The Concentration of Apolipoprotein A-I Decreases during Experimentally Induced Acute-Phase Processes in Pigs
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感染与免疫杂志 2005年第5期
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain
Danish Institute for Food and Veterinary Research, Copenhagen V, Denmark
French Agency for Food Safety, BP 53, Zopoles les Croix, 22440 Ploufragan, France
ABSTRACT
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.
TEXT
The acute-phase response is the answer of the organism to disturbances of its homeostasis due to infection, tissue injury, neoplastic growth, or immunological disorders, resulting in a whole array of systemic reactions, which induce changes in the concentrations of certain plasma proteins. These proteins are called acute-phase proteins (APP), and their concentrations can increase (positive APP) or decrease (negative APP) within hours or days during the process (4, 15, 18). In pigs, haptoglobin, C-reactive protein (CRP), serum amyloid A, and pig major acute-phase protein (pig MAP) are well-known positive APP. Their validity as markers of infection or inflammation has been confirmed (1, 9-12, 14, 17, 19). Nevertheless, little is known about the negative APP response in pigs, except for a small decrease in albumin and transferrin concentrations (19).
In previous studies investigating the pig APP response in a model of inflammation induced by turpentine injection, a protein with electrophoretic mobility alpha, preliminarily characterized as -lipoprotein, was detected as a major negative APP in pigs (19). Here, we purified the apolipoprotein A-I (ApoA-I), the major protein component of -lipoprotein or high-density lipoprotein (HDL) from normal pig sera. The change in its concentrations during the acute-phase response was evaluated in different experimental models.
ApoA-I purification and antiserum preparation. Pig serum lipoproteins were obtained by sequential ultracentrifugation (13) of blood serum. Very-low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) (density [d] < 1.035 g/ml), low-density lipoproteins (LDL) (1.035 g/ml < d < 1.080 g/ml), lighter HDL (1.080 g/ml < d < 1.160 g/ml), and the heavier HDL fraction (1.160 g/ml < d < 1.250 g/ml) were isolated in five sequential centrifugation steps performed with a Beckman 50.3 rotor at 10°C at 190,000 x g for 18 h for the first two steps (VLDL and IDL and LDL) and at 146,000 x g for 36 h for the others. The densities used were deduced from the work of Hollanders et al. (16) and adjusted by the addition of potassium bromide. Figure 1A shows the protein patterns from sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the different fractions obtained after the sequential ultracentrifugation of pig serum. The lighter HDL showed a major band of 28 kDa besides some minor faint bands of 51 and 64 kDa (Fig. 1A, lane 3). The band of 28 kDa was identified as ApoA-I by amino acid sequencing (Fig. 1B), carried out by the Institute of Animal Physiology and Genetics Research (Babraham, Cambridge, United Kingdom). ApoA-I is the major apolipoprotein of the HDL in all species studied, although its percentages in HDL can differ between species (16, 26). The band of 64 kDa was identified as albumin and eliminated by size exclusion chromatography (Sephadex G-150) (data not shown), obtaining an HDL for which ApoA-I represents 94% of the total protein as calculated by densitometry analysis of SDS-separated proteins. Specific antisera against ApoA-I were produced in rabbits by subcutaneous injection of this fraction, as previously described (20). The antisera obtained were solid-phase immunoadsorbed with a fraction of pig sera free of ApoA-I (3). The specificities of the antisera finally obtained are shown in Fig. 1C. These antisera were used to determine ApoA-I concentrations in pig serum by radial immunodiffusion (23). A serum previously calibrated with the isolated lighter HDL fraction after size exclusion chromatography was used as a standard. No differences in protein concentration were observed when denaturing agents were used (data not shown) (7, 28). Amounts of pig MAP, haptoglobin, and CRP were also determined by radial immunodiffusion (19).
