Comparison of paired serum and lithium heparin plasma samples for the measurement of serum amyloid A in horses using an automated turbidimetric immunoassay

This study aimed to evaluate whether equine serum amyloid A (SAA) concentrations could be reliably measured in plasma with a turbidimetric immunoassay previously validated for equine SAA concentrations in serum. Paired serum and lithium-heparin samples obtained from 40 horses were evaluated. No difference was found in SAA concentrations between serum and plasma using a paired t test (P = 0.48). The correlation between paired samples was 0.97 (Spearman’s rank P < 0.0001; 95% confidence interval 0.95–0.99). Passing-Bablok regression analyses revealed no differences between paired samples. Bland–Altman plots revealed a positive bias in plasma compared to serum but the difference was not considered clinically significant. The results indicate that lithium-heparin plasma samples are suitable for measurement of equine SAA using this method. Use of either serum or plasma allows for greater flexibility when it comes to sample collection although care should be taken when comparing data between measurements from different sample types.     

The use of receiver operating characteristic (ROC) analysis to assess the diagnostic value of serum amyloid A, haptoglobin and fibrinogen in traumatic reticuloperitonitis in cattle

Acute phase proteins (APPs) such as serum amyloid A (SAA), haptoglobin (Hp), and plasma fibrinogen (Fb) are diagnostic and prognostic biomarkers. This study determined the pattern of SAA in traumatic reticuloperitonitis (TRP) in cows, and compared the findings with Hp, and plasma Fb concentrations in the differential diagnosis of TRP using receiver operating characteristic (ROC) analysis. Blood samples were collected from 89 cows: 53 cases of TRP, 16 animals with other internal disorders and 20 healthy controls.SAA and Hp had a similar pattern and magnitude of changes in TRP. Using the ROC method, the optimal cut-off for the diagnosis of TRP was 68 μg/mL for SAA and 0.74 g/L for Hp, with 100% sensitivity and 86.1% specificity; either of these parameters may therefore be used as a sensitive and relatively specific tool for the differential diagnosis of TRP in cattle. The diagnostic accuracy of plasma Fb was significantly lower than either SAA or Hp (P < 0.01). The combined use of these APPs (whilst aware of their conditional interdependence) may not provide any additional diagnostic information than can be obtained using either SAA or Hp alone.     

Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions

Serum amyloid A (SAA) is a major acute phase protein in dogs. However, knowledge of qualitative properties of canine SAA and extent of its synthesis in extrahepatic tissues is limited. The aim of the study was to investigate expression of different SAA isoforms in serum and synovial fluid in samples obtained from dogs (n=16) suffering from different inflammatory or non-inflammatory conditions, which were either related or unrelated to joints. Expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. Serum amyloid A was present in serum from all dogs with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local production of these isoforms in the canine inflamed joint.     

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 amyloid A is not a marker for relapse of multicentric lymphoma in dogs

BACKGROUND: Serum amyloid A (SAA) is an acute phase protein whose concentration increases in inflammatory, infectious, and neoplastic conditions in animals and human beings. Multicentric lymphoma is a common cancer in dogs, and chemotherapy is indicated to attain long-term survival. However, frequent relapses lead to changes in chemotherapeutic protocols. OBJECTIVES: The aims of this study were to evaluate SAA as a marker for relapse of multicentric lymphoma in dogs and to determine whether chemotherapy induces changes in the concentration of SAA during treatment. METHODS: SAA was measured by an ELISA test in healthy control dogs (n=20), in healthy dogs receiving chemotherapy (n=8), and in dogs with lymphoma (n=20). All dogs receiving chemotherapy were randomly assigned to 2 treatment groups, one receiving cyclophosphamide, vincristine, and prednisone (CVP) and the other receiving vincristine, cyclophosphamide, methotrexate, and L-asparaginase (VCMA) protocols. SAA concentration was determined before chemotherapy at weeks 1-4 in healthy dogs receiving chemotherapy and in dogs with lymphoma, then every 3 weeks for 4 months in healthy dogs, and at relapse and in the sample prior to relapse in dogs with lymphoma. SAA was measured only once in the healthy control dogs. Results were analyzed using repeated measures ANOVA followed by Tukey multiple comparison tests to compare groups and weeks of treatment. RESULTS: Mean SAA concentration was significantly higher in dogs with lymphoma before chemotherapy compared with healthy and chemotherapy control dogs. No increase in SAA concentration was found at relapse. No differences were observed in SAA concentration based on type of chemotherapy protocol. CONCLUSIONS: SAA is not a marker of relapse in dogs with multicentric lymphoma, nor does chemotherapy regimen affect SAA concentration.     

