Canine serum thyroglobulin autoantibodies in health, hypothyroidism and non-thyroidal illness

Research in Veterinary Science 1999, 66, 243–246 Article No. rvsc.1998.0268, available online at http://idealibrary.com on

Canine serum thyroglobulin autoantibodies in health, hypothyroidism and non-thyroidal illness R.M. DIXON* University of Glasgow, Department of Veterinary Clinical Studies, Bearsden Road, Glasgow, G61 1QH C.T. MOONEY University College Dublin, Faculty of Veterinary Medicine, Department of Small Animal Clinical Studies, Ballsbridge, Dublin 4

SUMMARY Thyroglobulin autoantibody (TGAA) was measured in serum from dogs with hypothyroidism (n = 42), non-thyroidal illness (NTI) (n = 77) and clinically healthy dogs (n = 70) using a commercially available enzyme immunoassay kit. Precision studies were consistent with good intra-assay and inter-assay repeatability. TGAA positive results occurred in 15 of the 42 (36 per cent) hypothyroid and four healthy dogs of the remaining 147 animals resulting in a lower incidence of false positive results than obtained with previous TGAA assays. The presence of TGAA was not influenced by age, sex, neutering or pedigree status. Of the four apparently healthy TGAA-positive dogs, two had additional clinicopathological evidence of hypothyroidism. TGAA was positive in 43 per cent of hypothyroid dogs with unexpectedly normal serum cTSH concentrations and was particularly useful in the classification of these cases.

HYPOTHYROIDISM is a common endocrine disease of the dog. Approximately half of all naturally occurring cases result from lymphocytic thyroiditis (Gosselin et al 1981) a condition analogous to Hashimoto’s thyroiditis of humans. Like the human disease lymphocytic thyroiditis in the dog is considered to be immune mediated (Beale et al 1990) and a strong genetic predisposition has been proposed (Haines et al 1984b, Conaway et al 1985). In conjunction with appropriate clinical signs and clinicopathological abnormalities, evidence of thyroiditis as demonstrated by measurable circulating antibodies to thyroglobulin (TGAA) has been considered useful in supporting a diagnosis of hypothyroidism (Beale 1991). However many TGAA measurement techniques previously available were cumbersome to use and were associated in particular, with positive results in euthyroid dogs with non-thyroidal illness (Gosselin et al 1980, Haines et al 1984a, 1984b, Vollset and Larsen 1987, Beale et al 1990, Beale 1991). A commercial enzyme-linked immunosorbent assay (ELISA) kit is now available for canine serum TGAA measurement (Canine thyroglobulin autoantibody immunoassay kit, Oxford Laboratories, MI). This assay was recently evaluated by Nachreiner et al (1998) in order to determine a diagnostic threshold and was found to be useful in the identification of autoimmune thyroiditis in the dog. The purposes of this study were to more fully evaluate the potential value of this assay in the investigation of canine hypothyroidism by comparing results from clinically healthy dogs and those with confirmed hypothyroidism or non-thyroidal illnesses.

*Corresponding author

MATERIALS AND METHODS Case details and sample handling Blood samples were collected by jugular venipuncture. After clotting, samples were centrifuged and the serum harvested and stored at minus 20°C in plain glass tubes until analysis. The dogs were divided into three groups. Groups 1 and 2 consisted of 119 dogs referred to the University of Glasgow Department of Veterinary Clinical Studies for investigation of clinical or clinicopathological abnormalities consistent with hypothyroidism. In each of these dogs, serum thyrotropin (thyroid stimulating hormone, cTSH), total thyroxine (T4) and free T4 by equilibrium dialysis (fT4d) were measured by commercially available kit methods (canine TSH IRMA, Diagnostic Products; Magic T4, Chiron Diagnostics; Free T4 by equilibrium dialysis, Nichols Institute Diagnostics, respectively) and the dogs were classified as either hypothyroid (Group 1) or euthyroid (Group 2) based on the results of a bovine thyrotropin (TSH) response test as previously described (Dixon and Mooney, 1999). Group 1 (mean age 7·4; range 3 to 12 years) contained 24 female (12 neutered) and 18 male (two neutered) and group 2 (mean age 7·4; range 1 to 14 years) contained 36 female (20 neutered) and 41 male (eight neutered) dogs. Group 3 consisted of 70 clinically healthy dogs. This group was divided into two subsets, group 3A consisting of 19 dogs of a variety of breeds (mean age 4·9, range 0·5 to 12 years) and group 3B consisting of 51 bearded collies (mean age 3·9, range 0·5 to 11 years) being sampled at the request of their owners as part of a breeding program. Group 3A contained 10 female (six neutered) and nine male (six neutered) dogs and group 3B contained 32 female (five neutered) and 19 male (two neutered) dogs. TGAA

assay

TGAA was measured using a canine thyroglobulin autoantibody immunoassay, (Oxford Laboratories, MI) according

