/=1:100. All tested cases with necrotizing meningoencephalitis (NME: n=22) and granulomatous meningoencephalitis (GME: n=3) possessed the anti-astrocyte autoantibody, while the autoantibody was negative in most cases with other inflammatory CNS diseases. The autoantibody was also detected in 4 of 12 cases with brain tumors. Hence, examination of the autoantibody in the canine CFS would be significant for diagnosing NME and/or GME, as well as for understanding peritumoral events in cases with brain tumors.

Objective: To determine if antilens crystallin (ALC) serum and aqueous humor antibodies were present in normal dogs and dogs with cataracts, whether antibody incidence varied with stage of cataract, and whether antibody titer had a relationship to the presence of lens-induced uveitis., Methods: Serum and aqueous humor samples were obtained from normal dogs and dogs with cataracts. Lens crystallin was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and antilens crystallin antibodies were detected by Western immunoblot analysis. An indirect ELISA using crystallin protein as antigen was also used to detect antilens crystallin antibodies in serum and aqueous humor. Test groups included normal, incipient, immature, mature, hypermature and diabetic cataract., Results: SDS-PAGE identified bands with molecular weights of lens crystallin subunits. Western immunoblotting demonstrated reaction between canine serum and these protein bands. The five canine serum samples that reacted with crystallin subunits on Western blots had corresponding reactivity on the ELISA. All aqueous humor samples (30) were negative. Serum ALC antibodies were detected in 59.3% (16/27) of controls, 66.7% (16/24) of incipients, 50.0% (10/20) of immatures, 37.9% (11/29) of matures, 28.6% (6/21) of hypermatures, and 26.7% (4/15) of diabetics. Serum ALC antibodies were detected in 43.1% (47/109) of all cataract samples. There was a statistically significant negative association between the presence (P = 0.004) and maturity (P = 0.004) of cataract and presence of ALC serum antibodies. In the immature and hypermature cataract groups, there was a statistically significant negative association between ALC serum antibody titer and severity of uveitis (95% confidence interval)., Conclusions: There is a negative association between the presence (P = 0.004) and maturity (P = 0.004) of cataract and presence of ALC serum antibodies.

Circulating thyroglobulin autoantibody (TgAA) was analyzed using the Western immunoblot for determination of the dominant epitopes recognized by TgAA on tryptic peptides of canine thyroglobulin (cTg) in hypothyroid dogs. TgAA was measured in hypothyroid dogs, non-hypothyroid dogs with skin diseases and clinically normal dogs. Five of the 7 hypothyroid dogs, 1 of the 8 dogs with skin diseases and 1 of the 4 normal dogs were positive for TgAA. Four of the 5 TgAA-positive hypothyroid dogs were Golden Retrievers, and 3 of them showed high antibody titers. The sera of TgAA positive-dogs reacted to several peptides, and their patterns varied from sample to sample. Sera from 3 dogs with high titers of TgAA reacted broadly to high molecular weight peptides ranging from 45 to 90 kDa. These Western immunoblot patterns of the sera were disappeared after pretreatment with sufficient amount of intact cTg. All serum samples of both TgAA positive dogs and negative controls reacted to low molecular weight peptides ranging from 15 to 20 kDa. These immunoblot patterns of the sera were not disappeared even after pretreatment with sufficient amount of intact cTg. These findings show the possibility that the epitopes recognized by TgAA depend upon individual dogs with hypothyroidism and these autoantibodies recognize conformational epitopes on the cTg molecule.

A mongrel dog, aged 2 years, was found to have only a small number of sperm, immobilization of all sperm, and many sperm agglutinations in its ejaculates, and scrotal palpation revealed a small nodule in the left cauda epididymis. Addition of the dog's seminal plasma or serum to the semen of 2 normal dogs caused immobilization and agglutination of their sperm. Histological examination showed that the nodule was a sperm granuloma. Many lymphocytes were seen in the stroma around the sperm granuloma. Anti-sperm antibodies are presumed to be present in the semen and serum of the asthenozoospermic dog.

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.

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.