The acute phase protein serum amyloid A (SAA) as an inflammatory marker in equine influenza virus infection

The acute phase protein serum amyloid A (SAA) has proven potentially useful as an inflammatory marker in the horse, but the knowledge of SAA responses in viral diseases is limited. The aim of this study was to evaluate SAA as a marker for acute equine influenza A2 (H3N8) virus infection. This is a highly contagious, serious condition that inflicts suffering on affected horses and predisposes them to secondary bacterial infections and impaired performance. Seventy horses, suffering from equine influenza, as verified by clinical signs and seroconversion, were sampled in the acute (the first 48 h) and convalescent (days 11-22) stages of the disease, and SAA concentrations were determined. Clinical signs and rectal temperature were recorded. Secondary infections, that could have influenced SAA concentrations, were clinically suspected in 4 horses. SAA concentrations were higher in the acute stage than in the convalescent stage, and there was a statistically positive relationship between acute stage SAA concentrations and clinical signs and between acute stage SAA concentrations and maximal rectal temperature. Horses sampled early in the acute stage had lower SAA concentrations than those sampled later, indicating increasing concentrations during the first 48 h. There was a statistically positive relationship between convalescent SAA concentrations and degree of clinical signs during the disease process. The results of this investigation indicate that equine SAA responds to equine influenza infection by increasing in concentration during the first 48 h of clinical signs and returning to baseline within 11-22 days in uncomplicated cases.     

Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations

The aim of the present study was to evaluate whether equine serum amyloid A (SAA) concentrations could be measured reliably with a turbidometric immunoassay (TIA) developed for use with human serum. Intra- and inter-assay imprecision were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by linearity under dilution. The assay was subsequently used for measuring SAA concentrations in clinically healthy horses, horses with inflammatory diseases, horses with non-inflammatory diseases, and in horses before and after castration. In pools with low, intermediate and high SAA concentrations, the intra-assay imprecisions were 24.4%, 1.6% and 2.1%, and the inter-assay imprecisions were 33.2%, 4.6% and 6.5%. Slight signs of inaccuracy were observed, but these inaccuracies were negligible when considering the large dynamic range of the SAA response. The assay was able to detect the expected difference in SAA levels in different groups of horses. It was also able to demonstrate the expected dynamic changes in SAA after castration. In conclusion, equine SAA concentrations can be measured reliably using the TIA designed for human SAA.     

Evaluation of feline serum amyloid A (SAA) as an inflammatory marker

The concentration of feline serum amyloid A (fSAA) was determined by a direct enzyme-linked immunosorbent assay (ELISA) by using fSAA specific monoclonal antibodies, to evaluate the fSAA as an inflammatory marker in cats. The mean concentration +/- standard deviation of fSAA was found to be 0.60 +/- 1.06 microg/m l and 33.65 +/- 67.59 microg/ml in serum samples from normal cats (n=45) and cats (n=312) with various diseases and disorders, respectively. A significant difference (p<0.001) was found between the two groups. It was also found that the concentration of fSAA begins to increase rapidly at approximately 3-6 hr after spay, and increases up to significantly high levels in some disorders, like injury, renal failure, infectious diseases, etc.     

Expression of recombinant feline serum amyloid A (SAA) protein.

Feline serum amyloid A (SAA) cDNA clone was isolated from a hepatic mRNA of a mixed-breed cat. The feline SAA cDNA clone contains 333 nucleotides and deduced amino acid sequence shows 87.4%, 73.9%, and 65.8% homology with dog, human and mouse SAA respectively. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified in the feline cDNA clone. Recombinant feline SAA (rfSAA) was expressed at high levels using pGEX bacterial expression system. Cleavage from the fusion moiety, and purification using glutathione-sepharose yielded pure soluble form of rfSAA. Antibodies generated against rfSAA were specific for feline SAA and showed no cross-reactivity with human SAA. Furthermore, antibodies against human SAA did not react with feline SAA. These results indicate that antigenicity of feline SAA is totally different from human SAA.     

