Use of recombinant human thyroid-stimulating hormone for thyrotropin stimulation test in healthy, hypothyroid and euthyroid sick dogs

Recombinant human thyroid-stimulating hormone (rhTSH) was evaluated for the diagnosis of canine hypothyroidism, using TSH response tests. Phase I stimulation tests were performed in 6 healthy dogs weighing over 20 kg, using 50 and then 100 microg of freshly reconstituted rhTSH administered intravenously. In phase II, the same dogs were stimulated by using 100 microg of rhTSH frozen for 3 months at -20 degrees C. Phase III stimulation tests were performed by using 50 or 100 microg of freshly reconstituted or frozen rhTSH in healthy (n = 14), euthyroid sick (n = 11) and hypothyroid dogs (n = 9). A dose of 100 microg of rhTSH was judged more appropriate for dogs weighing more than 20 kg. Biological activity of rhTSH after freezing at -20 degrees C for up to 12 weeks was maintained. When stimulated, significant (P < 0.05) increases in total thyroxine concentration were observed only in healthy and euthyroid sick dogs. Results of this study show that the rhTSH stimulation test is able to differentiate euthyroidism from hypothyroidism in dogs.     

Use of recombinant human thyroid-stimulating hormone for thyrotropin stimulation test in euthyroid dogs

The purpose of this study was to evaluate the effects of the recombinant human thyroid-stimulating hormone (rhTSH) on serum total thyroxine (TT4) concentration in euthyroid dogs. Six healthy beagle dogs were used in each of the 3 phases of this study. Phase I: thyroid-stimulating hormone response tests were performed by using a total dose of 25 micrograms, 50 micrograms, and 100 micrograms of rhTSH, administered intravenously. Phases II and III: thyroid-stimulating hormone response tests were performed by using 50 micrograms of rhTSH administered by intramuscular and subcutaneous routes, respectively. In each phase and following all the administered doses of rhTSH, an increase in the serum TT4 concentration was noted, although it was not always significant. For phase I, there was a significant increase in serum TT4 concentrations. Based on this study, 50 micrograms was judged to be the optimal intravenous dose of rhTSH. For phases II and III, there was no significant increase in serum TT4 after the administration of rhTSH. Results of this study suggest that rhTSH could be a good substitute for bovine TSH, when used by the intravenous route, for the TSH stimulation test in dogs. Further studies are required to confirm its clinical usefulness.     

Effect of storage of reconstituted recombinant human thyroid-stimulating hormone (rhTSH) on thyroid-stimulating hormone (TSH) response testing in euthyroid dogs

Bovine thyrotropin (bTSH) stimulation testing has long been considered the gold standard for diagnosis of canine hypothyroidism. Unfortunately, bTSH is no longer commercially available. Recently, the use of recombinant human thyrotropin (rhTSH) to perform thyroid-stimulating hormone (TSH) stimulation testing in dogs was described. The cost of an rhTSH vial (1.1 mg) limits the practical use of this product. The study reported here was performed to determine the effects of storing rhTSH on the post-TSH increase of serum total (TT4) and free (FT4) thyroxine concentrations during TSH stimulation testing in 12 euthyroid Beagles in a crossover trial. Three TSH tests with recombinant human thyrotropin (rhTSH; 91.5 microg IV) were performed on each dog during 3 different periods: 1 with freshly reconstituted rhTSH (fresh); 1 with rhTSH, reconstituted and stored at 4 degrees C for 4 weeks (refrigerated); and 1 with rhTSH, reconstituted and frozen at -20 degrees C for 8 weeks (frozen). Blood samples for determination of TT4 and FT4 concentrations were collected before and 4 and 6 hours after rhTSH administration. There was no significant difference in TT4 or FT4 concentration after stimulation with fresh, refrigerated, and frozen rhTSH. Furthermore, there was no significant difference between TT4 or FT4 serum concentration observed 4 and 6 hours after rhTSH administration. In conclusion, reconstituted rhTSH can be stored at 4 degrees C for 4 weeks and at -20 degrees C for 8 weeks without loss of biological activity, allowing clinicians to perform more TSH response tests per vial.     

Comparison of biological activity and safety of recombinant canine erythropoietin with that of recombinant human erythropoietin in clinically normal dogs.