0034-5288/99/030243 + 04 $18.00/0

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R.M. Dixon, C.T. Mooney

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to the manufacturers instructions. The kit contains a 96-well polystyrene plate precoated with purified thyroglobulin. Diluted test sera samples and pre-supplied positive and negative control sera are added to the wells allowing any TGAA present to bind to the immobilised thyroglobulin. Anticanine immunoglobulin (IgG) conjugated to horseradish peroxidase (HRP) is then added and attaches to the TGAA. Following the addition of an enzyme substrate a colour change occurs which is proportional to the quantity of HRP and therefore sample TGAA concentration. A stop solution is added immediately prior to measurement of the optical density of each well at 450 nm. Plates were read using a commercial plate reader (MRX Microplate Reader, Dynex Technologies (UK) Ltd.). All test and validation samples were measured in duplicate and all standards in hexuplicate. Using the manufacturers instructions, optical density values greater than twice that of the negative control were considered positive whilst values less than two standard deviations above that of the negative control were considered negative with intermediate results being classified as equivocal. Inter-assay precision was determined by qualitative comparison of TGAA in aliquots of three samples (positive, negative and equivocal) measured in duplicate in six consecutive assay runs. Both the positive and negative aliquots gave positive and negative results respectively on every occasion. The equivocal aliquots gave five equivocal and one negative result. Intra-assay precision was determined by qualitative comparison of TGAA in 20 replicates each of two samples (positive and negative) in a single assay run. Each sample gave consistent results on each occasion.

TABLE 1: Serum hormone results (mean ± SD) in healty dogs and those with confirmed hypothyroidism or non-thyroidal illness (NTI).

Basal T4 Post-TSH T4 (nmol L–1) cTSH (ng ml–1) fT4d (pmol L–1)

*Healthy

Hypothyroid

NTI

24·3 ± 6·5 not done 0·29 ± 0·22 19·6 ± 5·5

7·8 ± 4·5 9·5 ± 6·8 2·2 ± 2·5 5·4 ± 3·4

22·2 ± 11·0 62·3 ± 33·2 0·41 ± 0·46 16·5 ± 9·25

*Results exclude the two dogs subsequently confirmed as hypothyroid.

TABLE 2: Principle disease categories affecting dogs with nonthyroidal illness. *The ‘other’ category includes one each of various conditions not otherwise represented in the table. Disease Type

Number of Dogs

Dermatological Obesity Non-thyroidal endocrine diseases Neurological Neoplasia Cardiovascular Musculoskeletal Gastrointestinal Ophthalmological Other*

20 (26%) 16 (21%) 10 (13%) 8 (10%) 6 (8%) 6 (8%) 2 (3%) 3 (4%) 2 (3%) 4 (5%)

TABLE 3: TGAA results in hypothyroid (group 1), euthyroid with non-thyroidal illness (group 2) and healthy (group 3) dogs.

Category Group 1 (Hypothyroid) Group 2 (Euthyroid) Group 3 (Healthy)

Number of Dogs

Negative

TGAA Result Equivocal

Positive

42 77 70

19 (45%) 72 (94%) 55 (79%)

8 (19%) 5 (6%) 11 (16%)

15 (36%) 0 (0%) 4 (6%)

Statistical analysis The effect of thyroid status, sex, neutering or pedigree status on TGAA results was determined by chi-squared analysis. Comparison of dog ages and thyroid hormone concentrations between groups was performed using a two-sample t-test. A P-value < 0·05 was considered significant.

