Characterisation of plasma acute phase protein concentrations in a high health boar herd

Acute phase proteins (APP) are used as markers of inflammation and sub-clinical disease and are considered potential biomarkers for pig health and welfare. However, reference ranges for their baseline concentrations are necessary before their use can be considered in routine herd health. In this study, C-reactive protein (CRP), haptoglobin (Hp), pig-major acute-phase protein (Pig-MAP) and transthyretin (TTR) baseline concentrations were determined in boars from a high health commercial herd and differences between seven commercial breeding lines within the herd were investigated. Reference ranges of 3.6-183 mg/L for CRP, 0.01-1.31 g/L for Hp, 0.32-2.9 g/L for Pig-MAP and 174-610 mg/L for TTR were found. Correlations were determined between Hp and CRP, Hp and Pig-MAP and CRP and Pig-MAP. Additionally, significant differences were found among the concentrations of CRP, Pig-MAP and TTR in seven commercial breeding lines.     

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.     

Acute phase proteins in cattle and swine: A review

The major acute phase proteins (APPs) in cattle are haptoglobin (Hp) and serum amyloid A (SAA), and in swine, are Hp, SAA, C-reactive protein (CRP), and Pig major acute phase protein (Pig-MAP). Many methodologic assays are presently available to measure these parameters, which are still being improved to increase their specificity, sensitivity, user-friendliness, and economic availability. In cattle, the main applications are the diagnosis and monitoring of frequent diseases such as mastitis and metritis in dairy cows and respiratory problems in young calves. In pigs, APPs are useful in the control of bacterial and viral infections, and they may be used at the slaughterhouse to monitor subclinical pathologies and improve food safety. The utility of APP in animal production must not be forgotten; optimization of protocols to improve performance, welfare, and nutrition may benefit from the use of APPs. Other sample types besides serum or plasma have potential uses; APP determination in milk is a powerful tool in the control of mastitis, saliva is a non-invasive sample type, and meat juice is easily obtained at the slaughterhouse. Increasing our knowledge of reference intervals and the influence of variables such as age, breed, sex, and the season is important. Finally, worldwide harmonization and standardization of analytical procedures will help to expand the use of APPs.     

Temporal changes in serum concentrations of acute phase proteins in newborn dairy calves

Age-dependent changes in blood concentrations of four bovine acute phase proteins (APPs), serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), haptoglobin (Hp) and alpha(1)-acid glycoprotein (AGP), were examined using two groups of newborn dairy calves. APP concentrations were monitored for either 3 weeks (Group A, n=13) or 2 months (Group B, n=13) after birth. Blood was collected at day 0-1 (Group A only), day 3 and then once or twice weekly until the end of the study. The possible transfer of colostral SAA as a source of SAA in the offspring was investigated by determining SAA isoforms in the serum of calves and in colostrum of their dams. Serum concentrations of all four APPs were high after birth, and concentrations showed a gradual decrease during the first 3 weeks of life. The lowest concentrations were at 21 days of age, after which concentrations stabilized. The calves synthesized adult SAA isotypes, and circulating SAA was not derived from colostrum. The results indicated that post-partum elevation of APPs is associated with the birth process and/or factors in colostrum and not necessarily with disease-related processes. This stresses the importance of considering a calf's age when interpreting APP concentrations in serum.     

Kinetics of the response of four positive acute phase proteins in pigs experimentally infected with toxigenic Pasteurella multocida

In the present study, acute phase proteins (APP) responses in pigs after infection with toxigenic strain of Pasteurella multocida (Pm) were evaluated. Twelve piglets from a herd free from toxigenic Pm were used. Six of them were infected intranasally with Pm. CRP, Hp, SAA and Pig-MAP concentrations were measured using commercial ELISA tests. CRP and Hp were significantly induced from 2 days post inoculation (dpi). The concentration of Hp in inoculated pigs remained elevated until the end of the study. The concentrations of SAA and Pig-MAP increased significantly from 3 dpi, and remained elevated to 5 or 7 dpi, respectively. Strong correlations were observed between concentration of Hp or SAA and changes in the lungs. No correlations were found between levels of APP in serum and changes observed in the turbinates. On the basis of our investigation, we cannot state that evaluation of APP concentrations in serum may provide useful information about severity of atrophic rhinitis. However, the concentration of all investigated APP increased after inoculation. Thus, APP-measurements may reveal ongoing infection. Monitoring of APP concentrations in the pig herds may help to pinpoint infected animals even before clinical signs are present. If found, these "APP-high" animals may be chosen next for pathogen-specific diagnostics. Early diagnosis and therapy may prevent the infection from spreading in the herd. Moreover, monitoring of APP concentrations in serum may be useful for selecting clinically healthy pigs before integration into an uninfected herd. Future studies should focus on the possibility of distinguishing infected and non-infected pigs under field conditions.     

Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection

The possible role of some acute phase proteins (APPs) and immunoglobulins in both the pathogenesis and diagnosis of feline infectious peritonitis (FIP) has been investigated. Serum protein electrophoresis and the concentration of haptoglobin (Hp), serum amyloid A (SAA), alpha(1)-acid glycoprotein (AGP), IgG and IgM were evaluated in cats exposed to feline coronavirus (FCoV) and in cats with FIP. The highest concentration of APPs was detected in affected cats, confirming the role of these proteins in supporting a clinical diagnosis of FIP. Repeated samplings from both FIP affected and FCoV-exposed cats showed that when FIP appeared in the group, all the cats had increased APP levels. This increase persisted only in cats that developed FIP (in spite of a decrease in alpha(2)-globulins) but it was only transient in FCoV-exposed cats, in which a long lasting increase in alpha(2)-globulins was observed. These results suggest that changes in the electrophoretic motility of APPs or APPs other than Hp, SAA and AGP might be involved in the pathogenesis of FIP or in protecting cats from the disease.     

Acute phase response in porcine reproductive and respiratory syndrome virus infection

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.     

The role of acute phase proteins in diagnosis and management of steroid-responsive meningitis arteritis in dogs

Acute phase proteins (APPs) have become an important tool in the diagnosis, management and prognosis of inflammatory diseases in humans and are developing a similar utility in domestic species. Steroid-responsive meningitis arteritis (SRMA) is a well-recognised inflammatory disease of the dog, the diagnosis of which remains unsatisfactory based on clinical criteria and non-specific laboratory investigations. In this prospective pilot study the authors examined the acute phase response throughout the course of SRMA in serum and cerebrospinal fluid (CSF) by evaluating three key stages in disease management: presentation, treatment response and putative relapse.Serum APPs were found to be of value in supporting the diagnosis of SRMA and monitoring its treatment. C-reactive protein (CRP), serum amyloid-A (SAA), alpha-1-acid glycoprotein (AGP) and haptoglobin (Hp) all exhibited an increase above our laboratory reference range in nine patients at initial presentation. During treatment APPs decreased significantly compared to presentation except Hp which increased (Wilcoxon–Signed–Rank-test: CRP, SAA and AGP P < 0.05). Serum CRP and SAA were also found to be of clinical value in the identification of putative relapse (seven cases), particularly in the light of unperturbed CSF parameters where APP concentrations were elevated. CSF APPs were found to be less reliable markers in the management of this disease. The results indicate that SRMA causes a significant APP response in dogs, which although not disease specific, is of value in supporting the diagnosis of SRMA.     

Acute phase protein response during subclinical infection of pigs with H1N1 swine influenza virus

In the present study acute phase proteins (APPs) responses in pigs after subclinical infection with H1N1 swine influenza virus (SwH1N1) were evaluated. Fourteen 5 weeks old, seronegative piglets, both sexes were used. Ten of them were infected intranasally with SwH1N1. C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA) and pig major acute phase protein (Pig-MAP) concentrations in serum were measured using commercial ELISAs. No significant clinical signs were observed in any of the infected pigs, however, all infected animals developed specific antibodies against SwH1N1 and viral shedding was observed from 2 to 5dpi. Only concentrations of Hp and SAA were significantly induced after infection, with mean maximum levels from days 1 to 2 post infection (dpi). The concentrations of CRP and Pig-MAP remained generally unchanged, however in half of infected pigs the concentration of CRP tended to increase at 1dpi (but without statistical significance). The results of our study confirmed that monitoring of APPs may be useful for detection of subclinically infected pigs. The use of SAA or Hp and Pig-MAP may be a valuable in combination [i.e. Hp (increased concentration) and Pig-MAP (unchanged concentration)] to detect subclinically SIV infected pigs, or to identify pigs actually producing a large amount of virus. Additional studies need to be done in order to confirm these findings.     