Turpentine-induced inflammation. In two independent experiments, six 20-week-old pigs (Large White x Landrace x Pietrain) were subcutaneously injected with 0.3 ml of turpentine oil/kg of body weight, as previously described (19). The time courses of the concentrations of the APP studied are shown in Fig. 2. One of the pigs was underresponsive to the treatment and has been excluded from the data shown below. The mean concentration of ApoA-I before the injection was 2.65 mg/ml (range, 2.06 to 3.18 mg/ml). After 12 h, the concentration of the protein began to decrease, showing minimum values, 50% lower than initial values, at 24 to 36 h postinjection (range, 1.15 to 1.52 mg/ml), and the values increased after that. The mean prechallenge concentration of pig MAP was 0.63 mg/ml, increasing by more than five times at days 2 and 3 postinjection and reaching values around 3 to 3.5 mg/ml. Haptoglobin showed a similar pattern, increasing by around five times. In the case of CRP, maximum values were obtained 1 to 2 days after the turpentine injection, showing a more transient response.
Experimental infection with Actinobacillus pleuropneumoniae. Three cross-bred, 13-week-old male pigs from a specific-pathogen-free herd were exposed to A. pleuropneumoniae serotype 5b, biotype 1 (14). Significant decreases in the concentrations of ApoA-I in the three pigs were observed (Fig. 3A, panel A-1). The mean concentration of ApoA-I before the infection was 2.3 mg/ml (range, 1.83 to 3.05 mg/ml), decreasing by about three times on days 2 to 3 after A. pleuropneumoniae infection (range, 0.54 to 0.98 mg/ml). The concentration of ApoA-I increased after that, to return to initial values at days 12 through 15 postinfection (p.i.). The values for pig MAP are shown in Fig. 3A (panel A-2) (14) as a control for the acute-phase response. All pigs were treated with Streptocillin vet (250 mg/ml dihydrostreptomycin and 200,000 IU/ml benzylpenicillinprocaine) at 23 h and at 28 h p.i. in order to decrease acute mortality after A. pleuropneumoniae infection (14). As shown by Lauritzen et al. (21), this can clearly influence the longer-term response of acute-phase proteins, following the clinical recovery of the animals that was caused by the treatment.
Experimental infection with Streptococcus suis. Eight 15-week-old pigs from a specific-pathogen-free herd were experimentally infected by intravenous injection of S. suis serotype 2 (strain 93) (17). The average ApoA-I concentration before challenge was 3.08 mg/ml (range, 2.38 to 3.9 mg/ml). A decrease of more than 50% was already observed in the first day p.i. (Fig. 3B, panel B-1), but minimum values (three to five times lower than the initial values; range, 0.5 to 1.23 mg/ml) were reached at days 2 and 3 p.i. and maintained until the end of the trial (with the exception of one pig). The mean prechallenge concentration of pig MAP (Fig. 3B, panel B-2) was 0.43 mg/ml (range, 0.36 to 0.69 mg/ml). Maximum levels, ranging from 12 to 40 times the initial values, were obtained at days 4 through 6 p.i. In the pig with a minor ApoA-I response, the increase of the pig MAP concentration was by only four times. This animal also showed minimal clinical symptoms (17).
All the experiments performed with animals were approved by the Animal Experimentation Committees or Ethical Committees in each institute and were compliant with all relevant European Union guidelines on animal experimentation.
In this work, we demonstrate that ApoA-I is a major negative APP in pigs. The sequential ultracentrifugation method described here, with two different steps to discriminate between lighter HDL and heavier HDL (2, 6, 30), allowed us to obtain a lipoprotein fraction for which ApoA-I represented more than 90% of the total protein. Decreases in the concentration of the protein of around 50% were observed after experimental inflammation. The levels of response of the other APP studied were, in general, lower than those in a previous study (19), probably because of the lower doses of turpentine used. The negative-APP nature of ApoA-I was confirmed in two experimental bacterial infections (A. pleuropneumoniae and S. suis) in which higher responses were observed. ApoA-I showed minimum concentrations ranging from three to five times lower than initial concentrations, an effect more pronounced with the S. suis infection. It could be speculated that the antibiotic treatment of the A. pleuropneumoniae-infected animals reduced the responses of both ApoA-I and pig MAP after the last antibiotic treatment (28 h p.i.), while, in contrast, the ApoA-I and pig MAP concentrations in S. suis-infected pigs remained at the maximum response levels or changed further after 24 h. Such an effect was noted previously by Lauritzen et al. (21) for other acute-phase proteins after A. pleuropneumoniae infection and antibiotic treatment. Decreases in ApoA-I concentrations during acute-phase processes in other species have been described (5, 8, 22, 24, 25, 27), suggesting that changes in the ApoA-I concentration could be involved in the modulation of some of the reactions that occur during inflammation (5, 30).