TABLE 4: Concentrations of serum basal T4, free T4, and cTSH in apparently healthy but TGAA-positive dogs. Animal Beardie 09 Beardie 14 Beardie 15 Rottweiler

Basal T4 (nmol L–1)

fT4d (pmol L–1)

cTSH (ng ml–1)

10·9 30·4 14·4 19·0

4·11 16·96 4·74 13·28

11·3 0·46 9·31 0·19

RESULTS Hormone concentrations from each group are summarised in Table 1. There was no significant effect of age, sex or neutering status on the likelihood of being hypothyroid. The distribution of the major diseases affecting dogs in group 2 is summarised in Table 2. Results of TGAA measurement in groups 1, 2 and 3 are summarised in Table 3. There was no significant difference in TGAA-positive, -equivocal or -negative results between groups 3A and 3B and therefore these were combined for analyses. Three of the four TGAA-positive dogs in this group were entire female bearded collies and the remaining one was an entire male rottweiler. Basal thyroid hormone analyses from these dogs are presented in Table 4 and supported either euthyroidism (n = 2) or hypothyroidism (n = 2) as evidenced by subnormal total T4 and fT4d and markedly elevated cTSH concentrations. There was a significant increase in TGAA-positive results in group 1 compared to group 2 and group 3 (P < 0·001 in each case) and in group 3 compared to group 2 (P = 0·02). There was a significant (P = 0·01) increase in TGAA-equivocal

results in group 1 compared to both group 2 and to groups 2 and 3 combined but there was no significant difference between group 2 and group 3. There was no significant difference in age, sex or neutering status between TGAA-positive and -negative animals within each group although the TGAA-positive hypothyroid dogs (mean age 6·6, range 3·0 to 10·6 years) tended (P = 0·09) to be younger than the TGAAnegative hypothyroid individuals (mean age 8·1, range 4·0 to 12·0 years). There was no significant difference in the prevalence of TGAA-positive results between pedigree and non-pedigree dogs both within each group and in the all the dogs as one group. There was no significant difference in basal total T4, cTSH or fT4d concentrations between TGAA-positive, -negative or -equivocal dogs within groups 1 and 2. There was no significant difference in hormone results between TGAA-negative and -equivocal dogs within group 3. Statistical analyses were not performed in TGAA-positive animals within group 3 because of subsequent confirmation of hypothyroidism in two of the four animals.

Thyroglobulin autoantibodies and thyroid function

In seven hypothyroid dogs with reference range cTSH concentrations, TGAA was positive in three (43 per cent), equivocal in one (14 per cent) and negative in three (43 per cent). In nine euthyroid dogs with elevated cTSH concentrations TGAA was equivocal in one (11 per cent), and negative in eight (89 per cent).

DISCUSSION Circulating canine TGAA were first identified using a tanned cell haemagglutination (TCH) method (Beierwaltes and Nishiyama 1968). Various other methods, mostly modifications of human assays have subsequently been used to identify canine TGAA (Gosselin et al 1980, Haines et al 1984a, Haines et al 1984b, Vollset and Larsen 1987, Vollset et al 1987, Thacker et al 1995). Passive haemagglutination tests (PHT) including the TCH, chromic chloride (CCM) and glutaraldehyde (GCH) methods have been commonly used but are time-consuming and cumbersome to perform (Vollset et al 1987). An ELISA method has previously been investigated (Haines et al 1984a) and although more convenient was not recommended for routine diagnostic use. The preparation of antigens and other assay components inhouse results in variation within and between laboratories and therefore the use of a commercially available species specific kit with minimal preparative steps is preferable. This study found the assay used to be precise and technically straightforward thereby assisting the routine laboratory classification of hypothyroid dogs. Whilst the lack of thyroid biopsy material prevented direct confirmation of thyroiditis in the animals under study, Nachreiner et al (1998) demonstrated excellent correlation of TGAA-positive results with histological evidence of thyroiditis using the same assay. The demonstration of TGAA in over a third of hypothyroid dogs is similar to previous reports of 24 per cent (GCH method) and 34 per cent (CCH method) of 45 hypothyroid dogs reported by Vollset and Larsen (1987) and 48 per cent (CCH method) and 24 per cent (TCH method) of 25 hypothyroid dogs reported by Gosselin et al (1980). Haines et al (1984a) used a modified ELISA method and found TGAA in 59 per cent of 34 hypothyroid dogs. Using the same assay as in the present study, Nachreiner et al (1998) found TGAA in 87 of 171 (51 per cent) hypothyroid dogs. However some of these animals were specifically selected for the presence of thyroid hormone autoantibodies which would bias the results in favour of TGAA positivity. The true prevalence of TGAA in hypothyroid dogs is therefore likely to be nearer that of the current study. None of the dogs with non-thyroidal illness including those with non-thyroidal endocrine diseases were positive for TGAA although five (6 per cent) were equivocal. These equivocal cases were spread between cardiovascular (n = 2), dermatological (n = 1), endocrinological (n = 1), and neurological (n = 1) diseases. The low prevalence of TGAA found in non-thyroidal illnesses is similar to the findings of Nachreiner et al (1998) who examined a total of 146 samples from dogs with calcium disorders, hyperadrenocorticism, diabetes mellitus, obesity and hypoadrenocorticism and reported TGAA-positive results in only eight (5 per cent) cases. No euthyroid dogs in the present study had recognised

