Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis

The aim of the study was to determine the intraarticular serum amyloid A (SAA) response pattern in horses with inflammatory arthritis. Inflammatory arthritis was induced by injection of lipopolysaccharide (LPS) into the radiocarpal joint of four horses. Serum and synovial fluid (SF) samples were collected before and at 4, 8, 12, 24, 48, 72, 96, and 144 h after injection. Concentrations of SAA were measured by immunoturbidometry, and expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. The LPS injection caused systemic and local clinical signs of inflammation. Serum amyloid A appeared in serum and SF within 8 h after LPS injection. Isoelectric focusing showed three major SAA bands with apparent isoelectric points (pI) of 7.9, 8.6, and >9.3 in serum and SF. Synovial fluid contained two additional isoforms with highly alkaline apparent pI values (apparent pI value extrapolated from standard curve = 10.0 and 10.2), which were not present in any of the serum samples. In conclusion, intraarticular injection of LPS induced systemic and local inflammatory responses in the horses. By demonstrating SF-specific SAA isoforms the results of the present study suggest that SAA is synthesized locally in the equine inflamed joint, similar to what has been demonstrated in humans previously. The marked local SAA synthesis suggests an important pathophysiological role in inflammatory arthritis.     

Concentrations of serum amyloid A in serum and synovial fluid from healthy horses and horses with joint disease

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.     

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis

Background: Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and α‐, β‐, and γ‐globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. Objectives: The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm‐blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Methods: Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi‐automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within‐run and within‐assay precision. Data from warm‐blooded and draught horses were compared using the Mann–Whitney U test. Results: Within‐run and within‐assay CVs were <5% for all protein fractions. No significant difference was found between warm‐blooded and heavy draught horses and so combined reference intervals (2.5–97.5%) were calculated for total protein (51.0–72.0 g/L), albumin (29.6–38.5 g/L), α₁‐globulin (1.9–3.1 g/L), α₂‐globulin (5.3–8.7 g/L), β₁‐globulin (2.8–7.3g/L), β₂‐globulin (2.2–6.0 g/L), and γ‐globulin (5.8–12.7 g/L) concentrations, and albumin/globulin ratio (0.93–1.65). Conclusion: Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.     

Serum amyloid A in equine health and disease

Serum amyloid A (SAA) is the major acute phase protein in horses. It is produced during the acute phase response (APR), a nonspecific systemic reaction to any type of tissue injury. In the blood of healthy horses, SAA concentration is very low, but it increases dramatically with inflammation. Due to the short half-life of SAA, changes in its concentration in blood closely reflect the onset of inflammation and, therefore, measurement of SAA useful in the diagnosis and monitoring of disease and response to treatment. Increases in SAA concentration have been described in equine digestive, reproductive and respiratory diseases and following surgical procedures. Moreover, SAA has proven useful for detection of some subclinical pathologies that can disturb training and competing in equine athletes. Increasing availability of diagnostic tests for both laboratory and field use adds to SAA's applicability as a reliable indicator of horses’ health status. This review article presents the current information on changes in SAA concentrations in the blood of healthy and diseased horses, focussing on clinical application of this biomarker.     

Diagnostic utility of measuring serum amyloid A with a latex agglutination turbidimetric immunoassay in bovine mastitis: Comparison with haptoglobin and alpha 1 acid glycoprotein

This study established the precision and accuracy of a modified latex agglutination turbidimetric immunoassay (LATIA) reagent, and evaluated the ability of the measurement of serum amyloid A (SAA) compared to haptoglobin and α1-acid glycoprotein, which are acute phase proteins (APPs), for diagnosis of clinical mastitis. Concentrations of APPs in cows with mastitis were significantly higher than those in healthy cow. Only the plasma SAA concentration in cows with clinical mastitis (44.90 mg/l; n=15) was significantly higher than that in those with subclinical mastitis (10.70 mg/l; n=16), enabling their diagnosis in contrast to other APPs. Thus, the SAA assay using a LATIA reagent is useful in assessing mastitis severity due to its higher sensitivity and specificity than other APP assays.     