Recent works have revealed the potential of the APP assay in the assessment of management quality, health, and welfare of animals in the pig production chain (9, 29). The use of a negative APP in combination with positive APP in an acute-phase index could have valuable applications when monitoring stages of disease (9, 29). Furthermore, a complete analysis of APP response would be useful when microbial or other inflammatory stimuli target different cytokine networks. As seen for positive APP, the concentration of ApoA-I showed some variation in normal sera (ranging from 1.83 to 3.90 mg/ml); however, the minimum was stable during acute-phase processes induced with different stimuli, being lower than 1 mg/ml. More studies will be carried out to gain more knowledge about the function of ApoA-I as the APP, as well as its performance in farm animals at different ages and in different breeds, and to evaluate its usefulness as a marker of the acute-phase response in pig production systems.
ACKNOWLEDGMENTS
This work was supported by European commission directorate general research shared cost number QLK5-2001-02219.
R. Carpintero holds a fellowship from Fundacion Cuenca Villoro.
REFERENCES
1. Alava, M. A., N. Gonzalez-Ramon, P. Heegaard, S. Guzylack, M. J. M. Toussaint, C. Lipperheide, F. Madec, E. Gruys, P. D. Eckersall, F. Lampreave, and A. Pieiro. 1997. Pig-MAP, porcine acute phase proteins and standardisation of assays in Europe. Comp. Haematol. Int. 7:208-213.
2. Angelo, M., C. E. Scanu, and P. Keim. 1975. Serum lipoproteins, p. 317-391. In F. W. Putnam (ed.), The plasma proteins, vol. 1. Academic Press, New York, N.Y.
3. Avrameas, S., and T. Ternynck. 1969. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6:53-66.
4. Baumann, H., and J. Gauldie. 1994. The acute phase response. Immunol. Today 15:74-80.
5. Burger, D., and J. M. Dayer. 2002. High-density lipoprotein-associated apolipoprotein A-I: the missing link between infection and chronic inflammation Autoimmun. Rev. 1:111-117.
6. Burstein, M., A. Fine, V. Atger, E. Wirbel, and A. Girard-Globa. 1989. Rapid method for the isolation of two purified subfractions of high density lipoproteins by differential dextran sulfate-magnesium chloride precipitation. Biochimie 71:741-746.
7. Bury, J., and M. Rosseneu. 1995. Quantification of human serum apolipoprotein AI by enzyme immunoassay. Clin. Chem. 31:247-251.
8. Cabana, V. G., S. S. Gidding, G. S. Getz, J. Chapman, and S. T. Shulman. 1997. Serum amyloid A and high density lipoprotein participate in the acute phase response of Kawasaki disease. Pediatr. Res. 42:651-655.
9. Eckersall, P. D. 2004. The time is right for acute phase protein assays. Vet. J. 168:3-5.
10. Eckersall, P. D., S. Duthie, M. J. Toussaint, E. Gruys, P. Heegaard, M. Alava, C. Lipperheide, and F. Madec. 1999. Standardization of diagnostic assays for animal acute phase proteins. Adv. Vet. Med. 41:643-655.
11. Gonzalez-Ramon, N., M. A. Alava, J. A. Sarsa, M. Pineiro, A. Escartin, A. Garcia-Gil, F. Lampreave, and A. Pineiro. 1995. The major acute phase serum protein in pigs is homologous to human plasma kallikrein sensitive PK-120. FEBS Lett. 371:227-230.
12. Gruys, E., M. J. Obwolo, and M. J. M. Toussaint. 1994. Diagnostic significance of the major acute phase proteins in veterinary clinical chemistry: a review. Vet. Bull. 64:1009-1018.
13. Havel, R. J., H. A. Eder, and J. H. Bragdon. 1955. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Investig. 34:1345-1353.