OBJECTIVE: To determine whether recombinant canine erythropoietin (rcEPO) stimulates erythropoiesis in dogs without causing the immunogenicity problem (ie, erythroid hypoplasia) associated with recombinant human erythropoietin (rhEPO). ANIMALS: 13 clinically normal dogs. PROCEDURE: Dogs were randomly assigned to 2 groups; 1 group (n = 6) received rhEPO, whereas the other group (7) received rcEPO. Both groups received SC injections of diluent for 4 weeks before initiating treatment with erythropoietin (100 U/kg of body weight, SC, 3 times/wk). Hematocrit and absolute reticulocyte count were monitored weekly, CBC were done monthly, and bone marrow aspirates for cytologic evaluation were obtained before and at 4, 8, 16, and 24 weeks during treatment. RESULTS: Weekly mean Hct and absolute reticulocyte count increased in both groups of dogs during the first 2 weeks of treatment. For dogs receiving rhEPO, precipitous decreases in reticulocyte number and more gradual decreases in Hct were associated with development of erythroid hypoplasia. Dogs receiving rhEPO developed erythroid hypoplasia by week 4 (n = 4), 8 (1), or 16 (1). With cessation of rhEPO treatment after diagnosis of erythroid hypoplasia, RBC production recovered 5 to 11 weeks (median, 7 weeks) later. In contrast, rcEPO treatment caused sustained increases in Hct and reticulocytosis. None of the dogs receiving rcEPO developed erythroid hypoplasia. CONCLUSIONS: rcEPO stimulated erythrocyte production in clinically normal dogs during a 24-week period without causing the erythroid hypoplasia encountered in rhEPO-treated dogs. CLINICAL RELEVANCE: Because rcEPO did not cause erythroid hypoplasia, rcEPO may represent an improved option, compared with rhEPO, for treatment of erythropoietin-dependent anemia in dogs.     

Evaluation of recombinant human thyroid-stimulating hormone to test thyroid function in dogs suspected of having hypothyroidism

OBJECTIVE: To evaluate the use of recombinant human (rh) thyroid-stimulating hormone (TSH) in dogs with suspected hypothyroidism. ANIMALS: 64 dogs with clinical signs of hypothyroidism. PROCEDURES: Dogs received rhTSH (75 microg/dog, IV) at a dose independent of their body weight. Blood samples were taken before and 6 hours after rhTSH administration for determination of total serum thyroxine (T(4)) concentration. Dogs were placed into 1 of 3 groups as follows: those with normal (ie, poststimulation values indicative of euthyroidism), unchanged (ie, poststimulation values indicative of hypothyroidism; no thyroid gland stimulation), or intermediate (ie, poststimulation values between unchanged and normal values) post-TSH T(4) concentrations. Serum canine TSH (cTSH) concentration was determined in prestimulation serum (ie, before TSH administration). RESULTS: 14, 35, and 15 dogs had unchanged, normal, and intermediate post-TSH T(4) concentrations, respectively. Basal T(4) and post-TSH T(4) concentrations were significantly different among groups. On the basis of basal serum T(4) and cTSH concentrations alone, 1 euthyroid (normal post-TSH T(4), low basal T(4), and high cTSH concentrations) and 1 hypothyroid dog (unchanged post-TSH T(4) concentration and low to with-in reference range T(4) and cTSH concentrations) would have been misinterpreted as hypothyroid and euthyroid, respectively. Nine of the 15 dogs with intermediate post-TSHT(4) concentrations had received medication known to affect thyroid function prior to the test, and 2 of them had severe nonthyroidal disease. CONCLUSIONS AND CLINICAL RELEVANCE: The TSH-stimulation test with rhTSH is a valuable diagnostic tool to assess thyroid function in selected dogs in which a diagnosis of hypothyroidism cannot be based on basal T(4) and cTSH concentrations alone.     

Use of recombinant human thyroid-stimulating hormone for thyrotropin-stimulation testing of euthyroid cats

OBJECTIVE: To evaluate response of euthyroid cats to administration of recombinant human thyroid-stimulating hormone (rhTSH). ANIMALS: 7 healthy cats. PROCEDURE: Each cat received each of 5 doses of rhTSH (0, 0.025, 0.050, 0.100, and 0.200 mg), IV, at 1-week intervals. Serum concentration of total thyroxine (TT4) and free thyroxine (fT4) was measured immediately before each injection (time 0) and 2, 4, 6, and 8 hours after administration of each dose. RESULTS: Overall TT4 response did not differ significantly among cats when administered doses were > or = 0.025 mg. Serum TT4 concentrations peaked 6 to 8 hours after administration for all doses > or = 0.025 mg. For all doses > or = 0.025 mg, mean +/- SEM TT4 concentration at 0, 6, and 8 hours was 33.9 +/- 1.7, 101.8 +/- 5.9, and 101.5 +/- 5.7 nmol/L, respectively. For all doses > or = 0.025 mg, mean fT4 concentration at 0, 6, and 8 hours was 38.7 +/- 2.9, 104.5 +/- 7.6, and 100.4 +/- 8.0 pmol/L, respectively. At 8 hours, the fT4 response to 0.025 and 0.050 mg was less than the response to 0.100 and 0.200 mg. Adverse reactions after rhTSH administration were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: The TSH stimulation test can be performed in cats by IV administration of 0.025 to 0.200 mg of rhTSH and measurement of serum TT4 concentrations at time of injection and 6 or 8 hours later. Clinical validation of the TSH stimulation test would facilitate development of additional tests of thyroid gland function, such as a TSH assay.     

Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Total serum IgE and IgA antibody levels in healthy dogs of different breeds and exposed to different environments

Total serum immunoglobulin (Ig) E and A levels were analysed in 233 healthy dogs as basis for comparison with atopic dogs in future studies. They were measured by ELISA in a group of non- colonised dogs of various breeds (group A) and three groups of colonised dogs including one German Shepherd and two Beagle kennels (groups B-D). IgE levels from non-colonised dogs were significantly higher than the ones of German Shepherds and Beagles C (P<0.05). IgA levels were alike in all groups except for the German Shepherds which displayed the lowest levels. Age and sex were not identified as common significant cofactors for IgE and IgA levels in all groups and IgE levels correlated negatively with IgA only in non-colonised dogs. In conclusion, IgE and IgA levels seem to be mainly influenced by genetic background. Thus use of total serum IgE as a diagnostic tool in the atopic dogs required extensive family data and therefore appears most suitable for research purposes within specific, well defined dog populations.     

Comparison of femoral inclination angle measurements in dysplastic and nondysplastic dogs of different breeds

In this study, inclination angle of the femoral head and neck was measured on 484 limbs of 242 dogs belonging to 7 breeds, examined for hip dysplasia. These inclination angles were compared according to age, sex and joint laxity, evaluated with Subluxation Index (SI) and Norberg angle (NA) results. The findings indicate that (a) there was a minimal (nonsignificant) difference in femoral inclination angle between the dysplastic and nondysplastic dogs belonging to 7 breeds; (b) although there was no significant difference in femoral inclination angle between the nondysplastic dogs belonging to 4 breeds (Pointer, Irish Setter, Golden Retriever and German Shepherd), a significant difference was observed between Doberman and Labrador, and between Anatolian Karabash and the other six breeds (p < 0.001). Age and sex did not affect the femoral neck angle.     

Secretion pattern of thyroid-stimulating hormone in dogs during euthyroidism and hypothyroidism

In as many as one third of dogs with primary hypothyroidism a plasma thyrotropin (TSH) concentration within the reference range for euthyroid dogs is found. To determine whether this is due to fluctuations in the release of TSH, the plasma profiles of TSH were analyzed in 7 beagle bitches by collecting blood samples every 10 min for 6 hr, both before and after induction of primary hypothyroidism. After induction of primary hypothyroidism, a 37-fold increase in mean basal plasma TSH concentration and a 34-fold increase in mean area under the curve for TSH were found. Analysis by the Pulsar program demonstrated pulsatile secretion of TSH in the hypothyroid state, characterized by relatively low amplitude pulses (mean [+/-SEM]) amplitude 41 +/- 3% of basal plasma TSH level) and a mean pulse frequency of 2.0 +/- 0.5 pulses/6 hr. In the euthyroid state, significant TSH pulses were identified in only 2 dogs. The mean basal plasma TSH level correlated positively (r = 0.84) with the mean amplitude of the TSH pulses, and correlated negatively (r = -0.88) with the TSH pulse frequency. The results of this study demonstrate pulsatile secretion of TSH in dogs during hypothyroidism and only small fluctuations in plasma TSH concentrations during euthyroidism. The findings also suggest that the low TSH values occasionally found in dogs with spontaneous primary hypothyroidism may in some cases in part be the result of ultradian fluctuations.     

