Tryptic peptides of canine thyroglobulin reactive with sera of patients with canine hypothyroidism caused by autoimmune thyroiditis

Canine thyroglobulin (cTg) was treated with trypsin at a ratio of trypsin to cTg of 1:100 (w/w). Tryptic peptides of cTg were analysed by Western immunoblotting for their reactivity to serum thyroglobulin autoantibodies (TgAA) from patients with TgAA-positive hypothyroidism and normal individuals. The sera of patients with TgAA-positive hypothyroidism reacted with several peptides: 43, 32.5 and 31 kDa; the sera of normal individuals did not bind these tryptic peptides. Some of the TgAA-positive sera of patients reacted with 25 kDa peptide in addition to three tryptic peptides above. This experiment was the first report about antigenic epitopes of cTg. These small tryptic peptides recognized by TgAA may be related with the induction of TgAA and may be useful as markers for autoimmune thyroid diseases in dog.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

Experimental thyroid autoimmunity in the dog

Thyroid autoimmunity was induced in 6 crossbred dogs by a single injection of thyroid extract in complete Freund's adjuvant alone, or followed by reimmunization with thyroid extract and incomplete Freund's adjuvant or peanut oil. Total thyroxine levels, autoantibodies to thyroglobulin and thyroid microsomal antigen and thyroid histopathological changes were sequentially monitored for up to 150 days. All dogs developed fluxuating levels of thyroid autoantibodies detectable by enzyme-linked immunosorbent-assay (ELISA). Histopathological damage was observed in one or more thyroid gland biopsies in 4 of the dogs, in 3 cases the lesions were of lymphocytic thyroiditis, in the fourth dog the lesion was a granulomatous reaction. Histopathological thyroid damage was observed in biopsies taken during periods of maximal autoantibody titer. Fixed immunoglobulin was not demonstrable in thyroid gland biopsies by direct immunofluorescence. In 5 dogs total thyroxine levels fell to below the normal range at some period during the study. However in only one dog did this correlate with a time when biopsy findings revealed histopathological thyroid damage. High levels of thyroid autoantibody may indicate histopathological thyroid damage in dogs, however lower levels of thyroid antibody may be found without evidence of histological damage or alteration in thyroid function.     

Lack of association between repeated vaccination and thyroiditis in laboratory Beagles

BACKGROUND: Intensive vaccination protocols have been suggested as partially responsible for an increased prevalence of autoimmune diseases in dogs in recent years. The aim of this study was to determine whether repeated routine vaccination in dogs is associated with an increased prevalence of thyroiditis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a prospective experimental study with 20 healthy purpose-bred Beagles. Five dogs were vaccinated with a multivalent vaccine and a rabies vaccine. Five dogs received only the multivalent vaccine, and 5 dogs received only the rabies vaccine. Five dogs were unvaccinated controls. The multivalent vaccine was administered at 8, 10, 12, 16, 20, 26, and 52 weeks of age and every 6 months thereafter. The rabies vaccine was administered at 16 and 52 weeks of age and then once a year. Blood samples were collected 1 week before euthanasia for evaluation of thyroid profiles and measurement of antibodies directed against canine thyroglobulin. Dogs were euthanized at 5.5 years of age, and the thyroid glands were evaluated histopathologically. Thyroiditis was present in 8 of 20 (40%) dogs at postmortem examination. No association was found between a dog being vaccinated and the prevalence of thyroiditis at postmortem examination. However, the power of the study to detect such an association was low because of the unexpected high prevalence of thyroiditis in the unvaccinated control dogs. Thyroid function tests were abnormal in 2 of 8 dogs with thyroiditis but were normal in all dogs without thyroiditis. CONCLUSIONS/SIGNIFICANCE: There was no evidence to support an association between routine vaccination and thyroiditis at postmortem examination in beagle dogs after repeated vaccination.     

Recognition pattern of thyroglobulin autoantibody from hypothyroid dogs to tryptic peptides of canine thyroglobulin

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.     

Prevalence of diagnostic characteristics indicating canine autoimmune lymphocytic thyroiditis in giant schnauzer and hovawart dogs