Acute phase proteins as indicators of calf herd health

The potential for using acute phase proteins (APPs) in the assessment of herd health was studied by examining the levels of serum haptoglobin, serum amyloid A (SAA) and plasma fibrinogen in relation to clinical findings and leukocyte counts in calves. Two groups of calves from conventional dairy farms were studied. The animals were examined 10 times during the first six weeks after introduction into a new environment. Haptoglobin, SAA and fibrinogen were analysed and weight gain, disease symptoms and treatments were recorded. Analysis of antibodies against viral infections was performed. An acute phase reaction (APR) score was established at each sampling by combining the APP results and total leukocyte counts. The health status differed between the two groups, although no manipulation of health had been performed, except that the group with a higher incidence of disease had a concurrent experimental infection with lungworm as part of another study. In the group with a higher incidence of disease, the mean weight gain was significantly lower, and the number of sampling days with elevated serum concentrations of APPs, and the mean maximum concentrations of haptoglobin and fibrinogen were significantly higher compared to the healthier group. The APR score was significantly higher at days 4 and 8 of the study in the group with a higher incidence of disease. The results indicate that measurement of APPs could be a useful tool for evaluation of health in calf herds.     

Serum and synovial fluid steady-state concentrations of trimethoprim and sulfadiazine in horses with experimentally induced infectious arthritis

The tarsocrural joints of 11 horses were inoculated with 1.2 to 2.16 x 10(6) viable Staphylococcus aureus organisms susceptible to a trimethoprim-sulfadiazine (TMP-SDZ) combination with minimal inhibitory concentration (MIC) of 0.25 micrograms of TMP/ml and 4.75 micrograms of SDZ/ml. Antimicrobial treatment consisted of oral administration of a TMP-SDZ combination--30 mg/kg of body weight given once daily (group-1 horses) or 60 mg/kg given as 30 mg/kg every 12 hours (group-2 horses). Paired serum and synovial fluid samples were obtained before intra-articular inoculation with the S aureus, after inoculation with S aureus but before antimicrobial treatment, and after inoculation at various hourly intervals after oral administration of the TMP-SDZ combination. The TMP-SDZ combination was administered daily in the 2 dosages for 21 days. Samples were collected after day 3 of repetitive drug administration so that drug steady-state concentration would have been achieved. Serum and synovial fluid samples were analyzed for TMP and SDZ concentrations. Administration of the TMP-SDZ combination at a dosage of 30 mg/kg once daily was not effective in maintaining TMP or SDZ concentrations above the MIC of TMP-SDZ for the S aureus (0.25 and 4.75 micrograms/ml for TMP and SDZ, respectively) in the infected synovial fluid or in maintaining adequate TMP concentration in the serum. The alternative use of the TMP-SDZ combination at a dosage of 60 mg/kg given as 30 mg/kg every 12 hours was effective in maintaining serum and synovial fluid concentrations of TMP and SDZ that were greater than the MIC for the infective organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Milk amyloid A: correlation with cellular indices of mammary inflammation in cows with normal and raised serum amyloid A

This study compared concentrations of amyloid A in bovine milk with the cell-based indicators of intramammary inflammation, somatic cell count and California Mastitis Test. Mammary quarter data pertaining to 180 cows were categorised according to concentrations of serum amyloid A in the cow of origin. Ranked correlation, ranked regression and receiver operator characteristics all demonstrated acceptable agreement between milk amyloid A concentrations and cell-based indices. There were some indications of reduction in this agreement, in cows with raised concentrations of serum amyloid A. However, there were also indications that serum amyloid A did not significantly influence milk amyloid A. The results of the current study indicate that milk amyloid A exhibits good correlation with established cell-based indicators of intramammary inflammation.     

