The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis.

The pig acute phase protein (APP) response to experimental Streptococcus suis (S. suis) infection was mapped by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pig-MAP) and the negative APPs albumin and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p.i.), until the pigs were killed and autopsied on day 14 p.i. Clinical signs (fever and lameness) were observed in four of the five inoculated pigs from day 2 p.i., and these pigs also had arthritic lesions at autopsy. CRP and SAA showed fast increases in serum concentrations, CRP being elevated from days 1 to 12 p.i. and peaking at 10 times the day 0-levels on day 1 p.i. SAA rose quickly to peak levels of 30-40 times the day 0-level on days 1-2 and returned to pre-inoculation level on day 5 p.i. Hp and pig-MAP showed slightly slower responses, both peaking around 5 days p.i. Hp was increased throughout the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes in albumin levels could be identified. One pig, showing clinical signs on day 2 only, also showed an APP response, although of a relatively short duration, whereas three pigs presenting clinical signs for several days had a more protracted acute phase response. Remarkably, the one pig showing no clinical signs and no arthritic lesions showed an APP response comparable to that of the other, clinically affected pigs. Thus, both acute clinical and subclinical S. suis infection could be revealed by the measurement of one or more of the APPs CRP, SAA, Hp, pig-MAP and Apo A-I. The combined measurement of two or three APPs, including proteins with slow and fast kinetics, should be used to achieve the highest sensitivity for the detection of ongoing S. suis infection during a prolonged time period. A diagnostic tool based on such APP-measurements could considerably improve strategic control procedures for this important infection.

Purification and determination of C-reactive protein and inter-α-trypsin inhibitor heavy chain 4 in dogs after major surgery through generation of specific antibodies.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) and C-reactive protein (CRP) have been isolated from acute phase dog sera by affinity chromatography with insolubilized polyclonal antibodies anti pig Major Acute phase Protein (Pig-MAP) and with p-Aminophenyl Phosphoryl Choline, respectively. Isolated proteins were used to prepare specific polyclonal rabbit antisera that have allowed quantifying their concentration in serum samples by single radial immunodifussion. Both proteins were quantified in sera from female dogs that had undergone ovariohysterectomy (OVH, n=9) or mastectomy (n=10). The observed increases in CRP concentrations showed that surgical traumas induced an acute phase response of a great magnitude in the dogs. In both surgeries a four-fold increase of ITIH4 concentrations was detected. It can be concluded that ITIH4 is a new positive acute phase protein in dogs, as reported in other species.

Fatty acids bound to alpha-fetoprotein and albumin during rat development.

The time-course levels and composition of the fatty acids bound to rat alpha-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n-3], either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n-3]. These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.

The effect of trypsin on bovine transferrin and lactoferrin.

The iron-saturated and iron-free (apo) forms of bovine transferrin and lactoferrin were digested with trypsin and the digests analysed by column chromatography and electrophoresis. Both of the iron-saturated proteins were more resistant to proteolysis than the corresponding apoproteins, and iron-transferrin was more resistant than iron-lactoferrin. Digestion of iron-transferrin yielded two iron-binding fragments with molecular weights of 32 000 and 38 500 whereas apotransferrin yielded only the larger fragment. In digests of lactoferrin, up to five different fragments with molecular weights ranging from 25 000 to 52 700 were detected, there being no obvious qualitative difference between digests of iron-lactoferrin and apolactoferrin. The susceptibility of apolactoferrin to tryptic digestion was only slightly reduced when apolactoferrin was complexed with beta-lactoglobulin, suggesting that complex-formation is not a mechanism for protecting lactoferrin against intestinal degradation. There was no immunological cross reaction between bovine transferrin or its digestion products against anti-lactoferrin antiserum, or vice-versa.

Major plasma proteins in pig serum during postnatal development.