Pig-major acute phase protein and haptoglobin serum concentrations correlate with PCV2 viremia and the clinical course of postweaning multisystemic wasting syndrome

The aim of the present longitudinal study was to assess the evolution of two acute phase proteins (APPs), pig-major acute phase protein (pig-MAP) and haptoglobin (HPT), in serum from pigs that developed postweaning multisystemic wasting syndrome (PMWS) in comparison to healthy and wasted non-PMWS affected pigs. In addition, evidence of infection with other pathogens and its relation with variations in APPs concentrations was also assessed. Fourteen independent batches of 100–154 pigs were monitored from birth to PMWS outbreak occurrence in 11 PMWS affected farms. Pigs displaying PMWS-like signs and age-matched healthy controls were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. At the moment of PMWS occurrence, pig-MAP and HPT concentration in PMWS affected pigs were higher than in healthy ones (p < 0.0001). No differences in APPs serum concentrations between subclinically PCV2-infected pigs and healthy non-PCV2-infected pigs (based on quantitative PCR on serum results) were detected. Results showed a significant correlation between PCV2 loads and both pig-MAP (R = 0.487–0.602, p < 0.0001) and HPT (R = 0.326–0.550, p < 0.05–0.0001) concentrations in serum of PMWS affected pigs, indicating that the acute phase response in PMWS affected pigs occurred concomitantly to PCV2 viremia. No other pathogen, apart from PCV2, was consistently related with variations in APPs concentrations. A ROC analysis, made to determine the capacity of discrimination of both APPs between PMWS affected and non-affected pigs, showed higher sensitivity and specificity values using pig-MAP compared to HPT. These results suggest that pig-MAP might be a better indicator of PMWS status than HPT. Moreover, the fact that APR occurred some weeks before the start of clinical signs suggests that APPs could provide valuable prognostic information for PMWS development.     

Diagnostic assessment of peritoneal fluid cytology in horses with abdominal neoplasia

Objective: To evaluate the diagnostic value of peritoneal fluid (PF) cytology for clinical diagnosis of abdominal neoplasia in horses. Material and methods: Ten horses with histopathologically confirmed abdominal neoplasia, in which a PF analysis was performed, were included in this retrospective study. PF was analyzed for total protein concentration and a nucleated cell count was performed. Using cytological criteria of malignancy, the PF samples were evaluated regarding their probability of malignancy. Results: Cytologic classification of cells according to criteria of malignancy allowed a positive cytologic diagnosis of neoplasia in 5 out of 10 peritoneal fluid samples. Malignant lymphoma was the most commonly diagnosed neoplasia (3/10) and could be identified by cytology in 2/3 cases. In 1/2 horses with plasma cell myeloma neoplastic cells were similarly found. Malignant melanoma (2/10) was diagnosed using cytology in one case (presence of melanin-containing cells). Cytological diagnosis of malignant neoplasia was established in the only horse with gastric squamous cell carcinoma, but the morphology of the identified tumour cells did not allow a specific diagnosis. Thus, a definitive diagnosis was achieved in 4/5 horses with proven abdominal neoplasia. The horses with adenocarcinoma (1/10) and haemangiosarcoma (1/10) had no evidence of neoplasia based on cytological findings. No relationship between total protein concentration or the nucleated cell count with the histolopathological diagnosis of abdominal neoplasia was found. Abnormal mitotic figures were considered of greater diagnostic value than the overall mitotic rate. Conclusion: The implementation of nuclear criteria of malignancy in the cytologic evaluation of PF samples allows the identification of neoplastic cells to an acceptable degree. For this purpose, the knowledge of the highly variable morphological features of mesothelial cells is essential. The absence of malignant cells does not rule out abdominal neoplasia. Clinical relevance: PF cytology should be considered as a valuable, minimally invasive, simple, and rapid diagnostic technique in horses with suspected abdominal neoplasia.     

Effects of ophthalmic disease on concentrations of plasma fibrinogen and serum amyloid A in the horse

Reasons for performing study: There is little scientific information available about the ability of ocular disease to cause a systemic inflammatory response. Horses are frequently affected with ocular disease and ensuring their systemic health prior to performing vision saving surgery under anaesthesia is essential for the successful treatment of ophthalmic disease. Hypothesis: Ocular disease will cause elevations in the concentration of the acute phase proteins fibrinogen and serum amyloid A in peripheral blood. Methods: Whole blood and serum samples were obtained from 38 mature horses with ulcerative keratitis or uveitis and no evidence of systemic disease, 9 mature horses with no evidence of ocular or systemic disease (negative controls) and 10 mature horses with systemic inflammatory disease and no evidence of ocular disease (positive controls). Blood samples were assayed for concentrations of the acute phase proteins fibrinogen and serum amyloid A. Results: Fibrinogen and serum amyloid A were significantly different in the positive control group compared to the negative control, corneal disease and uveitis groups (P<0.126). There was no significant difference between the negative control, corneal disease and uveitis groups (P<0.001). Conclusions: Ulcerative keratitis and anterior uveitis are not associated with elevated concentrations of the acute phase proteins fibrinogen and serum amyloid A in peripheral blood. Potential relevance: When the clinician is presented with a patient with ocular disease and elevated plasma fibrinogen or serum amyloid A concentrations, a nonocular inflammatory focus should be suspected.     