Molecular cloning and sequencing of equine cDNA encoding serum amyloid A (SAA)

The serum amyloid A (SAA) protein is a characteristic and sensitive acute phase reactant in all vertebrates investigated. We molecularly cloned the equine cDNA encoding SAA from the liver of a healthy horse by polymerase chain reaction (PCR). The cloned cDNA is 480 bases in length, and contains an open reading frame (ORF) of 387 nucleotides encoding a precursor SAA protein of 128 amino acids. The precursor of horse SAA seems to have an 18-residue signal peptide and differs from the reported amino acid sequences of the horse SAA by substitution of valine at residue 81. It shows high homology with SAA amino acid sequence of other species such as dog (80.6%), mink (77.5%), human (76.9%) and duck (71.9%). An insertion of eight amino acids at residues between 85 and 92, as compared to human SAA, has also been found in horse SAA. The availability of the equine SAA cDNA will provide a useful reagent for studying its role in diseased horses.     

Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response

A sensitive and precise immunoassay for equine serum amyloid A protein (SAA) was established and used to determine, for the first time, the circulating concentration of this protein in health and disease. As in other species, equine SAA was present only at trace levels in healthy animals but behaved as an extremely sensitive and rapidly responding acute phase reactant following most forms of tissue injury, infection and inflammation, objectively reflecting the extent and activity of disease. Measurements of SAA should make a significant contribution to diagnosis and management of viral and bacterial infection in horses, and routine serial assays could provide an objective criterion for monitoring prospectively the general health of horses in training and racing.     

Dynamics in serum of the inflammatory markers serum amyloid A (SAA), haptoglobin,fibrinogen and alpha2-globulins during induced noninfectious arthritis in the horse

Despite the importance of noninfectious joint diseases in equine medicine, little is known about the acute phase response which may be elicited if the local inflammatory process of noninfectious arthritis is sufficiently strong, Therefore the aim of this study was to monitor the systemic inflammatory response during experimentally-induced noninfectious arthritis by studying the dynamics in serum of the acute phase proteins serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins. Twenty-four Standardbred horses, age 3-7 years, found healthy on thorough clinical, radiological, haematological and serum biochemical examination, were injected aseptically into the right midcarpal joint with amphotericin B. Blood samples were drawn before induction of arthritis (0 h), and at 8, 16, 24, 36 and 48 h postinduction and then on Days 3, 4, 5 and 15 postinduction. All horses developed lameness with joint effusion and joint heat as well as increased respiratory rate, heart rate and body temperature. The lameness started to decline after 24-36 h and, in most animals, systemic signs disappeared on Day 2 postinjection. The concentration of the acute phase proteins increased following induction of arthritis. The SAA concentrations were higher than baseline concentrations from 16 h postinduction and were maximal at 36-48 h (227 times baseline concentration). The haptoglobin concentrations were higher than baseline concentrations from 24 h and were maximal at 48-96 h (1.14 times baseline concentration). The maximal concentrations of fibrinogen were seen between 36-72 h postinjection and increased on average 0.87 times from baseline concentrations. The fibrinogen concentrations were higher than baseline concentrations from 24 h postinjection. Alpha2-globulins concentrations showed a minor increase and increased 0.55 times from baseline concentrations. The markers had returned to baseline concentrations by Day 15. Our results demonstrate that amphotericin B-induced arthritis in a single joint gives rise to a systemic acute phase response measurable as increased concentrations in serum SAA, haptoglobin, fibrinogen and alpha2-globulins during the first 2 weeks of the condition and, thereby, that such an increase need not be indicative of infectious arthritis. Further research should be aimed at determining whether chronic noninfectious arthritis in the horse gives rise to increased acute phase protein concentrations in serum.     