The time-course of changes in the levels of albumin, alpha-fetoprotein (AFP), alpha(1)-protease inhibitor (alpha(1)-antitrypsin), alpha(1)-acid glycoprotein, fetuin, haptoglobin, transferrin, IgG and the major acute-phase protein (Pig-MAP) in the blood sera of pigs during the first days and weeks of life was investigated by quantitative radial immunodiffusion. The serum of newborn pigs before suckling was characterised by a very low concentration of total proteins (approximately 25 mg mL(-1)), low levels of albumin and transferrin and the lack of immunoglobulins. In contrast, alpha(1)-acid glycoprotein and fetuin are present at high levels (approximately 12 and 5 mg mL(-1) respectively). The results of the present study show that the piglets undergo a very rapid metabolic maturation with regard to serum proteins, evolving from a characteristic 'fetal' pattern to an 'adult' one. We have paid special attention to the evolution of haptoglobin and Pig-MAP, which are two important acute-phase proteins in pigs. The evolution of serum levels of these proteins suggests that piglets must overcome a moderate acute-phase situation during the first week of life.

The major acute phase serum protein in pigs is homologous to human plasma kallikrein sensitive PK-120.

A major acute phase protein (pig-MAP) has been isolated from the sera of pigs after turpentine injection. The protein is the pig counterpart of a recently cloned human serum protein denominated PK-120, which is a putative substrate for kallikrein [Nishimura et al., 1995 FEBS Lett. 357, 207-211]. The protein exists in other mammalian species and it is also an acute phase protein, at least in the rat. Pig-MAP shows homology, as PK-120, with the heavy chain 2 (HC-2) of the inter-alpha-trypsin inhibitor superfamily but does not possess trypsin inhibitory activity.

Incorporation of radiolabelled alphafetoprotein in the brain and other tissues of the developing rat.

The time course of uptake and autoradiographic localization of alphafetoprotein (AFP) were studied in the brain and other organs of fetuses, neonates and young rats injected with homologous radiolabelled AFP. Comparative data of radioactivity accumulation in the brain relative to that of several tissues (blood, liver, tongue, small intestine) showed bimodal patterns reflecting two periods of more active incorporation, with a maximum before the 16th day of fetal development. In brain autoradiographs, the strongest labelling was observed in 17-day-old fetuses 24 h after injection into the mother of [125I]AFP. The labelling included all regions of the brain. The results presented here give experimental support to the hypothesis that the presence of AFP in the developing nervous system of mammals and birds is primarily due to protein uptake rather than in situ synthesis.

The porcine acute phase response to infection with Actinobacillus pleuropneumoniae. Haptoglobin, C-reactive protein, major acute phase protein and serum amyloid A protein are sensitive indicators of infection.

In an experimental infection model mimicking acute Actinobacillus pleuropneumoniae (Ap) infection in swine (Sus scrofa) by aerosol inoculation, the development of a number of typical clinical signs was accompanied by a prototypic acute phase reaction encompassing fever and an acute phase protein response peaking at around 2 days after infection. Haptoglobin, C-reactive protein (CRP), and major acute phase protein (MAP) responded with large increases in serum levels, preceding the development of specific antibodies by 4-5 days. Serum amyloid A protein (SAA) was also strongly induced. The increase, kinetics of induction and normalization were different between these proteins. It is concluded that experimental Ap-infection by the aerosol route induces a typical acute phase reaction in the pig, and that pig Hp, CRP, MAP, and SAA are major acute phase reactants. These findings indicate the possibility of using one or more of these reactants for the nonspecific surveillance of pig health status.

Interaction of bovine lactoferrin with other proteins of milk whey.

The association between bovine lactoferrin and the major bovine whey proteins, beta-lactoglobulin, alpha-lactalbumin and albumin has been studied by immunochemical techniques, gel filtration and affinity chromatography in lacteal secretions and using purified proteins. Bovine lactoferrin is able to form non-covalent complexes with beta-lactoglobulin or albumin, with lactoferrin-protein molar ratios of 2:1 and 1:1 respectively. No association was detected with alpha-lactalbumin. Lactoferrin interacts with beta-lactoglobulin-Sepharose at low ionic strength, but not in the presence of 0.3 M NaCl, indicating that ionic interactions are important. The lack of association with alpha-lactalbumin suggests however a certain degree of specificity in this electrostatic interaction.

Effects of different antimicrobial treatments on serum acute phase responses and leucocyte counts in pigs after a primary and a secondary challenge infection with Actinobacillus pleuropneumoniae.