14. Heegaard, P. M., J. Klausen, J. P. Nielsen, N. Gonzalez-Ramon, M. Pineiro, F. Lampreave, and M. A. Alava. 1998. 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. Comp. Biochem. Physiol. B 119:365-373.
15. Heinrich, P. C., J. V. Castell, and T. Andus. 1990. Interleukin-6 and the acute phase response. Biochem. J. 265:621-636.
16. Hollanders, B., A. Mougin, F. N'Diaye, E. Hentz, X. Aude, and A. Girard. 1986. Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure. Comp. Biochem. Physiol. B 84:83-89.
17. Knura-Deszcza, S., C. Lipperheide, B. Petersen, J. L. Jobert, F. Berthelot-Herault, M. Kobish, and F. Madec. 2002. Plasma haptoglobin concentration in swine after challenge with Streptococcus suis. J. Vet. Med. B 49:240-244.
18. Kushner, I. 1988. The acute phase response: an overview. Methods Enzymol. 163:373-383.
19. Lampreave, F., N. Gonzalez-Ramon, S. Martinez-Ayensa, M. A. Hernandez, H. K. Lorenzo, A. Garcia-Gil, and A. Pineiro. 1994. Characterization of the acute phase serum protein response in pigs. Electrophoresis 15:672-676.
20. Lampreave, F., and A. Pineiro. 1982. Characterization of a new alpha-glycoprotein as the major serum component in later fetal and newborn pigs. Comp. Biochem. Physiol. B 72:215-219.
21. Lauritzen, B., J. Lykkesfeldt, M. T. Skaanild, O. Angen, J. P. Nielsen, and C. Friis. 2003. Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection. Res. Vet. Sci. 74:261-270.
22. Lindhorst, E., D. Young, W. Bagshaw, M. Hyland, and R. Kisilevsky. 1997. Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse. Biochim. Biophys. Acta 1339:143-154.
23. Mancini, G., A. O. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-254.
24. Oikawa, S., N. Katoh, F. Kawawa, and Y. Ono. 1997. Decreased serum apolipoprotein B-100 and A-I concentrations in cows with ketosis and left displacement of the abomasum. Am. J. Vet. Res. 58:121-125.
25. Oikawa, S., N. Kato, H. Itoh, T. Miyamoto, M. Konno, and T. Kajita. 1997. Decreased serum apolipoprotein A-I concentration in cows infected with Salmonella typhimurium. Can. J. Vet. Res. 61:182-186.
26. Schonfeld, G. 1979. Radioimmunoassays for human apo A-I and apoA-II as immunologic probes of the surface structure of HDL, p. 199-218. In K. Lippel (ed.), Report of the high density lipoprotein methodology workshop. NIH publication no. 79 1661. Department of Health and Human Services, National Institutes of Health, National Heart, Lung, and Blood Institute, Bethesda, Md.
27. Steel, D. M., and A. S. Whitehead. 1994. The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. Immunol. Today 15:81-88.
28. Steinberg, K. K., G. R. Cooper, S. R. Graiser, and M. Rosseneu. 1983. Some considerations of methodology and standardization of apolipoprotein A-I immunoassays. Clin. Chem. 29:415-426.
29. Toussaint, M. J. M., A. M. Van Ederen, and E. Gruys. 1995. Implication of clinical pathology in assessment of animal health and in animal production and meat inspection. Comp. Haematol. Int. 5:149-157.
30. Wang, M., and M. R. Briggs. 2004. HDL: the metabolism, function, and therapeutic importance. Chem. Rev. 104:119-137.(R. Carpintero, M. Pieiro,)
Danish Institute for Food and Veterinary Research, Copenhagen V, Denmark
French Agency for Food Safety, BP 53, Zopoles les Croix, 22440 Ploufragan, France
ABSTRACT
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.
