Objective—To determine serum amyloid A (SAA) concentrations in serum and synovial fluid from healthy horses and horses with joint disease and assess the effect of repeated arthrocentesis on SAA concentrations in synovial fluid.
Animals—10 healthy horses and 21 horses with various types of joint disease.
Procedures—Serum and synovial fluid samples were obtained from each horse. In 5 of the 10 healthy horses, arthrocentesis was repeated 9 times. Concentrations of SAA were determined via immunoturbidometry.
Results—Serum and synovial fluid SAA concentrations were less than the assay detection limit in healthy horses and did not change in response to repeated arthrocentesis. Synovial fluid SAA concentrations were significantly higher in horses with suspected bacterial joint contamination or infectious arthritis, or tenovaginitis than in healthy controls, and serum concentrations were significantly higher in horses with infectious conditions than in the other groups. Neither serum nor synovial fluid SAA concentrations in horses with low-inflammation joint conditions differed significantly from those in healthy controls. Concentrations of SAA and total protein in synovial fluid were significantly correlated.
Conclusions and Clinical Relevance—Synovial fluid SAA concentration was a good marker of infectious arthritis and tenovaginitis and appeared to reflect changes in inflammatory activity. The advantages of use of SAA as a marker include the ease and speed of measurement and the fact that concentrations in synovial fluid were not influenced by repeated arthrocentesis in healthy horses. Further study of the SAA response in osteoarthritic joints to assess its usefulness in diagnosis and monitoring of osteoarthritis is warranted.
Objective—To compare sensitivity and specificity of cytologic examination and 3 chromogen tests for detection of occult blood in cockatiel (Nymphicus hollandicus) excrement.
Animals—20 adult cockatiels.
Procedures—Pooled blood from birds was divided into whole blood and lysate aliquots. Excrement was mixed with each aliquot in vitro to yield 6 hemoglobin (Hb) concentrations (range, 0.375 to 12.0 mg of Hb/g of excrement). For the in vivo portion of the study, birds were serially gavaged with each aliquot separately at 5 doses of Hb (range, 2.5 to 40 mg/kg). Three chromogen tests and cytologic examination were used to test excrement samples for occult blood. Sensitivity, specificity, and observer agreement were calculated.
Results—In vitro specificity ranged from 85%to 100% for the 3 chromogen tests and was 100% for cytologic examination. Sensitivity was 0% to 35% for cytologic examination and 100% for the 3 chromogen tests on samples containing ≥ 1.5 mg of Hb/g of excrement. In vivo specificity was 100%, 90%, 65%, and 45% for cytologic examination and the 3 chromogen tests, respectively. Sensitivity was 0% to 5% for cytologic examination and ≥ 75% for all 3 chromogen tests after birds received doses of Hb ≥ 20 mg/kg. Observer agreement was lowest for cytologic examination.
Conclusions and Clinical Relevance—Chromogen tests were more useful than cytologic examination for detection of occult blood in cockatiel excrement. The best combination of sensitivity, specificity, and observer agreement was obtained by use of a chromogen test.
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 thyroidstimulating 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.
Procedure—Normal acid-base status was maintained in 8 dogs (group I), and metabolic acidosis was induced in 25 dogs. For 60 minutes, normocalcemia was maintained in group I and 10 other dogs (group II), and 10 dogs were allowed to become hypercalcemic (group III); hypocalcemia was then induced in groups I and II. Groups II and IV (5 dogs) were treated identically except that, at 90 minutes, the latter underwent parathyroidectomy. At intervals, variables including PaO2, Ca2+ concentration, arterial blood pH (pHa), and systolic blood pressure were assessed.
Results—In group II, PaO2 increased from baseline value (96 ± 2 mm Hg) within 10 minutes (pHa, 7.33 ± 0.001); at 60 minutes (pHa, 7.21 ± 0.02), PaO2 was 108 ± 2 mm Hg. For the same pHa decrease, the PaO2 increase was less in group III. In group I, hypocalcemia caused PaO2 to progressively increase (from 95 ± 2 mm Hg to 104 ± 3 mm Hg), which correlated (r = −0.66) significantly with a decrease in systolic blood pressure (from 156 ± 9 mm Hg to 118 ± 10 mm Hg). Parathyroidectomy did not alter PaO2 values.