O bjectives : To investigate prevalence of autoantibodies to thyroglobulin (TgAA) and/or elevated levels of thyroid stimulating hormone (TSH), indicating canine autoimmune lymphocytic thyroiditis (CLT) and/or hypothyroidism, in two high-risk dog breeds.
M ethods : A cohort study was conducted in two birth cohorts of giant schnauzer and hovawart dogs. The cohorts were three to four and six to seven years of age at the time of blood sampling and screening for TgAA and TSH levels. Blood sampling was accompanied by one initial and one follow-up questionnaire to the dog owners. A total number of 236 giant schnauzers and 95 hovawarts were included in the study.
R esults : Seventeen (7.2 per cent) giant schnauzers and three (3·2 per cent) hovawarts had been diagnosed as hypothyroid at the time of sampling. Out of the remaining dogs, 22 giant schnauzers (10·0 per cent) and nine hovawarts (10·1 per cent) had elevated TgAA and/or TSH levels. Prevalence of elevated TgAA and TSH levels varied with age.
C linical S ignificance : The high prevalence of diagnostic characteristics indicating CLT/hypothyroidism in these two breeds suggests a strong genetic predisposition. It would be advisable to screen potential breeding stock for TSH and TgAA as a basis for genetic health programmes to reduce prevalence of CLT in these breeds.     

Histologic and ultrastructural evaluation of thyroid lesions associated with hypothyroidism in dogs

Thyroid lesions in 16 pet dogs with hypothyroidism were evaluated by light and electron microscopy. Lymphocytic thyroiditis, found in seven dogs, was characterized by diffuse infiltration of the thyroid gland by lymphocytes, plasma cells and macrophages with formation of some lymphoid nodules and destruction of follicles, progressing to replacement of most of the thyroid by fibrous connective tissue. The basement membrane around follicles was thick and had electron-dense deposits. The morphology of the thyroid lesions and the presence of circulating thyroglobulin autoantibodies suggested that lymphocytic thyroiditis was immune-mediated. Idiopathic follicular atrophy, found in nine dogs, was characterized by loss of thyroid parenchyma and replacement by adipose connective tissue. Degeneration of individual follicular cells was present in the early stage, with exfoliation into the colloid and interfollicular area. Most of the thyroid gland consisted of adipose connective tissue with either interspersed small follicles or individual follicular cells that had dilated rough endoplasmic reticulum, large Golgi apparatus, and intracytoplasmic microfollicles in the advanced stage. Follicular atrophy was a degenerative lesion of follicular cells of unknown cause, not associated with inflammatory destruction in the thyroid gland.     

Characterization of and radioimmunoassay for canine thyroglobulin

Canine thyroglobulin (cTg) has been isolated and purified. It has similar electrophoretic patterns as Tg from other mammalian species. The main fraction had a MW of 660,000, whereas also fractions of a MW of approximately 1,300,000 (dimer) and 330,000 (subunit) were present. The iodine content was 0.8 to 1.0 % (w/w). cTg did not cross-react with antibodies against human Tg to a degree that would allow the use of a radioimmunoassay for human Tg for the determination of cTg in serum or plasma. Therefore a polyclonal antiserum was raised against cTg and a homologous radioimmunoassay was developed, which was sensitive (0.4 μg/l) and specific (cross-reactivity in cTg assay of human Tg, goat Tg, T₄, T₃, and DIT < 0.01 %).

Plasma Tg levels in normal dogs of both sexes and aged 3–15 years amounted to 192 ± 73 μg/l (mean ± SD, n=30). There was no relation between plasma Tg and T₄ levels.     

Neuromuscular abnormalities associated with hypothyroidism and lymphocytic thyroiditis in three dogs

Various degrees of persistent or paroxysmal paresis involving only the hindlimbs or all four limbs were observed in 3 dogs with hypothyroidism and lymphocytic thyroiditis. Clinical features included lethargy, obesity, alopecia, insidious and progressive paresis, hypotonia, and slow segmental reflexes in 2 dogs. Obesity, alopecia, paroxysmal paresis, and behavior change were observed in the third dog. Laboratory tests indicated that thyroid function was less than normal in all 3 dogs. Abnormal electromyographic potentials and slow motor nerve conduction velocities were found in each dog. Muscle biopsy specimen abnormalities included selective type-II myofiber atrophy in all dogs, whereas one dog had angular atrophy of type-I and type-II myofibers indicative of denervation. A substance that stained with para-aminosalicylic acid was observed within vacuoles of type-I myofibers in one dog. Lymphocytic thyroiditis characterized by lymphocytic infiltration of excised thyroid glands was observed in all dogs.     

Passive hemagglutination for detection of autoantibodies against thyroglobulin in dogs

Primary hypothyroidism in dogs is mainly caused by autoimmune thyroiditis or idiopathic follicular atrophy of the thyroid gland (Gosselin et al. 1982). Serologically, autoimmune thyroiditis can be diagnosed by detection of autoantibodies against thyroid antigens in hypothyroid dogs (Mizejewski et al. 1971, Gosselin et al. 1980). Passive hemagglutination test (PHT) has been the serological test, most widly used for detection of antibodies (Mizejewski et al. 1971, Gosselin et al. 1982, Vollset & Larsen 1987).     