Pharmacokinetics and pharmacodynamics of protamine zinc recombinant human insulin in healthy dogs

Protamine zinc insulins are generally considered to be long acting, with slow absorption from subcutaneous tissue. Protamine zinc recombinant human insulin (PZIR) may be useful to treat diabetic dogs. The purpose of this study was to describe the pharmacokinetics and pharmacodynamics of PZIR in dogs. PZIR was administered subcutaneously to 10 healthy Beagles using an incomplete crossover design, at doses of 0.3 or 0.5 U/kg (each n=5), 0.8 U/kg (n=10), or 0.8 U/kg at three separate sites (n=6). Insulin and glucose concentrations were measured over 24 h. The shapes of insulin and glucose curves were variable among dogs, and the relationship between insulin dose, concentration, and glucose-lowering effect was nonlinear. For single-site 0.8 U/kg, median (range) onset of action was 3.5 h (0.5-10 h), time to glucose nadir was 14 h (5 to >24 h), and duration of action was >24 h (16 to >24 h). Mathematical model predictions of times to 50% and 90% insulin absorption, and fraction of insulin absorbed in 24 h, were not significantly different among protocols. Results confirm the tendency toward a late onset and long duration of action for PZIR in dogs. This insulin may be an alternative treatment option for diabetic dogs.     

Aldosterone-to-renin and cortisol-to-adrenocorticotropic hormone ratios in healthy dogs and dogs with primary hypoadrenocorticism

In dogs with primary hypoadrenocorticism, hypocortisolism and hypoaldosteronism usually are present, but these deficiencies also may occur in isolated forms. The diagnosis is commonly made by measuring plasma cortisol concentration before and after stimulation with ACTH, thereby ignoring aldosterone. In search of an alternative approach that would include assessment of glucocorticoid and mineralocorticoid production, 2 pairs of endocrine variables were measured: (1) plasma concentration of cortisol and ACTH, and (2) plasma aldosterone concentration and plasma renin activity. In addition, the cortisol-to-ACTH ratio (CAR) and the aldosterone-to-renin ratio (ARR) were calculated. Reference intervals were established in a population of 60 healthy dogs. In these dogs, CAR ranged from 1.1 to 26.1 and ARR ranged from 0.1 to 1.5. The variables were compared with those of 22 dogs with spontaneous primary hypoadrenocorticism. Plasma concentration of cortisol and ACTH in both groups of dogs overlapped, whereas CAR did not. Similarly, plasma aldosterone concentration and plasma renin activity overlapped, whereas ARR did not. These observations indicate that measurement of these endogenous variables (in one blood sample) allows the specific diagnoses of primary hypocortisolism and primary hypoaldosteronism.     

Comparison of two radiographic techniques for evaluation of hip joint laxity in 10 breeds of dogs

OBJECTIVE: To evaluate hip joint laxity in 10 breeds of dogs via 2 radiographic techniques. ANIMALS: 500 clinically normal dogs. DESIGN: Prospective study. PROCEDURE: Radiographs obtained via routine hip joint evaluations performed in sedated dogs of 10 popular breeds were randomly selected from a database. Measurements of distraction index (DI) and hip-extended index (HEI) on 1 hip joint radiograph randomly chosen from each dog were made. RESULTS: Mean age of dogs was 20.7 months. Mean HEI was 0.17 (range, 0.0 to 0.72) and mean DI was 0.44 (range, 0.07 to 0.96). Borzois had uniformly tight hip joints as judged by use of both methods and were considered the gold standard by which hip joint laxity was judged (all Borzois had DI < or = 0.32). Overall, DI was significantly greater than HEI. Within each breed, mean DI was always greater than mean HEI. Significant differences were detected among breeds for HEI; however, compared with DI, the magnitude of differences among breeds was less. CONCLUSIONS AND CLINICAL RELEVANCE: Distraction radiography detected the greatest range and magnitude of passive hip laxity in the 10 breeds of dogs. The difference in values between breeds known to have high prevalence of canine hip dysplasia and those in Borzois was greater for DI than for HEI. Breeds must be evaluated individually because of inherent differences in hip joint laxity.     

Comparison of the pharmacokinetic profiles of two different amphotericin B formulations in healthy dogs

This study was conducted to compare the pharmacokinetic profiles of conventional (Fungizone^®) and liposomal amphotericin B (AmBisome^®) formulations in order to predict their therapeutic properties, and evaluate their potential differences in veterinary treatment. For this purpose, twelve healthy mixed breed dogs received both drugs at a dose of 0.6 mg/kg by intravenous infusion over a 4‐min period in a total volume of 40 ml. Blood samples were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 24, 48, 72 and 96 hr after dosing, and concentrations of drug in plasma were determined by high‐performance liquid chromatography (HPLC). Pharmacokinetics was described by a two‐compartment model. Although both formulations were administered at the same doses (0.6 mg/kg), the plasma pharmacokinetics of liposomal amphotericin B differed significantly from those of amphotericin B deoxycholate in healthy dogs (p < .05). Liposomal amphotericin B showed markedly higher peak plasma concentrations (approximately ninefold greater) and higher area under the plasma concentration curve values (approximately 14‐fold higher) compared to conventional formulation. It is concluded that AmBisome^® reached higher plasma concentration and lower distribution volume and had a longer half‐life compared to Fungizone^®, and therefore, AmBisome^® is reported to be an appropriate and effective choice for the treatment of systemic mycotic infections in dogs.     