Conclusions and Clinical Relevance—Induction of hypocalcemia and metabolic acidosis each increased PaO2 in anesthetized dogs, whereas acidosis-induced hypercalcemia attenuated that increase. In anesthetized dogs, development of metabolic acidosis or hypocalcemia is likely to affect ventilatory control.
Objective—To compare 4 assay procedures for prediction of passive transfer status in lambs.
Animals—Thirty-one 1-day-old Sardinian lambs.
Procedure—Serum IgG concentration was determined by use of single radial immunodiffusion. The following were determined: serum total protein concentration as measured by refractometry (ie, refractometry serum total protein concentration), serum total protein concentration as determined by the biuret method (ie, biuret method serum total protein concentration), serum γ-globulin concentration as determined by serum protein electrophoresis, and serum γ-glutamyltransferase (GGT) activity as measured by spectrophotometry. Accuracy of these assays for estimation of serum IgG concentration in 1-day-old lambs was established by use of linear regression analysis.
Results—Refractometry serum total protein concentration, biuret method serum total protein concentration, and serum γ-globulin concentration were closely and linearly correlated with serum IgG concentration. The natural logarithm (ln) of serum GGT activity was closely and linearly correlated with serum IgG concentration (ln). Refractometry serum total protein concentration, biuret method serum total protein concentration, and γ-globulin concentration accounted for approximately 85%, 91%, and 95% of the variation in serum IgG concentration, respectively. Serum GGT activity (ln) accounted for approximately 92% of the variation in serum IgG concentration (ln).
Conclusions and Clinical Relevance—For prediction of passive transfer status in 1-day-old lambs, serum GGT activity or biuret method serum total protein concentration determination will allow for passive transfer monitoring program development. Immediate refractometry serum total protein concentration determination is beneficial in making timely management and treatment decisions. Serum γ-globulin concentration determination can be used as a confirmatory test.
Objective—To use in vitro assays to evaluate the effects of a novel immunosuppressive agent, FTY720, on biological functions (migration, phagocytosis, and production of reactive-oxygen species [ROS]) of feline peripheral neutrophils and determine the cytotoxic effects of FTY720 on feline peripheral neutrophils.
Sample Population—Peripheral neutrophils obtained from 8 healthy cats.
Procedure—Peripheral neutrophils were isolated from blood samples obtained from the 8 cats and exposed to the phosphorylated form of FTY720 (FTY720-P). A fluorescence-based in vitro evaluation of migration was performed. Phagocytosis of microbes and production of ROS were evaluated by use of a 2-color flow cytometry system. Samples of whole blood obtained from the cats were incubated with various concentrations of FTY720-P, fluorescein-labeled Staphylococcus aureus, and dihydroethidium. Cytotoxic effects were evaluated by use of propidium iodide staining.
Results—Addition of FTY720-P caused a slight non-significant decrease in phagocytosis and production of ROS by feline peripheral neutrophils. Migration activity of feline peripheral neutrophils was significantly increased by the addition of FTY720-P. Addition of FTY720-P at concentrations considered for clinical use did not increase the death rate of feline peripheral neutrophils.
Conclusions and Clinical Relevance—FTY720 does not inhibit critical functions of feline peripheral neutrophils in vitro.
Objective—To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions.
Animals—28 clinically normal horses.
Procedure—Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (μCa) and magnesium (μMg) fractions. Serum concentrations of iCa and iMg were measured in the ultrafiltrate by use of selective electrodes. Serum concentration of complexed calcium (cCa) or magnesium (cMg) was calculated by subtracting iCa or iMg from μCa or μMg, respectively.