Serum Amyloid A and Haptoglobin Concentrations and Liver Fat Percentage in Lactating Dairy Cows with Abomasal Displacement

Background: There has been increased interest in measuring the serum concentration of acute phase reactants such as serum amyloid A [SAA] and haptoglobin [haptoglobin] in periparturient cattle in order to provide a method for detecting the presence of inflammation or bacterial infection.
Objectives: To determine whether [SAA] and [haptoglobin] are increased in cows with displaced abomasum as compared with healthy dairy cows.
Animals: Fifty-four adult dairy cows in early lactation that had left displaced abomasum (LDA, n = 34), right displaced abomasum or abomasal volvulus (RDA/AV, n = 11), or were healthy on physical examination (control, n = 9).
Materials and Methods: Inflammatory diseases or bacterial infections such as mastitis, metritis, or pneumonia were not clinically apparent in any animal. Jugular venous blood was obtained from all cows and analyzed. Liver samples were obtained by biopsy in cattle with abomasal displacement.
Results: [SAA] and [haptoglobin] concentrations were increased in cows with LDA or RDA/AV as compared with healthy controls. Cows with displaced abomasum had mild to moderate hepatic lipidosis, based on liver fat percentages of 9.3 ± 5.3% (mean ± SD, LDA) and 10.8 ± 7.7% (RDA/AV). [SAA] and [haptoglobin] were most strongly associated with liver fat percentage, r _s=+0.55 ( P < .0001) and r _s=+0.42 ( P = .0041), respectively.
Conclusions and Clinical Importance: An increase in [SAA] or [haptoglobin] in postparturient dairy cows with LDA or RDA/AV is not specific for inflammation or bacterial infection. An increase in [SAA] or [haptoglobin] may indicate the presence of hepatic lipidosis in cattle with abomasal displacement.     

Serum Protein Fraction in Mature Horses and Relationship With Metabolic and Hematological Parameters

This study was conducted to determine the effect of age on serum protein fractions and their relationship with metabolic and hematological profiles in mature horses. Twenty-five mature Italian Saddle horses (mean age 13.6 ± 4.8 years) fed the same diet (grass hay and concentrate) were stratified according to age as first maturity, M1 (≤10 years old); second maturity, M2 (>10 and <15 years old); and old, O (>15 years), to be monitored every 28 days for a continuous period of 140 days. Horses in group O had higher plasma protein and thiol concentrations and white blood cell and neutrophil counts than the other two groups. Serum α₂-globulin concentrations were positively correlated with total plasma cholesterol (r = 0.514; P < .001), alkaline phosphatase (r = 0.430; P < .001), aspartate aminotransferase (r = 0.339; P < .001), ceruloplasmin (r = 0.321; P < .001), glutamic pyruvate transaminase (r = 0.444; P < .001), reactive oxygen metabolites (r = 0.426; P < .001), and blood neutrophil counts (r = 0.344; P < .01), and negatively with plasma bilirubin (r = −0.522; P < .001) and creatinine (r = −0.400; P < .001). These results suggest differences in hematological and metabolic profile in Italian Saddle horses after 15 years of age, resulting mainly from changes in plasma proteins and inflammatory mediators. The α₂-globulins fraction seems a quick but reliable marker of an inflammatory situation that, successively, should be better investigated with specific metabolites or enzymes.     

A Comparison of Elevated Blood Parameter Values in a Population of Thoroughbred Racehorses

