Expression of α1-acid glycoprotein and lipopolysaccharide binding protein in visceral and subcutaneous adipose tissue of dairy cattle

Adipose tissue is an endocrine compartment that plays an important role in immune defence by producing and releasing a wide range of proteins, including acute phase proteins (APPs). The liver is the main organ of APP synthesis, although extrahepatic production has also been reported. In the present study, expression of two APPs in dairy cattle, lipopolysaccharide binding protein (LBP) and α1-acid glycoprotein (AGP), was determined in four visceral (pericardial, mesenteric, omental and retroperitoneal) and three subcutaneous (withers, tail head and sternum) adipose tissue depots. mRNA expression was evaluated using qualitative and quantitative PCR, protein profiles were assessed by Western blot analysis and cellular localisation was determined by immunohistochemistry. The presence of LBP and AGP was demonstrated at mRNA and protein levels in all seven adipose tissue depots. Expression of AGP and LBP suggests that they may have roles as local and systemic inflammatory adipokines.     

Acute phase protein changes in calves during an outbreak of respiratory disease caused by bovine respiratory syncytial virus

Bovine acute phase proteins (APPs), lipopolysaccharide binding protein (LBP), serum amyloid A (SAA), haptoglobin (Hp) and alpha₁-acid glycoprotein (AGP) were evaluated as inflammatory markers during an outbreak of bovine respiratory disease (BRD) caused by bovine respiratory syncytial virus (BRSV). Calves (n = 10) presented mild to moderate signs of respiratory disease. Secondary bacterial infections, Pasteurella multocida and Mycoplasma dispar as major species, were detected in tracheobronchial lavage samples. Concentrations of SAA and LBP increased at week 1 had the highest values at week 3 and decreased at week 4 of outbreak. Some calves had high Hp concentrations at week 3, but AGP concentrations did not rise during respiratory disease. Higher SAA, LBP and Hp concentrations at a later stage of BRD (week 3) were associated with the low BRSV-specific IgG₁ production, suggesting that these calves had enhanced inflammatory response to the secondary bacterial infection. In conclusion, APPs (especially SAA and LBP) are sensitive markers of respiratory infection, and they may be useful to explore host response to the respiratory infections in clinical research.     

Widespread expression of SAA and Hp RNA in bovine tissues after evaluation of suitable reference genes

The serum amyloid A (SAA) and haptoglobin (Hp) are the most prominent acute phase proteins (APPs) in cow. Liver mainly produces APPs, but extra hepatic expression has also been demonstrated in some tissues. The major aim of the present study was to assess the constitutive SAA and Hp mRNA expression by quantitative PCR (qPCR) in a wide panel of 33 bovine tissues, including gastrointestinal tract, respiratory system, urogenital system, mammary gland, hematopoietic system, central nervous system, eye, thyroid and heart. Normalization of gene expression in different samples requires reference genes, which are stably expressed. Therefore, seven reference genes were investigated (ACTB, GAPDH, HMBS, SDHA, YWHAZ, SF3A1, EEF1A2) and three genes, namely SF3A1, HMBS and ACTB, were selected after assessing their stability with geNorm? and NormFinder^© softwares.The qPCR analysis confirmed liver as the principal source of SAA and Hp, but also identified both APPs’ mRNA in almost all tissues.The highest expression rate of SAA was found in thyroid, followed by pancreas and submandibulary gland. Hp mRNA expression was detected at high concentration in pancreas and submandibulary gland.The present data indicated a widespread expression of SAA and Hp also in non pathological conditions, thus envisaging a possible role as immunomodulatory and protective molecules. To understand where SAA and Hp come from is the prerequisite to their utilization as Acute Phase Reaction biomarkers.     

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.     

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.     

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.     

Identification of the bovine alpha1-acid glycoprotein in colostrum and milk

Alpha1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. This paper presents the detection of bovine AGP (boAGP) in mammary secretions (colostrum and milk) and mammary gland tissue. Bovine AGP was detected by Western blotting in all the samples analysed, and could be quantified in colostrum at 162 (+/- 63.7) microg/mL and 114.5 (+/- 67.8) microg/mL during the first 12 h and 24 h respectively. In mature milk, the boAGP concentration clearly decreased and was no longer detectable using the Radial Immunodiffusion (RID) technique. The concentration of mature milk boAGP was therefore semi-quantified using an anion-exchange chromatographic procedure that allowed the concentration of the protein to be determined. The presence of AGP in bovine milk was confirmed by the internal sequence analysis performed following purification to homogeneity of the protein from milk. The concentration of AGP in bovine milk with low SCC (< 250,000) was very similar to that from bovine milk with high SCC (> 250,000). In order to investigate the origin of AGP in bovine milk, a search for mRNA was carried out in somatic cells and mammary gland tissue: mRNA expression of the boAGP gene was detected in mammary gland tissue, but not in somatic cells. Finally, the cDNA sequence of the boAGP was determined, and is hereby presented.     

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.     