Serum iron and plasma fibrinogen concentrations as indicators of systemic inflammatory diseases in horses

BACKGROUND: Detection of systemic inflammation, which is important for proper diagnosis and prompt treatment, can be challenging. HYPOTHESIS: Measurement of plasma iron concentration is a sensitive method for detecting systemic inflammation in horses compared with measurements of plasma fibrinogen concentration, a traditional marker for inflammation in the horse. ANIMALS: Ninety-seven horses hospitalized with diseases causing systemic inflammation, 22 horses with localized inflammation, and 12 clinically normal horses were included in this study. METHODS: A retrospective study was made on hospitalized horses that had both plasma iron and fibrinogen concentrations measured on hospital admission. RESULTS: Plasma iron concentration was lower in horses with systemic inflammation (64 +/- 45 microg/dL) than the reference interval minimum (105 microg/dL) and were significantly lower (P = .001) than the value in a group of horses with local inflammation (123 +/- 45 microg/dL) and in healthy transported horses (143 +/- 29 microg/dL). Low plasma iron and high fibrinogen concentrations were both sensitive indicators of systemic inflammation in horses with sensitivity of 90 and 82%, respectively. There was a similar correlation between either continued decreases in iron concentration (Rsp of 0.239) or increases in fibrinogen concentration (Rsp of 0.280) during hospitalization and a worse prognosis. CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of plasma iron concentration better reflected acute inflammation than did fibrinogen concentration.     

酮病和健康奶牛血清中触珠蛋白和血清淀粉样蛋白A浓度的比较研究

血清中触珠蛋白(HP)和血清淀粉样蛋白A(SAA)浓度的变化与奶牛代谢性疾病(如酮病、脂肪肝等)密切相关.为了明确酮病奶牛和健康奶牛血清中触珠蛋白和血清淀粉样蛋白A浓度差异,本试验运用酶联免疫吸附试验(ELISA),分别测定了酮病和健康奶牛血清中触珠蛋白和血清淀粉样蛋白A浓度,结果显示,酮病奶牛血清中触珠蛋白浓度和SAA浓度极显著高于(P＜0.01)健康奶牛.结果表明,酮病奶牛普遍存在炎症等病理过程,这一结果可为进一步阐明奶牛酮病的发病机制提供理论依据.     

Clinical evaluation of serum alcohol dehydrogenase activity in horses with acute intestinal obstruction

Objectives – To measure serum alcohol dehydrogenase (ADH) activity in horses with acute intestinal obstruction and to determine the diagnostic and prognostic utility of this analyte. Design – Prospective observational study. Setting – University Veterinary Hospital. Animals – Thirty healthy horses (control group) and 77 horses with acute intestinal obstruction, including 36 horses with nonstrangulating obstruction (23 with left ventral colon impaction and 13 with left dorsal displacement [G1], 22 with small intestinal strangulation [G2], and 19 with colon torsion [G3]). Interventions – Serum ADH activity was assayed spectrophotometerically in all horses. Serum lactate concentration and hepatic enzyme (aspartate aminotransferase, gamma‐glutamyl transferase, glutamate dehydrogenase) activities were measured using an automatic analyzer. Measurements and Main Results – The median [interquartile range] serum ADH activity in healthy horses was 10.5 [8.7 – 11 U/L]. ADH activity was significantly increased (P<0.05) in G1=16.5 [13.8 – 18 U/L], G2=40 [20 – 74.9 U/L], and G3=63.2 [40 – 78 U/L] compared with healthy controls. Aspartate aminotransferase and glutamate dehydrogenase activities were also significantly increased in G3 in comparison with controls. ADH activity was correlated with serum lactate concentration in G1 and G3, respectively (P<0.01, r=0.55 and 0.8). Other liver enzymes did not show any significant correlation with lactate. ADH activity was directly related to the probability of strangulation; odds ratio=1.11. ADH activity >20 U/L had 80.6% specificity and 80.5% sensitivity for discriminating horses with strangulating obstruction. Twelve horses euthanized before surgery were excluded from the outcome analysis. Increasing ADH activity was associated with nonsurvival; odds ratio=1.03. ADH activity <80 U/L had 94.44% specificity and 66.67% sensitivity for survival. Conclusion – Serum ADH activity may be a useful clinical parameter in detecting intestinal strangulation in horses and may provide some prognostic value in horses with acute intestinal obstruction.