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calcium disorders although cases with hyperadrenocorticism (n = 4), diabetes mellitus (n = 4), obesity (n = 9), and hypoadrenocorticism (n = 2) were present and negative for TGAA. Earlier studies reported a considerably higher prevalence of autoantibodies in dogs with a variety of non-thyroidal illnesses particularly endocrinopathies. Haines et al (1984b) found TGAA in 43 per cent of 65 dogs with non-thyroidal endocrine disease and 13 per cent of 1057 hospitalised dogs with various non-endocrine diseases. Of 40 dogs with non-thyroidal illnesses reported by Gosselin et al (1980), 12 per cent were positive for TGAA by CCH although all were negative when TCH was used. The difference in methodologies reported is undoubtedly a major reason for the conflicting published results and the availability of the assay used in this study should help standardise future studies. TGAA-positive and -equivocal results respectively occurred in four (6 per cent) and 11 (16 per cent) of 70 apparently healthy dogs. Two of these TGAA-positive dogs had additional biochemical evidence suggestive of hypothyroidism as did two of three apparently healthy TGAA-positive dogs reported by Nachreiner et al (1998). This suggests that the presence of circulating TGAA in apparently healthy animals could be an early indicator of thyroid disease as it is in humans (Gordin and Lamberg 1975) and may be associated with extreme elevations in cTSH concentrations as was found in the two dogs in this study. However, because biochemical euthyroidism was demonstrated in the remaining two dogs, a TGAA-positive result should not be presumed to co-exist with thyroid dysfunction. Caution is advised in over-interpretation of such results and additional clinical and clinicopathological monitoring for hypothyroidism is advised before a firm diagnosis is made. In a previous study, two of 11 TGAA-positive euthyroid dogs followed for 18 months developed biochemical but not clinical evidence of hypothyroidism (Haines et al 1984b) although other similar cases showed no such progression during this time. The possible subsequent development of hypothyroidism in TGAA-positive healthy dogs could not be supported or refuted in the present study because of limited follow-up information. These studies are currently ongoing to address such a possibility. The importance of TGAA-equivocal results has not been addressed in previous studies (Nachreiner et al 1998). The manufacturers recommend re-testing at a later date, although there is no data available to suggest whether an individual is likely to become TGAA-positive or -negative with time. In the present study, thyroid hormone concentrations were not significantly different in TGAA-equivocal, negative or -positive cases within each group, which is not supportive of a progressive change in TGAA status from negative through equivocal to positive as the transition from euthyroidism to hypothyroidism occurs. However, such results may be influenced by the small number of cases within each category. TGAA-equivocal results were more prevalent amongst hypothyroid than euthyroid dogs, suggesting that investigation of thyroid function should be undertaken as recommended for TGAA-positive dogs. Lengthier longitudinal case studies may help in clarifying the association of TGAA with the stage of thyroid disease which is more clearly defined in humans (Gordin and Lamberg 1975).

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There was no influence of pedigree status on the prevalence of abnormal TGAA results. Previous studies have documented thyroiditis within particular breeds and family lineages (Conaway et al 1985, Haines et al 1984b). In the present study, other than bearded collies, there were relatively few individuals of each breed represented preventing identification of such a breed-predisposition. There was no evidence of sex or neutering status influencing TGAA which agrees with the findings of other studies (Beale et al 1990, Haines et al 1984b, Vollset and Larsen 1987). The TGAA-positive hypothyroid dogs tended to be younger than the TGAA-negative hypothyroid dogs as has been found previously (Nachreiner et al 1998). This is presumably related to the earlier onset of hypothyroidism in individuals at risk from lymphocytic thyroiditis (Feldman and Nelson 1996). Serum cTSH is now routinely measured alongside total T4 or fT4d to establish a diagnosis of hypothyroidism. However a subset of hypothyroid dogs have reference range cTSH concentrations and euthyroid dogs can have cTSH results within the hypothyroid range (Dixon and Mooney 1999, Peterson et al 1997, Scott-Moncrieff et al 1998). TGAA measurement correctly identified thyroid abnormalities in 43 per cent of hypothyroid dogs with reference range cTSH concentrations. This confirms the value of TGAA measurement in these cases because whilst a negative TGAA result cannot exclude hypothyroidism a positive result strongly supports it. The presence of TGAA in a similar proportion of hypothyroid dogs with the expected rise in serum cTSH concentration to those with unexpectedly ‘normal’ cTSH results is strong evidence that central hypothyroidism is not a common cause of such discordant results.