Antimicrobial activity of red-tailed phascogale (Phascogale calura) serum

Antimicrobial substances in serum include circulating complement proteins and acute phase proteins (APPs). We identified gene sequences for APPs, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) in marsupial genomes. Hp and SAA levels were measured in red-tailed phascogale (Phascogale calura) sera using commercially available assays. Hp levels were higher in males than females, while SAA levels suggest the phascogales used in this study were healthy. Serum was co-cultured with four bacterial species. Bacterial growth was inhibited after incubation at 37 °C, however effectiveness differed with bacteria and incubation time. The least amount of bacterial growth was noticed after introduction to K. pneumoniae, and most when introduced to P. aeruginosa. Despite marsupials not having mature immune tissues at birth, and unable to mount specific immune responses, this study suggests other immune strategies, such as APPs in serum likely aid marsupials in their defence against pathogens.     

Preliminary studies of serum acute-phase protein concentrations in hematologic and neoplastic diseases of the dog

Serum concentrations of acute-phase proteins (APPs): haptoglobin (Hp), ceruloplasmin (Cp), serum amyloid A (SAA), and C-reactive protein (CRP) were determined in healthy dogs (n = 15) and dogs with different diseases grouped as acute inflammation (I, n = 12), hematologic neoplasias (HT, including leukemia and lymphoma, n = 16), nonhematologic neoplasias (NHT, including epithelial, mesenchymal, and mixed, n = 20), and autoimmune hemolytic anemia (AIHA, n = 8). SAA and CRP were analyzed using commercially available enzyme-linked immunosorbent assay (ELISA) kits, and Hp and Cp were measured using colorimetric methods, all previously validated for use in dogs. Increased concentrations of all APPs were observed in all groups of diseased dogs, but statistical significance only was observed with Hp (I, P < .001; HT, P < .05), Cp (I, P < .05; AIHA, P < .01), and CRP (I, P < .001; HT, P < .001; AIHA, CRP P < .05). High variability in individual APPs within each group of diseases was found with no significant differences between leukemia and lymphoma as well as among different types of neoplasia. The AIHA group had smaller increases in Hp, SAA, and CRP but higher concentrations of Cp. When follow-up of individual cases was possible, a decrease in APPs generally was found in cases with favorable outcome. The results of this study suggest that neoplasia and hematologic diseases such as AIHA should be considered as possible causes of mild increases in APPs in dogs. Measurement of APPs may be helpful to assess clinical evolution and monitor treatment of these processes.     

Porcine acute phase protein concentrations in different diseases in field conditions

Five acute phase proteins (APPs) [C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), pig-MAP and albumin] were measured in pigs with naturally occurring infections by porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae, and in animals with tail and ear bites, arthritis and other acute inflammatory processes. Healthy specific pathogen-free (SPF) pigs were used as controls. In PRRSV-infected pigs, all APPs with the exception of pig-MAP exhibited significant changes compared with controls. In animals affected with ADV only Hp presented changes of statistical significance, whereas pigs with PCV2 showed marked modifications in all APPs tested. Animals affected with Mycoplasmosis showed concentrations of all positive APPs significantly above levels obtained in SPF pigs, though albumin concentrations did not differ from controls. Finally, all APPs studied showed substantial changes in pigs with acute inflammation. The results indicated that an acute phase response was developed in the different diseases studied, this response being higher in animals with clinical signs and concurrent bacterial processes. Haptoglobin would be the APP that better reflects pathological states; however, to get more complete and valuable information it might be advisable to perform APPs profiles including another APP, such as CRP or SAA.     

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.     

An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E₂ (PGE₂) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE₂ at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.     

Evaluation of some acute phase proteins in cattle naturally infected with Babesia bigemina

Bovine babesiosis due to Babesia bigemina infection induces systemic inflammation, evidenced by increased sialic acid (SA) levels and declined cholinesterase activity. The current study was undertaken to assess further indicators of the systemic inflammation in the naturally infected cattle. To this end, serum levels of some selected acute phase-proteins (APPs) including serum amyloid A (SAA), haptoglobin (Hp), ceruloplasmin (Cp), and fibrinogen were measured. Additionally, sensitivity and specificity of the APPs were calculated by receiver operating characteristic curve. The correlation among APPs, SA and cholinesterase activity were also assessed. Our previous blood specimens were used to measure APPs. Briefly, the diseased animals were divided into two groups according to the parasitemia: 12 mildly (20 % <) and 8 severely (20 %>) infected animals. Moreover, 10 healthy animals as the control were included. The levels of all APPs were measured to be significantly elevated in a parasitemia burden-dependent fashion as compared to the control. Furthermore, all the APPs showed 100 % specificity, but only SAA and Cp had 100 % sensitivity. A strong and positive correlation was calculated between the APPs and SA; however, cholinesterase activity was inversely correlated with AAPs and SA. In conclusion, inflammatory reactions play a pivotal role in the pathogenesis of bovine babesiosis and APPs can be considered as the potential indicators of inflammation.     

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.     

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.