In racing Thoroughbred horses, blood cell counts and key biochemistry parameters are used to monitor horse health during training. The most common measure is total white blood cell (WBC) count, usually coupled with estimates of the relative abundance of the five main types of WBC. However, WBC can go down and up when challenged, making interpretation difficult. In contrast, a large majority of health issues that impact training should trigger an inflammatory response. In this study, we test the potential for two inflammatory biomarkers, fibrinogen and serum amyloid A (SAA), to provide more reliable indicators of health issues across a large sample of horses in training. We find that although WBC and other cell counts are generally correlated with each other and other biochemistry parameters across their full range of values, fibrinogen and SAA exhibit the greatest concordance among the top 15% of values. Moreover, horses with the top WBC values do not overlap significantly with those having the top fibrinogen and SAA values. Because most horses are healthy, these patterns suggest that natural variation in cell counts and biochemistry largely occlude values that might indicate health issues. In contrast, the subset of unusual horses with elevated levels of both fibrinogen and SAA are strongly suggestive of the expected handful of animals with minor, undetected issues. We conclude that fibrinogen and SAA have excellent potential as biomarkers and are likely to be more informative about conditions relevant to horses in training compared with the widely used WBC.     

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.     

Analytical validation of commercially available methods for acute phase proteins quantification in pigs

The aim of this study was to validate commercially available methods for porcine haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and major acute phase protein (Pig-MAP) determinations. Intra and inter assay coefficients of variation (CVs) were lower than 20% in all cases with exception of inter assay CVs for CRP and Pig-MAP assays with samples of low acute phase proteins concentration, and for SAA assay at any acute phase proteins concentration. All methods showed good linearity and detection limits were low enough to detect APPs levels in healthy animals. Hp and SAA were very affected by haemolysis. Lipaemia influenced mainly on SAA determination. Over 15-fold increase was observed in CRP and SAA concentrations after artificially induced inflammation by a single subcutaneous dose of turpentine, whereas Hp and Pig-MAP increased less than 5-fold.     

Serum and synovial fluid concentrations of ampicillin trihydrate in calves with suppurative arthritis

Eight calves with suppurative arthritis were each given a single intramuscular injection of ampicillin trihydrate at a dose of 10 mg/kg. Ampicillin concentrations were measured serially in serum and in suppurative and normal synovial fluid over a 24-hour period. The mean peak serum concentration was 2.5 +/- 0.54 micrograms/ml 2 hours after injection. The highest concentration in normal synovial fluid was 3.5 +/- 0.40 micrograms/ml at 4 hours and the highest concentration in suppurative synovial fluid was 2.7 +/- 0.58 micrograms/ml at 2 hours. Overall mean ampicillin concentration in normal synovial fluid for the first 8 h (2.9 +/- 0.32 micrograms/ml) was significantly different from that in suppurative synovial fluid (2.1 +/- 0.33 micrograms/ml) and serum (1.9 +/- 0.30 micrograms/ml; p less than 0.05).     

Steroid Responsive Meningitis-Arteritis: A Prospective Study of Potential Disease Markers, Prednisolone Treatment, and Long-Term Outcome in 20 Dogs (2006–2008)

Background: Previous multidrug studies have identified the value of prednisolone in treating steroid responsive meningitis-arteritis (SRMA) and the potential value of acute phase proteins (APPs) and immunoglobulin A (IgA) in diagnosis and monitoring.
Hypothesis: (1) Prednisolone monotherapy is a successful immunosuppressive modality in the treatment of SRMA; (2) protein markers are useful in identifying the potential for relapse.
Animals: Twenty client-owned dogs with SRMA presented to the University of Glasgow Small Animal Hospital between May 2006 and May 2008.
Methods: A prospective, observational study: CBC, biochemistry, and cerebrospinal fluid (CSF) analyses were performed. C-reactive protein (CRP), serum amyloid-A, α-1-acid glycoprotein, and haptoglobin (Hp) were assessed in the serum. IgA concentrations were determined in the serum and CSF.
Results: Clinical resolution of SRMA was achieved in all 20 dogs. Serum CRP concentration remained increased at remission in 16/20 dogs whereas CSF cytology was within normal limits in 20/20 dogs. Serum APPs decreased significantly on treatment ( P < .05) except Hp, which remained unaltered. Serum and CSF IgA concentrations remained increased for the duration of treatment.
Conclusions and Clinical Importance: The prednisolone regimen presented was successful in treating SRMA without the need for additional drugs. Serum APPs are of use in the diagnosis and management of SRMA, particularly in relation to identifying relapse. Serum and CSF IgA concentrations remain increased throughout disease, aiding in diagnosis but not contributing to the management of SRMA.