The susceptibility to an initial challenge and a re-challenge inoculation with Actinobacillus pleuropneumoniae was analysed in pigs that were treated with antimicrobials of different efficacies following the first exposure to A pleuropneumoniae. In brief, 30 nine-week-old specific pathogen-free pigs were allocated to five groups of six. After acclimatisation, four groups were inoculated with A pleuropneumoniae serotype 2. At the onset of clinical signs, three of the groups of pigs were treated with enrofloxacin, tetracycline or penicillin. A fourth group served as the inoculated control and the fifth group as a control group that had not been inoculated. On day 28, all five groups were re-challenged with the same strain of A pleuropneumoniae serotype 2 as had been used in the first inoculation. No treatments were carried out at this time. The acute phase responses and differential leucocyte counts were monitored in detail after both inoculations. Leucocytosis and acute phase responses in the forms of serum amyloid A, pig-major acute phase protein and haptoglobin were recorded in all of the inoculated groups after the onset of clinical signs following the first inoculation. A porcine mannan-binding lectin-A response was less evident in the pigs. Acute phase responses resembling those of the first inoculation were observed in the pigs that had not previously been inoculated and in the pigs treated with enrofloxacin. Acute phase responses were not recorded in the other three groups, where the pigs had seroconverted to A pleuropneumoniae serotype 2 following the first inoculation.

Acute-phase protein response in pigs experimentally infected with Haemophilus parasuis.

The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10(9)CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-immunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.

Inter-alpha-trypsin inhibitor heavy chain 4 as a marker of acute rejection in pancreas allotransplantation in pigs.

BACKGROUND/AIMS: An early, simple, and reliable marker for acute pancreatic allograft rejection is not available. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an interleukin-6-dependent acute-phase positive protein that can act as an anti-inflammatory protein. We studied the response of the ITIH4 in pigs undergoing pancreas allotransplantation (PT) and evaluated this protein as a biomarker for acute graft rejection. METHODS: PT with enteric drainage of the exocrine secretion and systemic venous drainage was performed on 12 Landrace pigs. No immunosuppression was administered. Serum concentrations of glucose, amylase, lipase, insulin, C-peptide, and ITIH4 were determined daily. RESULTS: The response of ITIH4 to PT was early, intense, and prolonged, with 2 peaks in serum concentration. The first peak, which started on day 1 and reached maximum (around 6 mg/dL) on day 3, was attributed to the systemic acute phase response to surgical stress. The second peak, which exceeded the first peak and reached maximum (>8 mg/dL) on day 6, began when the recipients were still normoglycemic, and preceded onset of the diabetic state caused by acute graft rejection by an average of 4 days. CONCLUSION: Serum ITIH4 could help to predict subclinical acute graft rejection after PT in pigs.

Pig acute-phase protein levels after stress induced by changes in the pattern of food administration.

A total of 240 pigs, 74 days old, half boars and half females, were included in a trial designed to assess the effect of the stress caused by changes in the pattern of food administration on the concentration of acute phase proteins (APP) and productive performance parameters. Half of the animals (pigs fed ad libitum, AL group) had free access to feed, while the rest were fed following a disorderly pattern (DIS group), in which animals had alternating periods of free access to feed and periods of no feeding, when food was removed from the feeder. The periods of free access to feed (two daily periods of 2-h duration) were randomly assigned, and varied from day to day. Total feed supplied per day was identical in both groups, and exceeded the minimal amount required for animals of these ages. Pen feed intake, individual body weights and the main positive pig APP pig major acute phase protein (Pig-MAP), haptoglobin, serum amyloid A (SAA), C-reactive protein (CRP), and the negative APP apolipoprotein A-I (ApoA-I) and transtherytin were determined every 2 weeks during the period 76 to 116 days of age. Animals fed ad libitum had better average daily gain (ADG) than DIS animals in the whole experimental period (P < 0.01) but the differences in ADG were only produced in the two first experimental sub-periods (60 to 74 and 74 to 116 days of age), suggesting that the stress diminished when the animals get used to the DIS feeding. Interestingly differences in ADG between DIS and AL pigs were due to males, whereas no differences were observed between females. The same differences observed for ADG were found for APP. DIS males had higher Pig-MAP concentration than AL males at 74 and 116 days of age, lower ApoA-I concentration at 74 days of age and higher haptoglobin and CRP concentration at 116 days of age (P < 0.05). The results obtained in this trial show an inverse relationship between weight gain and APP levels, and suggest that APP may be biomarkers for the evaluation of distress and welfare in pigs.

The concentration of apolipoprotein A-I decreases during experimentally induced acute-phase processes in pigs.