TEXT
The acute-phase response is the answer of the organism to disturbances of its homeostasis due to infection, tissue injury, neoplastic growth, or immunological disorders, resulting in a whole array of systemic reactions, which induce changes in the concentrations of certain plasma proteins. These proteins are called acute-phase proteins (APP), and their concentrations can increase (positive APP) or decrease (negative APP) within hours or days during the process (4, 15, 18). In pigs, haptoglobin, C-reactive protein (CRP), serum amyloid A, and pig major acute-phase protein (pig MAP) are well-known positive APP. Their validity as markers of infection or inflammation has been confirmed (1, 9-12, 14, 17, 19). Nevertheless, little is known about the negative APP response in pigs, except for a small decrease in albumin and transferrin concentrations (19).
In previous studies investigating the pig APP response in a model of inflammation induced by turpentine injection, a protein with electrophoretic mobility alpha, preliminarily characterized as -lipoprotein, was detected as a major negative APP in pigs (19). Here, we purified the apolipoprotein A-I (ApoA-I), the major protein component of -lipoprotein or high-density lipoprotein (HDL) from normal pig sera. The change in its concentrations during the acute-phase response was evaluated in different experimental models.
ApoA-I purification and antiserum preparation. Pig serum lipoproteins were obtained by sequential ultracentrifugation (13) of blood serum. Very-low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) (density [d] < 1.035 g/ml), low-density lipoproteins (LDL) (1.035 g/ml < d < 1.080 g/ml), lighter HDL (1.080 g/ml < d < 1.160 g/ml), and the heavier HDL fraction (1.160 g/ml < d < 1.250 g/ml) were isolated in five sequential centrifugation steps performed with a Beckman 50.3 rotor at 10°C at 190,000 x g for 18 h for the first two steps (VLDL and IDL and LDL) and at 146,000 x g for 36 h for the others. The densities used were deduced from the work of Hollanders et al. (16) and adjusted by the addition of potassium bromide. Figure 1A shows the protein patterns from sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the different fractions obtained after the sequential ultracentrifugation of pig serum. The lighter HDL showed a major band of 28 kDa besides some minor faint bands of 51 and 64 kDa (Fig. 1A, lane 3). The band of 28 kDa was identified as ApoA-I by amino acid sequencing (Fig. 1B), carried out by the Institute of Animal Physiology and Genetics Research (Babraham, Cambridge, United Kingdom). ApoA-I is the major apolipoprotein of the HDL in all species studied, although its percentages in HDL can differ between species (16, 26). The band of 64 kDa was identified as albumin and eliminated by size exclusion chromatography (Sephadex G-150) (data not shown), obtaining an HDL for which ApoA-I represents 94% of the total protein as calculated by densitometry analysis of SDS-separated proteins. Specific antisera against ApoA-I were produced in rabbits by subcutaneous injection of this fraction, as previously described (20). The antisera obtained were solid-phase immunoadsorbed with a fraction of pig sera free of ApoA-I (3). The specificities of the antisera finally obtained are shown in Fig. 1C. These antisera were used to determine ApoA-I concentrations in pig serum by radial immunodiffusion (23). A serum previously calibrated with the isolated lighter HDL fraction after size exclusion chromatography was used as a standard. No differences in protein concentration were observed when denaturing agents were used (data not shown) (7, 28). Amounts of pig MAP, haptoglobin, and CRP were also determined by radial immunodiffusion (19).
Turpentine-induced inflammation. In two independent experiments, six 20-week-old pigs (Large White x Landrace x Pietrain) were subcutaneously injected with 0.3 ml of turpentine oil/kg of body weight, as previously described (19). The time courses of the concentrations of the APP studied are shown in Fig. 2. One of the pigs was underresponsive to the treatment and has been excluded from the data shown below. The mean concentration of ApoA-I before the injection was 2.65 mg/ml (range, 2.06 to 3.18 mg/ml). After 12 h, the concentration of the protein began to decrease, showing minimum values, 50% lower than initial values, at 24 to 36 h postinjection (range, 1.15 to 1.52 mg/ml), and the values increased after that. The mean prechallenge concentration of pig MAP was 0.63 mg/ml, increasing by more than five times at days 2 and 3 postinjection and reaching values around 3 to 3.5 mg/ml. Haptoglobin showed a similar pattern, increasing by around five times. In the case of CRP, maximum values were obtained 1 to 2 days after the turpentine injection, showing a more transient response.