Serum acute phase proteins as biomarkers of pleuritis and cranio-ventral pulmonary consolidation in slaughter-aged pigs

The purpose of this study was to investigate the relationship between the existence of lung lesions in pigs at slaughter and the concentration of the serum acute phase proteins (APP), haptoglobin (Hp), pig-major acute protein (Pig-MAP) and C-reactive protein (CRP). A total of 24 pig farms were selected out of a larger farm database previously screened to study risk factors associated with pleuritis and cranio-ventral pulmonary consolidation (CVPC) lesions at slaughter-aged pigs in Spain. The farms were classified as “pleuritis negative (P−) or positive (P+)” and as “CVPC negative (M−) or positive (M+)” and divided into four groups according to a 2X2 factorial design (P−M−, P−M+, P+M−, P+M+). Also at slaughter, blood from 20 randomly selected pigs from each farm was collected. Obtained serum samples were used to measure acute phase proteins. All APP concentrations were significantly higher for M+ farms than for M− ones. However, only Hp and Pig-MAP showed significantly higher concentrations for P+ farms than for P− ones. Pig-MAP was the most sensitive biomarker since it was able to clearly discriminate between P−/P+ and M−/M+ groups (p < 0.001 in both cases). Hp was an excellent marker for pleuritis and good for CVPC lesions. CRP was able to discriminate for CVPC lesions but not for pleuritis. The present results indicate that Pig-MAP and, possibly Hp, may be used as potential markers to characterise and discriminate respiratory lesions in swine herds at slaughter.     

Acute phase proteins in cattle after exposure to complex stress

Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin concentrations were obtained before (0-sample) and at 8, 24 and 48 hours after the start of transportation. Upon arrival the animals gave the impression of being anxious, and they appeared to have difficulty coping with isolation and with being tied on the slippery floors of the research stable. Serum concentrations of SAA and haptoglobin increased significantly in response to the stressors (P < 0.01 and 0.05 at 48 hours, respectively). Additionally, the animals had transient neutrophilia at 8 and 24 hours (P < 0.05). In conclusion, the results of the study suggest that SAA and haptoglobin may serve as markers of stress in adult cattle.     

Temporal changes in concentrations of serum amyloid-A and haptoglobin and their associations with weight gain in neonatal reindeer calves

Age-related changes in serum concentrations of two acute phase proteins (APPs), haptoglobin (Hp) and serum amyloid-A (SAA) were investigated in newborn reindeer calves. Repeated blood samples were obtained from 51 reindeer calves at ages 0-32 days (2-4 samples from each calf). An increase of SAA concentrations was observed during the first 2 weeks of life. However, by the end of the observation period, SAA concentrations had decreased to levels below those of the first week. Serum Hp concentrations increased throughout the observation period. SAA concentrations in the second week had a negative association with weight gain during the entire study period (4 months). These time-related changes in APP concentrations suggest that these proteins have a role in the defence and adaptation mechanisms of newborn reindeer calves. Possible reasons for these changes include the presence of APP mediators in the colostrum, exposure to environmental pathogens after birth and age-related changes in hepatic synthesis of APP.     

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.     

Bovine alpha-1 acid glycoprotein can reduce the chemotaxis of bovine monocytes and modulate CD18 expression

The acute phase protein alpha(1)-acid glycoprotein (AGP--Orosomucoid) is a lipocalin with immunomodulatory functions. The present study provides evidence that the plasma glycoforms of AGP inhibit the migration of bovine monocytes in response to classical chemoattractants. The inhibition is specific, since neutrophils are apparently not affected. To investigate the molecular basis of this finding, the expression of the molecules mostly involved in chemotaxis, including CD18, CD11b and CD47 was studied. It was found that the incubation of activated monocytes with acute phase concentration of AGP (0.9 mg/mL) induces a down-regulation of CD18, and has no apparent influence on CD11b and CD47. RT-PCR expression studies on CD18, CD11b and CD47 mRNA revealed that AGP treatment does not modify the expression rate of these genes. Since AGP treatment is related to a down-regulation of CD18 on the surface of the monocytes, the authors suggest that one of its possible functions consists in specifically reducing the firm adhesion phase of bovine monocytes to the endothelium.     