In this work, apolipoprotein A-I (ApoA-I) was purified from pig sera. The responses of this protein after sterile inflammation and in animals infected with Actinobacillus pleuropneumoniae or Streptococcus suis were investigated. Decreases in the concentrations of ApoA-I, two to five times lower than the initial values, were observed at 2 to 4 days. It is concluded that ApoA-I is a negative acute-phase protein in pigs.

Characterization of a new alpha-glycoprotein as the major serum component in later fetal and newborn pigs.

1. Using crossed immunoelectrophoresis, fetuin, alpha-fetoprotein, albumin, transferrin, alpha1-antitrypsin and a fetospecific-like alpha-glycoprotein, were identified as the main serum components in fetal pig (Sus scrofa domesticus). 2. Fetuin was found to be a trypsin inhibitor, but not a chymotrypsin inhibitor. 3. The fetospecific-like alpha-glycoprotein, not previously reported, accounted for 50% of the total serum proteins in newborn pigs. This protein, however, was a minor component of the adult serum.

Concentrations of major plasma proteins in serum and whole-tissue extracts of porcine fetuses during development.

In extracts from fetuses up to 32 days of gestation, the major serum proteins were fetuin, alpha-fetoprotein and alpha 1-antitrypsin, but albumin was not detected. The concentration of all proteins rose with age until 40-50 days of gestation; and then the serum concentration of alpha-fetoprotein (2.9 mg ml-1), alpha 1-antitrypsin (4.4 mg ml-1) and transferrin (2.6 mg ml-1) fell progressively to about 1 mg ml-1 at birth, whereas those of fetuin, albumin and alpha 1-acid glycoprotein increased. The patterns of serum proteins in fetuses at about the middle of gestation were similar in extracts and sera. At birth, the major proteins were alpha 1-acid glycoprotein and fetuin, which accounted for 45 and 18% of serum proteins, respectively. Albumin represented only 7% of serum proteins at this age. For most of the second gestational period, the six quantified proteins accounted for about 85% of total serum proteins. In early gestation, a significant proportion of serum proteins was intracellular.

The major serum protein of fetal and newborn pigs: biochemical properties and identification as a fetal form of alpha 1-acid glycoprotein.

The major protein component of fetal pig serum, has been immunologically identified as alpha 1-acid glycoprotein (orosomucoid). Amino acid composition and total carbohydrate content (around 38% by weight) were similar in the adult and fetal forms of alpha 1-acid glycoprotein. These forms differ, however, in the proportion of individual monosaccharides. Fucose, represented the 1.5% (by weight) in the fetal protein, and the 2.5% in its adult counterpart. The latter was more susceptible to neuraminidase and also possesses a higher mannose/galactose ratio than the fetal form. Insolubilized Concanavalin A (Con A) retained 80% of the adult protein, whereas the fetal form was mostly Con A-non reactive. The proportion of this -non reactive fraction, as revealed by crossed immuno-affino-electrophoresis experiments, was age-dependent and varied from 62% at fetal age of 50-60 days to 80% at birth.

The effect of trypsin and chymotrypsin on the antibacterial activity of complement, antibodies, and lactoferrin and transferrin in bovine colostrum.

The effect of trypsin and chymotrypsin on antibacterial factors in bovine colostrum has been studied. Endogenous complement in colostrum was extremely sensitive to both enzymes. IgM was attacked by chymotrypsin but not by trypsin. Trypsin slowly attacked IgG1, causing loss of biological activity due to cleavage of both light and heavy chains. IgG1 was only very slightly attacked by chymotrypsin. Lactoferrin and transferrin in the iron-free state were both susceptible to proteolysis, but the iron saturated forms were more resistant and tended to give rise to stable iron-binding fragments.

Interactions of different albumins and animal sera with insolubilized Cibacron Blue, Evaluation of apparent affinity constants.

1. A high concentration Cibacron Blue-Sepharose derivative has been used to study the affinity chromatography of albumin from eight animal species. 2. The apparent affinity constants for albumin varies between 3.9 x 10(4) M-1 and 0.9 x 10(4) M-1, in the order: Human greater than rabbit greater than horse greater than pig = dog greater than bovine greater than rat greater than chicken. 3. Other serum proteins were also bound to the gel, particularly lipoproteins and alpha 2-macroglobulin.