Experimental infection with Actinobacillus pleuropneumoniae. Three cross-bred, 13-week-old male pigs from a specific-pathogen-free herd were exposed to A. pleuropneumoniae serotype 5b, biotype 1 (14). Significant decreases in the concentrations of ApoA-I in the three pigs were observed (Fig. 3A, panel A-1). The mean concentration of ApoA-I before the infection was 2.3 mg/ml (range, 1.83 to 3.05 mg/ml), decreasing by about three times on days 2 to 3 after A. pleuropneumoniae infection (range, 0.54 to 0.98 mg/ml). The concentration of ApoA-I increased after that, to return to initial values at days 12 through 15 postinfection (p.i.). The values for pig MAP are shown in Fig. 3A (panel A-2) (14) as a control for the acute-phase response. All pigs were treated with Streptocillin vet (250 mg/ml dihydrostreptomycin and 200,000 IU/ml benzylpenicillinprocaine) at 23 h and at 28 h p.i. in order to decrease acute mortality after A. pleuropneumoniae infection (14). As shown by Lauritzen et al. (21), this can clearly influence the longer-term response of acute-phase proteins, following the clinical recovery of the animals that was caused by the treatment.
Experimental infection with Streptococcus suis. Eight 15-week-old pigs from a specific-pathogen-free herd were experimentally infected by intravenous injection of S. suis serotype 2 (strain 93) (17). The average ApoA-I concentration before challenge was 3.08 mg/ml (range, 2.38 to 3.9 mg/ml). A decrease of more than 50% was already observed in the first day p.i. (Fig. 3B, panel B-1), but minimum values (three to five times lower than the initial values; range, 0.5 to 1.23 mg/ml) were reached at days 2 and 3 p.i. and maintained until the end of the trial (with the exception of one pig). The mean prechallenge concentration of pig MAP (Fig. 3B, panel B-2) was 0.43 mg/ml (range, 0.36 to 0.69 mg/ml). Maximum levels, ranging from 12 to 40 times the initial values, were obtained at days 4 through 6 p.i. In the pig with a minor ApoA-I response, the increase of the pig MAP concentration was by only four times. This animal also showed minimal clinical symptoms (17).
All the experiments performed with animals were approved by the Animal Experimentation Committees or Ethical Committees in each institute and were compliant with all relevant European Union guidelines on animal experimentation.
In this work, we demonstrate that ApoA-I is a major negative APP in pigs. The sequential ultracentrifugation method described here, with two different steps to discriminate between lighter HDL and heavier HDL (2, 6, 30), allowed us to obtain a lipoprotein fraction for which ApoA-I represented more than 90% of the total protein. Decreases in the concentration of the protein of around 50% were observed after experimental inflammation. The levels of response of the other APP studied were, in general, lower than those in a previous study (19), probably because of the lower doses of turpentine used. The negative-APP nature of ApoA-I was confirmed in two experimental bacterial infections (A. pleuropneumoniae and S. suis) in which higher responses were observed. ApoA-I showed minimum concentrations ranging from three to five times lower than initial concentrations, an effect more pronounced with the S. suis infection. It could be speculated that the antibiotic treatment of the A. pleuropneumoniae-infected animals reduced the responses of both ApoA-I and pig MAP after the last antibiotic treatment (28 h p.i.), while, in contrast, the ApoA-I and pig MAP concentrations in S. suis-infected pigs remained at the maximum response levels or changed further after 24 h. Such an effect was noted previously by Lauritzen et al. (21) for other acute-phase proteins after A. pleuropneumoniae infection and antibiotic treatment. Decreases in ApoA-I concentrations during acute-phase processes in other species have been described (5, 8, 22, 24, 25, 27), suggesting that changes in the ApoA-I concentration could be involved in the modulation of some of the reactions that occur during inflammation (5, 30).
