Primary bovine hepatocytes in the study of cytokine induced acute-phase protein secretion in vitro.

To investigate the utility of primary cultures of bovine hepatocytes for compartmentalized acute phase protein studies the secretion of serum amyloid-A (SAA) and haptoglobin (Hp) was measured after stimulation by pro-inflammatory cytokines (recombinant human IL-6 (rhIL-6) and recombinant human tumour necrosis factor-alpha (rhTNF-alpha)). During the incubation period of the experiment, the SAA and Hp secretion into the culture medium increased (P < 0.05). SAA concentrations showed an additional increase following treatment with each of the cytokines (P < 0.01). Hp concentrations remained unchanged, whereas incubation with a combination of both resulted in a significant increase of the medium concentration of both SAA (P < 0.01) and Hp (P < 0.05). From these findings it is concluded that primary bovine hepatocytes can be used for in vitro studies on acute-phase protein secretion.

First evidence for the existence of multiple isoforms of bovine serum amyloid-A (apoSAA).

Bovine serum amyloid-A (SAA) was purified from acute-phase high density lipoprotein (HDL) by affinity chromatography and subsequent gel filtration chromatography. The identity of the isolated protein was checked by Western blotting following SDS-urea-PAGE using antisera raised against the purified protein fraction (SAA) and Amyloid A (AA). The antiserum raised against the purified SAA stained Congo red positive regions in the kidney of an AA-amyloidotic cow and reacted on Western blot with an AA-related protein of approximately 14 kDa. Moreover, it immunostained two to three bands, of approximately 14 kDa, present in serum from diseased cows, proportionally to the serum SAA concentration as measured by ELISA. Isoelectric focusing of the purified bovine SAA fraction revealed three major (pI 5.5, 6.0, 6.4) and three minor (pI 4.8, 5.0, 7.3) isoforms and two-dimensional SDS-urea-PAGE confirmed the identity of the major isoforms. Isoelectric focusing of SAA isolated from sera, obtained from cows affected with different diseases, showed a variable ratio of the isoforms. In SAA isolated from serum obtained from a cow suffering from spontaneous AA-amyloidosis only one isoform (pI 4.8) was detectable. It is concluded that the results give first evidence for the existence of multiple isoforms of bovine SAA, occurring in different plasma concentration ratios during different diseases.

Influence of physical stress on the plasma concentration of serum amyloid-A (SAA) and haptoglobin (Hp) in calves.

The influence of physical stress on the plasma concentration of the acute-phase proteins serum amyloid-A (SAA) and haptoglobin (Hp) was studied in 10 calves. Two different stress levels were created by housing two groups of five calves, each on different types of floor. The stress level was assessed by studying videotapes of the animals, and, subsequently, by quantifying the problems related with moving across the pens and the time the calves spent lying down and standing. Plasma concentrations of Hp, SAA, aldolase, and cortisol were measured in blood samples obtained by jugular venepuncture. Plasma SAA concentrations were significantly (p < 0.001) elevated in animals housed on the floor type associated with the highest level of physical stress, although the concentrations were within the normal range for healthy adult cattle. Hp concentrations were not elevated. The floor type did not alter the stress related biochemical variables aldolase and cortisol. It is concluded that plasma SAA concentrations rise upon physical stress, whereas Hp concentrations do not change. The absence of a significant difference in aldolase or cortisol concentrations indicates that the difference in the level of neuro-endocrine stress between the animals housed on the two floor types is only minimal. Consequently, SAA is suggested to be a sensitive variable to assess physical welfare in calves.

Serum amyloid-A (SAA) and haptoglobin (Hp) plasma concentrations in newborn calves.

The concentrations of 2 major bovine acute-phase proteins, haptoglobin (Hp) and serum amyloid-A (SAA), were measured in plasma obtained shortly after birth from 22 healthy calves. In a separate group of diseased calves (n = 8), Hp and SAA concentrations were measured to determine whether newborn calves (up to 4 d old) are able to produce SAA and Hp. In blood samples taken directly after birth, the Hp plasma concentrations were all below the limit of detection. The mean SAA concentration was independent of weight (r = 0.063), degree of acidosis (r = -0.125), sex (p > 0.05), and were not different in calves born after different types of obstetrical help (p > 0.05). In the group of diseased calves, an increased Hp concentration was measured in only 2 of 8 animals, whereas the mean SAA concentration was significantly higher (p < 0.05) than in the healthy newborn calves. These data suggest that prenatal stress due to parturition does not form a stimulus for the production of acute-phase proteins in the fetal calf. The low Hp plasma concentrations might indicate that either it takes a few days to establish a detectable concentration of this protein, or that Hp production is not fully developed in newborn calves.

Concentrations of serum amyloid-A (SAA) and haptoglobin (HP) as parameters of inflammatory diseases in cattle.

The concentrations of the acute-phase proteins, serum amyloid-A (SAA) and haptoglobin (Hp), were determined in the plasma of healthy cows (n = 25) and cows with spontaneous acute (n = 6), subacute (n = 37), or chronic (n = 7) inflammatory diseases. The plasma concentration of SAA alone, Hp alone, and the Hp/SAA ratio, differed significantly (p < 0.001) between healthy animals and animals with inflammatory diseases. Plasma Hp concentrations in the group of cows with acute inflammatory diseases were significantly (p < 0.01) different from those in the group with chronic inflammatory diseases. Moreover, the Hp/SAA ratio in chronic inflammatory diseases was significantly different from this ratio in acute (p < 0.01) and subacute (p < 0.05) inflammatory diseases. It is therefore concluded that the plasma concentrations of SAA and Hp and the Hp/SAA ratio are useful parameters to distinguish healthy animals from animals with inflammation and can be helpful in distinguishing between acute and chronic of inflammatory diseases.