Experimental autoimmune thyroiditis. II. An immunocytochemical and ultrastructural study of the lesion.

The early steps of thyroid damage in experimentally induced autoimmune thyroiditis in mice were studied. Mice were immunized with 150 micrograms of rat thyroglobulin followed by 20 micrograms of bacterial LPS 3 h later on day 0 and 7. Thyroid glands were obtained on days 14, 21 and 28 in order to be studied by light microscopy, electron microscopy and immunocytochemical techniques. The initial lesion was a focal inflammatory infiltrate composed of lymphocytes and lymphoblasts, and plasma cells as the damage progressed. In some cases an increase in neutrophils, macrophages and eosinophils was also seen. The infiltrate became multifocal and eventually diffuse and in few cases germinal center-like structures were seen. The cellular infiltrate corresponded to IgG, IgM and IgA bearing B cells and to a lesser degree Thy 1.2, Lyt 1 and Lyt 2 bearing T cells. Follicular alterations corresponded to different degenerative states of the follicular cells that in some cases was seen without the participation of lymphoid cells. Another mechanism observed was the migration of small lymphoid cells and plasma cells between the follicular cells.     

Spontaneous thyroiditis in BB Wistar diabetic rats

Spontaneous lymphocytic thyroiditis was observed at necropsy in 36 BB Wistar diabetic rats (63.2%) and in eight of their nondiabetic siblings (42.1%). The incidence of thyroiditis decreased both with age and with longer duration of diabetes. All rats with pancreatic insulitis (a manifestation of the onset of diabetes) also had thyroiditis. BB Wistar rats with insulitis had more severe lymphocytic thyroiditis, characterized by lymphocytic, plasmacytic, and macrophage infiltration of thyroid interstitium and follicles. A milder, mostly perivascular and interstitial lymphocytic thyroiditis was characteristic of lesions in rats which did not have insulitis. The histological appearance of the thyroiditis suggests that these rats may be subject to autoimmune disease at the onset of diabetes which involves sites other than just the pancreas.     

Thyroid carcinosarcoma in a dog

An adult male mixed breed dog developed pain, on swallowing, that lasted for 1 week. Physical exam and radiographs revealed a subcutaneous mass in the cervical area and three metastatic nodules in the lung. The cervical mass was surgically removed and fixed in 10% buffered formalin. Histopathologic diagnosis was carcinosarcoma supported by positive immunohistochemistry (IHC) results for cytokeratin and vimentin. IHC for thyroglobulin proved that the tissue of origin was the thyroid gland. This is the fifth canine case of thyroid carcinosarcoma to be documented, but the first one to be confirmed by specific cell markers. It is an extremely rare neoplasm that also occurs in people.     

Comparison of thyroid analytes in dogs aggressive to familiar people and in non-aggressive dogs

A cross-sectional study was performed in order to examine the association between canine aggression to familiar people and serum concentrations of total thyroxine (TT4), free thyroxine (fT4), thyroxine autoantibodies (T4AA), total triiodothyronine (TT3), free triiodothyronine (fT3), triiodothyronine autoantibodies (T3AA), thyroid stimulating hormone (TSH), and thyroglobulin autoantibodies (TgAA). The subjects were 31 dogs historically aggressive to familiar people and 31 dogs with no history of aggression. Behavioral evaluation and physical examination were completed for each dog in addition to a complete blood count, serum chemistry panel, TT4, fT4 by equilibrium dialysis, TT3, fT3, TgAA, T3AA, and T4AA. Significant differences were found between the two groups with respect to only T4AA, which was increased in the aggressive group, but the concentrations for both groups were within the normal reference range. There were no differences between the two groups in the thyroid analytes most commonly measured by veterinary practitioners evaluating thyroid function in dogs. The results of this study revealed no significant difference between aggressive and non-aggressive dogs in the thyroid concentrations most commonly used to diagnose canine hypothyroidism.     

Some immunopathological aspects of aged Praomys (Mastomys) natalensis

$\text{Praomys (Mastomys) natalensis}$ develops a wide variety of autoantibody specificities with age; of these only those to thyroid colloid were associated with a histopathological lesion e.g. thyroiditis. An inverse relationship was found between the mean number of autoantibodies and the occurrence of lymphoreticular tumors. It appears that $\text{Mastomys}$ is a better model for studies on the general mechanisms of autoimmunity than for investigating specific autoimmune diseases per se.     

Clinical hypothyroidism associated with trimethoprim-sulfadiazine administration in a dog

Treatment of a 9-year-old spayed female mixed-breed dog with trimethoprim-sulfadiazine for a prolonged period resulted in clinical signs of hypothyroidism, and results of thyroid gland function tests were indistinguishable from those associated with endogenous hypothyroidism. Drug-induced hypothyroidism was diagnosed on the basis of history, normal thyroid uptake of sodium pertechnetate, and complete recovery of thyroid gland function after administration of trimethoprim-sulfadiazine was discontinued.     