Acute phase proteins in response to Dictyocaulus viviparus infection in calves

Three experiments were carried out to examine the acute phase response, as measured by the acute phase proteins (APP) haptoglobin, serum amyloid A (SAA) and fibrinogen, in calves infected with lungworm, Dictyocaulus vivparus. In addition, eosinophil counts were analysed. Three different dose models were used in 3 separate experiments: 1) 250 D. viviparus infective third stage larvae (L3) once daily for 2 consecutive days, II) 100 D. viviparus L3 once daily for 5 consecutive days, and III) 2000 L3 once. All 3 dose regimes induced elevated levels of haptoglobin, SAA and fibrinogen, although there was considerable variation both between and within experiments. A significant increase was observed in all 3 APP at one or several time points in experiment I and III, whereas in experiment II, the only significant elevation was observed for fibrinogen at one occasion. The eosinophil numbers were significantly elevated in all 3 experiments. The results show that lungworm infection can induce an acute phase response, which can be monitored by the selected APP. Elevated APP levels in combination with high numbers of eosinophils in an animal with respiratory disease may be used as an indicator of lung worm infection, and help the clinician to decide on treatment. However, high numbers of eosinophils and low levels of APP do not exclude a diagnosis of lungworm. Thus, lungworm infection may not be detected if measurements of APP are used to assess calf health in herds or individual animals.     

The acute phase response in calves experimentally infected with bovine viral diarrhoea virus and/or Mannheimia haemolytica

The aim of this investigation was to study differences and similarities in the acute phase response of calves experimentally infected in the respiratory tract with either bovine viral diarrhoea virus (BVDV) or Mannheima haemolytica (Mh), or with a combination of both (BVDV/Mh). A non-inoculated control group was also included. The acute phase response was measured by serum or plasma concentrations of the acute phase proteins (APPs) haptoglobin, serum amyloid A (SAA) and fibrinogen, and of cortisol, prostaglandin F2alpha-metabolite and interferon-alpha (IFN-alpha) activity. Clinical symptoms were also recorded and were most severe in the BVDV/Mh group. The symptoms were mild to moderate in the BVDV group, while none, or very mild symptoms were observed in the Mh group. In all inoculated groups, a significant acute phase response was observed, with elevated values of haptoglobin, SAA and fibrinogen, while the control group remained unaffected throughout the study. In general, the magnitude of the response was similar, but the duration of elevated concentrations of APPs was significantly longer in the BVDV/Mh group than in the BVDV group, reflecting the duration of the clinical symptoms. However, in the single infection groups, the APP response and the clinical symptoms were not correlated. The IFN-alpha activity increased in all BVDV-inoculated animals, but no response in cortisol and PGF2alpha-metabolite concentrations was observed after infection. Basal levels of serum concentrations of haptoglobin, SAA and fibrinogen were established and may be used for evaluating calf health in herds. The duration of elevated haptoglobin, SAA and fibrinogen values did not differ significantly within groups indicating that their value as indicator of disease is equal.     

Haptoglobin and serum amyloid A in milk from dairy cows with chronic sub-clinical mastitis

New tools are needed to detect chronic sub-clinical mastitis, especially in automatic milking systems. Haptoglobin and serum amyloid A (SAA) are the two most sensitive bovine acute phase proteins, and their concentrations increase in milk from cows with clinical mastitis and in milk from cows with experimentally induced chronic sub-clinical Staphylococcus aureus mastitis. The aim of this study was to further evaluate the potential for haptoglobin and SAA in milk as indicators of chronic sub-clinical mastitis. Quarter milk samples were collected from 41 cows with a mean composite milk somatic cell count (CSCC) above 300,000 cells/mL during at least two months prior to sampling. Quarter milk samples were also taken from eleven cows with a mean CSCC below 80,000 cells/mL during at least two previous months. These samples were analysed for haptoglobin, SAA, adenosine triphosphate (ATP) activity and bacterial growth. The samples were grouped according to their ATP, haptoglobin and SAA status. ATP+ samples had ATP > 2 x 10(-10) mol/mL, Hp+ and SAA+ samples had detectable levels of haptoglobin (> or = 0.3 mg/L) and SAA (> or = 0.9 mg/L), respectively. In udder quarter samples from healthy cows, 42 out of 44 samples belonged to the ATP-Hp-SAA- group. Among cows with chronic sub-clinical mastitis, the ATP+Hp+SAA+ group contained 66 out of 164 samples while 44 samples belonged to the ATP+Hp-SAA- group. Detectable levels of haptoglobin and SAA were found in 92 and 80 samples, respectively. Growth of udder pathogens was detected in 28 samples and Staphylococcus aureus was the most common bacteria. In conclusion, haptoglobin and SAA concentrations below the detection limit were considered as good indicators of healthy udder quarters. A substantial variation in haptoglobin and SAA concentrations in milk was observed in udder quarters with chronic sub-clinical mastitis.     

Differential effects of alpha1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production

During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). alpha1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.