ACKNOWLEDGEMENTS The authors would like to thank Mrs Elizabeth Gault, Dr Elizabeth Aughey, Miss Jan MacDonald and Mr David Irvine for their assistance with this study.

REFERENCES BEALE K.M. (1991) Thyroid pathology and serum antithyroglobulin antibodies in hypothyroid and healthy dogs (abstract). Journal of Veterinary Internal Medicine 5, 128. BEALE K.M., HALLIWELL R.E.W., & CHEN C.L. (1990) Prevalence of antithyroglobulin antibodies detected by enzyme-linked immunosorbent assay of canine serum. Journal of the American Veterinary Medical Association 196, 745–748. BEIERWALTES W.H. & NISHIYAMA R.H. (1968) Dog thyroiditis: Occurrence and similarity to Hashimoto’s struma. Journal of Endocrinology 83, 501–508. CONAWAY D.H., PADGETT G.A., & NACHREINER R.F. (1985) The familial occurrence of lymphocytic thyroiditis in borzoi dogs. American Journal of Medical Genetics 22, 409–414. DIXON R.M. & MOONEY C.T. (1999) Evaluation of serum free thyoxine and thyrotropin concentrations in the diagnosis of canine hypothyroidism. Journal of Small Animal Practice 40, 72–78.. FELDMAN E.C. & NELSON R.W. (1996) Hypothyroidism. In: Canine and Feline Endocrinology and Reproduction, 2nd edn. W.B. Saunders Co., Philadelphia. pp. 68–117. GORDIN A. & LAMBERG B.A. (1975) Natural course of symptomless autoimmune thyroiditis. Lancet ii, 1234–1238. GOSSELIN S.J., CAPEN C.C. & MARTIN S.L. (1981) Histologic and ultrastructural evaluation of thyroid lesions associated with hypothyroidism in dogs. Veterinary Pathology 18, 299–309. GOSSELIN S.J., CAPEN C.C., MARTIN S.L. & TARGOWSKI S.P. (1980) Biochemical and immunological investigations on hypothyroidism in dogs. Canadian Journal of Comparative Medicine 44, 158–168. HAINES D.M., LORDING P.M. & PENHALE W.J. (1984a) The detection of canine autoantibodies to thyroid antigens by enzyme-linked immunosorbent assay, haemagglutination and indirect immunofluorescence. Canadian Journal of Comparative Medicine 48, 262–267. HAINES D.M., LORDING P.M. & PENHALE W.J. (1984b) Survey of thyroglobulin autoantibodies in dogs. American Journal of Veterinary Research 45, 1493–1497. NACHREINER R.F., REFSAL K.R., GRAHAM P.A., HAUPTMAN J. & WATSON G.L. (1998) Prevalence of autoantibodies to thyroglobulin in dogs with nonthyroidal illness. American Journal of Veterinary Research 59, 951–955. PETERSON M.E., MELIAN C. & NICHOLS R. (1997) Measurement of serum total thyroxine, triiodothyronine, free thyroxine and thyrotropin concentrations for the diagnosis of hypothyroidism in dogs. Journal of the American Veterinary Medical Association 211, 1396–1402. SCOTT-MONCRIEFF J.C., NELSON R.W., BRUNER J.M. & WILLIAMS D.A. (1998) Comparison of serum concentrations of thyroid-stimulating hormone in healthy dogs, hypothyroid dogs, and euthyroid dogs with concurrent disease. Journal of the American Veterinary Medical Association 212, 387–391. THACKER E.L., DAVIS J.M., REFSAL K.R. & BULL R.W. (1995) Isolation of thyroid peroxidase and lack of autoantibodies to the enzyme in dogs with autoimmune thyroid disease. American Journal of Veterinary Research 56, 34–38. VOLLSET I. & LARSEN H.J. (1987) Occurrence of autoantibodies against thyroglobulin in Norwegian dogs. Acta Veterinaria Scandinavica 28, 65–71. VOLLSET I., LARSEN H.J. & KNOEVELSRUD T. (1987) Passive hemagglutination for detection of autoantibodies against thyroglobulin in dogs. Acta Veterinaria Scandinavica 28, 117–119. Accepted December 4, 1998