Experimental autoimmune thyroiditis. I. Participation of IgA, IgM and IgG in the development of the disease

Many factors have been shown to be important in the pathogenesis of experimentally induced autoimmune thyroiditis (EAT) in mice. However, the role of thyroid antibodies has not been clearly established. EAT was induced with rat Tg + LPS in RK mice. Two injections were given i.v. on days 0 and 7 and the antibody titre against rat and syngeneic Tg was determined in the weeks thereafter. Determination of the cellular infiltration of the thyroid, the presence of IgA, IgG and IgM in thyroid sections and electron microscope analysis of electro-dense deposits were also undertaken. Results show that (1) RTg + LPS is a potent inducer of autoimmunity with high antibody titer to RTg and MTg and 100% of the mice showed some degree of thyroid infiltrate on days 21 and 28; (2) immunofluorescence staining revealed that initially IgM is the main immunoglobulin and is later replaced by IgG. IgA is constantly present throughout the experimental period; (3) electro-dense deposits were found almost exclusively between the capillary basement membrane and the follicular basement membrane.     

A comparison of the spring-rise phenomenon in the faecal nematode-egg counts of housed sheep with that of sheep grazing infective pasture

Abstract

A ram lamb with congenital goitre showed signs of respiratory distress at birth. All previously affected lambs from this flock had been born dead or had died soon after birth. The serum free-thyroxine level at birth was low (9.8pmol/?) but treatment with oral L-thyroxine resulted in an increase in thyroxine blood levels to above normal and regression of the goitre. Four weeks after withdrawal of thyroid supplementation and when the lamb was eight weeks old, the radio iodide uptake mechanism of the thyroid glands was shown to be intact but the iodide was not organified. Low normal peroxidase activity was demonstrated in fresh thyroid tissue but gel electrophoresis demonstrated an absence of normal thyroglobulin from extracts of the goitrous thyroid. This lamb and the other affected lambs of this flock have an inherited defect in thyroglobulin synthesis and it is likely that the respiratory distress was associated with the low levels of thyroid hormone affecting foetal lung development.     

A defect in thyroglobulin synthesis in an inherited ovine goitre: possible neonatal respiratory distress syndrome

A ram lamb with congenital goitre showed signs of respiratory distress at birth. All previously affected lambs from this flock bad been born dead or bad died soon after birth. The serum free-thyroxine level at 1 birth was low (9.8 pmol/l) but treatment with oral L-thyroxine resulted in an increase in thyroxine blood levels to above normal and regression of the goitre. Four weeks after withdrawal of thyroid supplementation and when the lamb was eight weeks old, the radio iodide uptake mechanism of the thyroid glands was shown to be intact but the iodide was not organified. Low normal peroxidase activity was demonstrated in fresh thyroid tissue but gel electrophoresis demonstrated an absence of normal thyroglobulin from extracts of the goitrous thyroid. This lamb and the other affected lambs of this flock have an inherited defect in thyroglobulin synthesis and it is likely that the respiratory distress was associated with the low levels of thyroid hormone affecting foetal lung development.     

Rat lymphocytic thyroiditis associated with ingestion of an immunosuppressive compound

Lymphocytic thyroiditis was induced in young Wistar rats by feeding them the immunosuppressive compound frentizole [1-(6-methoxy-2-benzothiazolyl)-3-phenyl urea] for 1 year. About half of the rats given 0.060 and 0.150% frentizole in the diet had lymphocytic thyroiditis. The incidence of thyroiditis was low in the group given the high dose because of severe anemia and hepatic disease which resulted in increased mortality. Reversibility of the thyroid lesion was indicated by reduced incidence rates at 15 and 18 months after treatment was stopped at 1 year. The thyroiditis was characterized by interstitial infiltrates of many lymphocytes and plasma cells and fewer macrophages with mild degenerative changes in the follicular epithelium. This inflammatory cell infiltrate was generally diffuse but occasionally was multifocal, particularly in thyroid glands of rats late in the reversibility phase of the study. The inflammatory cell infiltrate caused the thyroid glands to be several times normal size. Sera from rats with lymphocytic thyroiditis contained hemagglutinating antibody against rat perfect correlation between the presence of anti-thyroglobulin antibody and enlarged thyroid glands. Fine granular deposits of IgG and complement were identified in some areas of the follicular basement membrane. We concluded that the lymphocytic thyroiditis was immunologically mediated, at least in part, by anti-thyroglobulin antibody.