Serum amyloid A3 (SAA3), not SAA1 appears to be the major acute phase SAA isoform in the pig

The acute-phase serum amyloid A (SAA) protein family comprises two main circulating (systemic) isoforms, SAA1 and SAA2, synthesised in liver and one local isoform, SAA3, produced in extrahepatic tissues. Systemic and local SAA show structural differences, which suggests different functions. In the pig, AA-amyloidosis is extremely uncommon, and the structural protein in swine has characteristics of systemic SAA. The only pig SAA sequences published so far, either derived form hepatic or extrahepatic sites have been designated SAA2, but the translated protein shows the properties of SAA3 proteins. The aim of this study was to characterise all the porcine SAA isoforms by sequencing from cDNA and genomic DNA obtained form multiple porcine tissues. Primer pairs were designed to amplify presumably all isoforms of SAA firstly and then specifically for each isotype. Results show that the only isotype isolated and sequenced both from hepatic and extrahepatic tissues correspond to a SAA3-like amino acid sequence. No SAA1-like sequences were identified, which could be indicative of the gene being very rare and consistent with the observed resistance to AA-amyloidosis. Finally, it is concluded that the pig is unique among other species in that the main circulating hepatic SAA isotype shows the characteristics of local highly alkaline SAA. This likely precludes a function as apolipoprotein.     

Evaluation of a commercially available apparatus for measuring the acute phase protein serum amyloid A in horses

The aim of this study was to investigate the reliability of an immunoturbidometric assay for measuring the acute phase protein serum amyloid A (SAA) in horses in clinical practice. The assay was compared to a previously validated assay, and overlap performance was assessed by measuring the concentration of SAA in clinically healthy horses and horses with inflammatory and non-inflammatory diseases. In pools of serum with low and high SAA concentrations the assay's intra-assay coefficients of variation were 11.7 per cent and 4.6 per cent, and its interassay coefficients of variation were 9.1 per cent and 5.6 per cent, respectively. Slight inaccuracies were observed, but they were negligible in comparison with the range of the SAA response. The assay systematically underestimated the concentrations of SAA in comparison with the results of the validated assay. The assay detected the expected difference in SAA concentrations between the healthy and diseased horses.     

A radial immunodiffusion enzyme assay for detection of antibody to equine rhinopneumonitis virus (EHV-1) in horse serum

A radial immunodiffusion enzyme assay (RIDEA) was developed for detection and quantitation of antibodies to equine herpes virus-1 (EHV-1) in horse sera. The detection and quantitation of EHV-1 antibody levels were based on the diameter of the radial diffusion zone of specific antibody in each serum sample reacting with EHV-1 antigen. The circular zone was made visible using peroxidase-conjugated rabbit anti-horse immunoglobulin G and a substrate containing hydrogen peroxide. The results of the RIDEA were compared with those of virus neutralization (VN) and enzyme-linked immunosorbent assay (ELISA) and found to be highly correlated. The relative sensitivity and specificity (percentage of agreement with VN test) were found to be 98.2 and 92.5%, respectively. Because the test procedure is relatively easy to perform, the RIDEA could be used as a field test to detect antibodies to EHV-1 in horses.     

A direct enzyme immunoassay for the measurement of furosemide in horse plasma

A new enzyme immunoassay (EIA) for the measurement of furosemide in horse plasma is described. The lower limit of detection of this EIA method was 7.8 ng/ml. The intra-and inter-assay coefficients of variation ranged from 2.5% to 4.9% and 7.5% to 9.8%, respectively. Cross-reactivity with other compounds was not observed. There was a high correlation (r2=0.987) between the high-performance liquid chromatography and EIA results obtained for furosemide concentrations in horse plasma. These results indicate that the newly developed EIA method is useful for the quantitative analysis of furosemide in horse plasma.     

Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: a field study

REASONS FOR PERFORMING THE STUDY: Early recognition of excessive inflammation and infectious complications after surgery, leading to early institution of therapy, reduces post operative discomfort and facilitates recovery. Because serum amyloid A (SAA) is a highly sensitive marker of inflammation, measurements of SAA and other acute phase reactants in the equine surgical patient may be valuable in assisting clinical assessment of post operative inflammation. OBJECTIVES: To investigate changes in inflammatory markers after castration and to correlate levels of acute phase reactants with clinical severity of inflammation after castration. METHODS: Leucocyte numbers and blood levels of iron, SAA and fibrinogen were determined before castration and on Days 3 and 8 post operatively in 2 groups of horses; Group 1 (n = 11) had mild post operative inflammation and an uncomplicated recovery and Group 2 (n = 7) had local clinical signs of moderate to severe inflammation. RESULTS: Both groups had elevated serum SAA levels at Day 3 post operatively. In Group 1 concentrations had returned to preoperative levels by Day 8, whereas in Group 2 concentrations remained elevated. Plasma fibrinogen concentrations in serum increased to equal levels in both groups and stayed elevated throughout the study period. Serum iron concentrations of Group 1 did not change in response to castration, whereas concentrations in Group 2 decreased below preoperative levels on Day 8. Leucocyte numbers remained unchanged during the post operative period in both groups. CONCLUSIONS: Serum SAA and iron profiles reflected the course of inflammation and their levels correlated with the clinical severity of inflammation. In contrast, fever and changes in leucocyte numbers, which are usually considered to be hallmarks of inflammation and infection, were not useful for monitoring post operative recovery. POTENTIAL RELEVANCE: Measurements of SAA and iron may improve post operative monitoring. As sustained inflammation may indicate that the surgical wound has become infected, SAA and iron measurements may facilitate early recognition and hence early treatment of infection.     

Late-stage mediators of the inflammatory response: identification of interleukin-1 and a casein-degrading enzyme in equine acute inflammatory exudates

Interleukin-1 and a casein-degrading enzyme have been identified in an experimental system for studying acute inflammation in the horse. The levels of both the cytokine and the proteinase increased over the first 24 hours following initiation of the inflammatory response, and remained at high levels through to the last sample collected at 48 hours. This is in marked contrast to prostaglandin E2 concentrations which were low initially, peaked at four to eight hours and had returned to low levels by 12 to 24 hours. It is likely that interleukin-1 and various proteinases are involved in the later stages of the inflammatory response, such as the tissue remodelling associated with wound repair, and control of this cytokine may be important in the progression from acute to chronic inflammation.     

The association between plasma levels of acute phase proteins, haptoglobin, alpha-1 acid glycoprotein (AGP), pig-MAP, transthyretin and serum amyloid A (SAA) in Large White and Meishan pigs

During infection, the acute phase response triggers the release of acute phase proteins (APP), alpha-(1) acid glycoprotein (AGP), serum amyloid A (SAA) and Pig-MAP into the circulation, accompanied by a decrease in plasma levels of transthyretin. We quantified the association between these APP in 26 apparently healthy pigs from two breeds, 13 Large White and 13 Meishan (16 male; 10 female). There was a significant correlation between plasma levels of haptoglobin and Pig-MAP (r=0.57; p<0.05), but no significant associations between any of the other APP tested. We also measured the relationship between PigMAP, transthyretin and SAA, and the proportions of peripheral blood mononuclear sub-sets, CD8(+) cells, CD4(+) cells, CD11R1(+) cells, MHC DQ(+) cells, and monocytes. There were correlations between both plasma levels of Pig-MAP and the proportion of monocytes (r=0.55; p<0.05) and plasma levels of transthyretin and the proportion of MHC DQ(+) cells (r=0.40; p<0.01). Breed and sex influenced plasma levels of Pig-MAP but not plasma levels of transthyretin. Overall, these results suggest closer links between the mechanisms that regulate the release haptoglobin, Pig-MAP and monocytes compared to those that regulate the release of AGP, SAA and transthyretin.     

Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon

A study aimed at validating a human progesterone enzyme immunoassay kit was carried out on cattle at Bambui, Cameroon. Progesterone ELISA Kits (EH-511) were obtained from Clinpro International. Forty-one cows were selected, of which 19 were pregnant and 22 within 14 days post partum. Blood samples were analysed and progesterone levels were deduced from a curve obtained from standard absorbance values (A ₄₅₀). Results show that 95.5% of postpartum cows had progesterone levels below 1 ng/ml, with the highest level being 0.75 ng/ml. The mean level was 0.5 ± 0.26 ng/ml. The cows in the ‘pregnant group’ had progesterone levels ranging from 3.5 to 17.5 ng/ml. This kit can be used for measuring progesterone levels in cattle. Levels of 1 ng/ml for two consecutive samples or one sample at or above 3 ng/ml are an indication of the presence of corpus luteum, while cows below 1 ng/ml will be in anoestrus.     

Serological diagnosis of influenza A infections in the horse by enzyme immunoassay. Comparison with the complement fixation test

An enzyme immunoassay (EIA) using horseradish peroxidase and a type-specific antigen is described for the detection and quantitation of anti-influenza antibodies in the horse. Compared with the complement fixation (CF) test (using the same antigen), EIA proved to be superior with respect to sensitivity and reliability. The internal variation of EIA was low and thus small titres in EIA can be considered of diagnostic significance. However, no strict correlation with CF was observed. The use of an immunoconjugate against equine IgM in parallel with IgG would certainly improve the sensitivity of the test, especially in early stages of infection.     

The use of divalent cation chelating agents (EDTA/EGTA) to reduce non-specific serum protein interaction in enzyme immunoassay

An indirect enzyme immunoassay (ELISA) for detection of bovine antibody toBrucella abortus was modified by the addition of divalent chelating agents to the serum diluent. This addition resulted in an increase in specificity from 96.0% in the regular assay to 99.4% in the modified procedure. Of the 15 715 sera initially tested by the indirect ELISA, 691 that had given positive reactions were selected for retesting in the indirect ELISA with EDTA/EGTA added. The buffered plate antigen test (BPAT) correctly identified 98.6% of the samples as negative. The addition of chelating agents did not alter the sensitivity of the indirect ELISA, which correctly classified 609 sera from animals from whichB. abortus had been isolated as positive. The sensitivity of the BPAT was 97.8%.Abbreviations ABTS 2,2-azinobis(3-ethylbenzthiazoline sulphonic acid) - BPAT buffered plate antigen test - EDTA ethylenediaminetetraacetic acid disodium salt - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ELISA enzyme-linked immunosorbent assay - IgG₁ immuno- globulin G₁ - IgM immunoglobulin M - Tris tris(hydroxymethyl)aminomethane     

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.     

Staphylococcus aureus lipotechoic acid induces differential expression of bovine serum amyloid A3 (SAA3) by mammary epithelial cells: Implications for early diagnosis of mastitis

Mastitis is one of the most costly diseases of agriculturally important animals and is a common problem for lactating cows. Current methods used to detect clinical and especially subclinical mastitis are either inadequate or problematic. Pathogens such as the gram-positive bacterium Staphylococcus aureus or the gram-negative bacterium Escherichia coli typically cause mastitis. E. coli induces clinical mastitis, whereas, S. aureus causes a subclinical, chronic infection of the mammary gland. In this study we report the differential expression and secretion of mammary-derived serum amyloid A3 (SAA3) by bovine mammary epithelial cells following stimulation with the S. aureus cell wall component, lipotechoic acid (LTA). Two-dimensional immunoblot analyses confirmed that bovine SAA3 is the predominant SAA isoform produced by LTA stimulated mammary epithelial cells. Our previous study showed that bovine SAA3 is also differentially expressed in response to the gram-negative bacterial endotoxin lipopolysaccharide. Collectively, these data indicate that the local production of SAA3 by mammary epithelial cells in response to either gram-positive or gram-negative bacterial components may provide a sensitive indicator for early detection and treatment of mastitis in vivo, minimizing chronic cases of infection, the spread of mastitis to other animals, and economic losses.