Purification and quantitative measurement of bovine serum amyloid-A.

Bovine serum amyloid-A (b-SAA) was purified from a pool of acute phase serum using hydrophobic interaction chromatography and gel filtration. Serum was applied at a low salt concentration to a phenyl-sepharose column and SAA was eluted with a gradient of 0 to 6 M guanidine-HCl. Fractions containing SAA were pooled, concentrated and further purified by gel filtration on Superose-12. The concentration of SAA in bovine serum was quantified by an indirect ELISA using rabbit anti-human SAA and horseradish peroxidase conjugated donkey anti-rabbit IgG. Dilutions of an acute phase bovine serum sample were used as working standards. The SAA concentration of this standard was determined by comparison with purified b-SAA on SDS-polyacrylamide gel electrophoresis followed by densitometry at 590 nm. The assay detection limit was 3 micrograms ml-1; the intra-assay coefficient of variation was 4 per cent and interassay coefficients of variation were 5.5 per cent and 7.2 per cent at 66 and 178 micrograms ml-1 SAA, respectively. In calves experimentally infected with Pasteurella haemolytica type A1 the ELISA was able to detect a 10-fold increase of SAA within 24 hours of inoculation.

Peripartum acute-phase protein serum amyloid-A concentration in plasma of cows and fetuses.

Concentration of the acute-phase protein serum amyloid-A (SAA) was measured by means of an indirect ELISA method, in plasma of 3 pregnant cows and in plasma taken by cannula from another 4 pregnant cows and, by separate cannula, from their fetuses. Blood samples were taken daily from approximately 72 hours before until 72 hours after parturition. After parturition, SAA concentration increased significantly (P < 0.05) in maternal plasma. In fetal plasma, only a nonsignificant increase was found at time of delivery. The concentration of maternal SAA started to increase within the first 24 hours after delivery, reaching a peak value between 24 and 48 hours after delivery. In the aforementioned plasma samples from the 4 pregnant cows and their fetuses, the concentration of maternally derived cortisol increased nonsignificantly after parturition. The concentration of fetally derived cortisol was significantly (P < 0.05) increased at parturition (t = 0), compared with the initial fetal cortisol concentration at 120 hours before delivery. Peripartum concentration of maternal SAA increased and maternal cortisol remained low, whereas fetal SAA concentration remained low and fetal cortisol concentration increased. These findings indicate possible suppressive action of fetal cortisol on fetal SAA production. However, it might be argued that the main cause of the difference in SAA concentration is the difference in tissue damage between cows and fetuses at parturition.

Relationship between pathological findings and values of haematological and blood-chemistry variables in apparently healthy finishing pigs at slaughter.

The present study was performed to study possibilities of early decision making for appropriate conveyor-line at future slaughtering of normal, clinically healthy finishing pigs. Blood was collected at slaughter from barrows (n = 112). A meticulous examination for subclinical pathological lesions was performed, revealing 5 groups of subjects listed in order of increasing disease-activity: 1--no real disease-activity; 2--with mild subchronic lesions; 3--with subacute lesions; 4--with abscesses; and 5--with fibrinous-necrotic lesions. Significant differences for values of erythrocyte sedimentation rate (ESR), protein, albumin, globulins, and plasma viscosity appeared to occur in this series. It is suggested that measuring acute phase reactants in blood of slaughtered pigs in the near future may reveal appropriate modern tools for meat inspection and predicting slaughtered animal quality.

Failure of adrenocorticotrophic hormone to release serum amyloid A in cattle.

The objective of this study was to determine whether adrenocorticotrophic hormone plays a direct role in the secretion of serum amyloid A (SAA) in cattle. Seven lactating Holstein-Friesian cows were given either saline or 25 iu adrenocorticotrophic hormone (ACTH) intravenously at 09.00 in a two factor crossover design. Plasma cortisol concentrations had increased significantly by one hour after the injection of ACTH, whereas the SAA level had not increased by 24 hours after injection. It is concluded that in cattle adrenocorticotrophic hormone has no direct influence on the release of SAA by the hepatocyte.

[Acute phase proteins as diagnostic aid in veterinary medicine]. / Acute fase-eiwitten als diagnostisch hulpmiddel in de diergeneeskunde.

The acute phase response is the general nonspecific defence mechanism induced by noxious stimuli, which precedes the specific defence mechanism of the immune response. In addition to other changing physiological parameters, the concentration of a number of proteins changes significantly during the acute phase response. These acute phase proteins were studied in detail in human subjects. However, the knowledge of acute phase proteins in domestic animals, is limited. This knowledge is reviewed in the present paper. Attention is directed to C-reactive protein (CRP), serum amyloid-A (SAA) and haptoglobin (Hp). These three proteins promise to be the most useful ones in the routine diagnosis of diseases of animals, which is discussed at the end of the paper.