Recent works have revealed the potential of the APP assay in the assessment of management quality, health, and welfare of animals in the pig production chain (9, 29). The use of a negative APP in combination with positive APP in an acute-phase index could have valuable applications when monitoring stages of disease (9, 29). Furthermore, a complete analysis of APP response would be useful when microbial or other inflammatory stimuli target different cytokine networks. As seen for positive APP, the concentration of ApoA-I showed some variation in normal sera (ranging from 1.83 to 3.90 mg/ml); however, the minimum was stable during acute-phase processes induced with different stimuli, being lower than 1 mg/ml. More studies will be carried out to gain more knowledge about the function of ApoA-I as the APP, as well as its performance in farm animals at different ages and in different breeds, and to evaluate its usefulness as a marker of the acute-phase response in pig production systems.
ACKNOWLEDGMENTS
This work was supported by European commission directorate general research shared cost number QLK5-2001-02219.
R. Carpintero holds a fellowship from Fundacion Cuenca Villoro.
REFERENCES
1. Alava, M. A., N. Gonzalez-Ramon, P. Heegaard, S. Guzylack, M. J. M. Toussaint, C. Lipperheide, F. Madec, E. Gruys, P. D. Eckersall, F. Lampreave, and A. Pieiro. 1997. Pig-MAP, porcine acute phase proteins and standardisation of assays in Europe. Comp. Haematol. Int. 7:208-213.
2. Angelo, M., C. E. Scanu, and P. Keim. 1975. Serum lipoproteins, p. 317-391. In F. W. Putnam (ed.), The plasma proteins, vol. 1. Academic Press, New York, N.Y.
3. Avrameas, S., and T. Ternynck. 1969. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6:53-66.
4. Baumann, H., and J. Gauldie. 1994. The acute phase response. Immunol. Today 15:74-80.
5. Burger, D., and J. M. Dayer. 2002. High-density lipoprotein-associated apolipoprotein A-I: the missing link between infection and chronic inflammation Autoimmun. Rev. 1:111-117.
6. Burstein, M., A. Fine, V. Atger, E. Wirbel, and A. Girard-Globa. 1989. Rapid method for the isolation of two purified subfractions of high density lipoproteins by differential dextran sulfate-magnesium chloride precipitation. Biochimie 71:741-746.
7. Bury, J., and M. Rosseneu. 1995. Quantification of human serum apolipoprotein AI by enzyme immunoassay. Clin. Chem. 31:247-251.
8. Cabana, V. G., S. S. Gidding, G. S. Getz, J. Chapman, and S. T. Shulman. 1997. Serum amyloid A and high density lipoprotein participate in the acute phase response of Kawasaki disease. Pediatr. Res. 42:651-655.
9. Eckersall, P. D. 2004. The time is right for acute phase protein assays. Vet. J. 168:3-5.
10. Eckersall, P. D., S. Duthie, M. J. Toussaint, E. Gruys, P. Heegaard, M. Alava, C. Lipperheide, and F. Madec. 1999. Standardization of diagnostic assays for animal acute phase proteins. Adv. Vet. Med. 41:643-655.
11. Gonzalez-Ramon, N., M. A. Alava, J. A. Sarsa, M. Pineiro, A. Escartin, A. Garcia-Gil, F. Lampreave, and A. Pineiro. 1995. The major acute phase serum protein in pigs is homologous to human plasma kallikrein sensitive PK-120. FEBS Lett. 371:227-230.
12. Gruys, E., M. J. Obwolo, and M. J. M. Toussaint. 1994. Diagnostic significance of the major acute phase proteins in veterinary clinical chemistry: a review. Vet. Bull. 64:1009-1018.
13. Havel, R. J., H. A. Eder, and J. H. Bragdon. 1955. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Investig. 34:1345-1353.
14. Heegaard, P. M., J. Klausen, J. P. Nielsen, N. Gonzalez-Ramon, M. Pineiro, F. Lampreave, and M. A. Alava. 1998. 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. Comp. Biochem. Physiol. B 119:365-373.
15. Heinrich, P. C., J. V. Castell, and T. Andus. 1990. Interleukin-6 and the acute phase response. Biochem. J. 265:621-636.
16. Hollanders, B., A. Mougin, F. N'Diaye, E. Hentz, X. Aude, and A. Girard. 1986. Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure. Comp. Biochem. Physiol. B 84:83-89.
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