Results—Mean ±SE serum tCa concentration was 3.26 ± 0.06 mmol/L. Calcium fractions were as follows: pCa, 1.55 ± 0.03 mmol/L (47.4 ± 0.9%); iCa, 1.58 ± 0.03 mmol/L (48.5 ± 0.7%); and cCa, 0.13 ± 0.02 mmol/L (4.1 ± 0.9%). Serum tMg concentration was 0.99 ± 0.04 mmol/L. Magnesium fractions were as follows: pMg, 0.33 ± 0.04 mmol/L (33.3 ± 4.2%); iMg, 0.57 ± 0.02 mmol/L (57.6 ± 1.7%); and cMg, 0.09 ± 0.02 mmol/L (9.1 ± 1.9%). Refrigeration (4°C) did not affect iCa values, whereas iMg declined by 8% after 120 hours. Neither iCa nor iMg was affected by freezing (−20°C).
Conclusions and Clinical Relevance—In equine serum, iMg is less stable than iCa; thus, when serum samples are not going to be analyzed promptly, freezing may be preferable to refrigeration for storage.
Objective—To develop an assay to measure canine von Willebrand factor (vWF):collagen-binding activity (CBA) to screen for type 2 von Willebrand disease (vWD) in dogs.
Sample Population—293 plasma samples submitted for analysis of canine vWF antigen (vWF:Ag) and 12 control plasma samples from dogs with inherited type 2 or 3 vWD.
Procedure—Bovine collagens were evaluated for suitability as binding substrate for vWF. Assay sensitivity to depletion, proteolytic degradation, or a genetic deficiency of high-molecular-weight vWF were determined. Amounts of vWF:Ag and vWF:CBA were measured. The ratio of vWF:Ag to vWF:CBA was used to discriminate between type 1 and type 2 vWD.
Results—An assay for canine vWF activity was developed by use of mixed collagen (types I and III). When vWF:Ag was used to subtype vWD, 48% of the dogs were classified as clinically normal, 9% as indeterminate, and 43% as type 1 vWD. Inclusion of vWF activity resulted in reclassification of 5% of those identified as type 1 to type 2 vWD. However, vWF:CBA of the reclassified dogs was not persistently abnormal, a finding compatible with acquired type 2 vWD. Some Doberman Pinschers had lower antigen-to-activity ratios than other breeds with type 1 vWD, suggesting that Doberman Pinschers have more functional circulating vWF.
Conclusions and Clinical Relevance—Analysis of canine vWF activity should be included among the vWF-specific assays used to confirm type 2 vWD. The prevalence of inherited forms of type 2 vWD in screened dogs is lower than acquired forms that can result secondary to underlying disease.
Objective—To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood.
Sample population—Blood samples from 10 dogs.
Procedure—Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine.
Results—A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference.
Conclusions and Clinical Relevance—Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.
Objectives—To measure urine concentrations of sulfated glycosaminoglycans (GAGs), determine optimal storage conditions for urine samples, establish a reference range, and determine whether there is correlation between 24-hour total urine GAG excretion and the GAG-to-creatinine ratio (GCR).
Animals—14 healthy adult dogs.
Procedure—Single urine sample GAG concentrations and GCRs were measured in samples collected from 14 healthy dogs at the start of the 24-hour collection period. Twenty-four–hour total urine GAG excretions were determined from urine collected during a 24-hour period in the same 14 dogs. Total sulfated GAG concentrations were also measured in urine from these dogs after the urine had been stored at 4°C and -20°C for 1, 7, and 30 days.
Results—Urine GAG concentrations were not significantly different from baseline values after urine was stored at 4°C for up to 1 day and -20°C for up to 30 days. Neither single urine sample GAG concentration (R, 0.422) nor GCR (R, 0.084) was an adequate predictor of 24-hour total urine GAG excretion.
Conclusions and Clinical Relevance—Results of this study provide data that can be used to establish a reference range for 24-hour total urine GAG excretion in dogs and adequate conditions for sample storage. Contrary to findings in humans, there was no significant linear correlation between 24-hour total urine GAG excretion and single urine sample GCR in dogs, limiting clinical use of the single urine sample test.