Hemodynamic evaluation of the right portal vein in healthy dogs of different body weights

Background

Doppler ultrasonography is an important tool for evaluating hepatic portal hemodynamics. However, no study in dogs of different body weights, in the range encountered in routine clinical veterinary practice, has been reported. It can be difficult to obtain an ideal insonation angle when evaluating the main portal vein, so evaluation of the right portal vein branch has been described in humans as an alternative. The aim of this study was to analyze, through Doppler ultrasonography, the hemodynamics in the right portal vein branch in dogs of different body weights.

Methods

Thirty normal dogs were divided in three groups by weight, in order to establish normal values for mean velocity, flow volume and portal congestion index of the right portal vein branch by means of Doppler ultrasonography.

Results

In all dogs ideal insonation angles were obtained for the right portal vein branch. The average velocity was similar in the three groups, but the portal congestion index and the flow volume differed, showing that the weight of the dog can influence these values.

Conclusion

Doppler ultrasonography for the evaluation of flow in the right branch of the portal vein could be a viable alternative, or complement, to examining the main vessel segment. This is especially so in those animals in which an ideal insonation angle for examination of the main portal vein is hard to obtain. In addition, the weight of the dog must be considered for the correct evaluation of the portal system hemodynamics, particularly for portal blood flow and the congestion index.     

Effects of deracoxib and aspirin on serum concentrations of thyroxine, 3,5,3'-triiodothyronine, free thyroxine, and thyroid-stimulating hormone in healthy dogs

OBJECTIVE: To evaluate the effects of deracoxib and aspirin on serum concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3), free thyroxine (fT4), and thyroid-stimulating hormone (TSH) in healthy dogs. ANIMALS: 24 dogs. PROCEDURE: Dogs were allocated to 1 of 3 groups of 8 dogs each. Dogs received the vehicle used for deracoxib tablets (PO, q 8 h; placebo), aspirin (23 to 25 mg/kg, PO, q 8 h), or deracoxib (1.25 to 1.8 mg/kg, PO, q 24 h) and placebo (PO, q 8 h) for 28 days. Measurement of serum concentrations of T4, T3, fT4, and TSH were performed 7 days before treatment (day -7), on days 14 and 28 of treatment, and 14 days after treatment was discontinued. Plasma total protein, albumin, and globulin concentrations were measured on days -7 and 28. RESULTS: Mean serum T4, fT4, and T3 concentrations decreased significantly from baseline on days 14 and 28 of treatment in dogs receiving aspirin, compared with those receiving placebo. Mean plasma total protein, albumin, and globulin concentrations on day 28 decreased significantly in dogs receiving aspirin, compared with those receiving placebo. Fourteen days after administration of aspirin was stopped, differences in hormone concentrations were no longer significant. Differences in serum TSH or the free fraction of T4 were not detected at any time. No significant difference in any of the analytes was detected at any time in dogs treated with deracoxib. CONCLUSIONS AND CLINICAL RELEVANCE: Aspirin had substantial suppressive effects on thyroid hormone concentrations in dogs. Treatment with high dosages of aspirin, but not deracoxib, should be discontinued prior to evaluation of thyroid function.     

Opinions of veterinarians regarding aggression in different breeds of dogs

Aggression in 108 dog breeds and six crosses was ranked by 185 members of the Companion Animal Society of the New Zealand Veterinary Association. The Rottweiler and German Shepherd were categorised as extremely aggressive and the Akita, Basengi, Chihuahua (long-coated and short-coated), Chow Chow, Cocker Spaniel, Shar pei and Welsh Corgi (Cardigan and Pembroke) were classified as very aggressive. A chi-squared test of data from the twenty most popular breeds showed that significant differences were perceived between the breeds. Cocker Spaniels, Pembroke Welsh Corgis, German Shepherds and Rottweilers were more aggressive than Staffordshire Bull Terriers, Border Collies and Doberman Pinchers, which were more aggressive than Cavalier King Charles Spaniels, Bichon Frise, English Springer Spaniels, German Shorthaired Pointers, Weimaraners, Labrador Retrievers, Golden Retrievers, Newfoundlands, Dalmatians, Boxers, Bulldogs, Bull Mastiffs and Rough Collies.     

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.