• View in gallery
    Figure 1—

    Mean ± SD serum concentrations of haptoglobin (diamonds), SAA (squares), and CRP (triangles) in 4 pigs before and after inoculation with the CSF virus Alfort 187 strain. Day of inoculation was designated as day 0. 'Within an APP, value differs significantly (P < 0.05; Mann-Whitney U test), compared with the preinoculation (PI) value.

  • View in gallery
    Figure 2—

    Mean ± SD serum concentrations of haptoglobin (diamonds), SAA (squares), and CRP (triangles) in 4 pigs before and after inoculation with the ASF virus Spain 70 isolate. See Figure 1 for remainder of key.

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Serum concentrations of C-reactive protein, serum amyloid A, and haptoglobin in pigs inoculated with African swine fever or classical swine fever viruses

Pedro J. Sánchez-CordónDepartamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014, Córdoba, Spain
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda Puerta de Hierro s/n, 28040, Madrid, Spain

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José J. CerónDepartamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100, Murcia, Spain

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Alejandro NúñezPathology Department, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, KT15 3NB Surrey, England

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Silvia Martínez-SubielaDepartamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100, Murcia, Spain

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Miriam PedreraDepartamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014, Córdoba, Spain

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José L. Romero-TrevejoDepartamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014, Córdoba, Spain

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María R. GarridoDepartamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014, Córdoba, Spain

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José C. Gómez-VillamandosDepartamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba, Edificio de Sanidad Animal, Campus de Rabanales, 14014, Córdoba, Spain

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Abstract

Objective—To determine serum concentrations of the selected acute-phase proteins (APPs) haptoglobin, serum amyloid A (SAA), and C-reactive protein (CRP) in pigs experimentally inoculated with classical swine fever (CSF) and African swine fever (ASF) viruses.

Animals—8 crossbred (Large White × Landrace) 10-week-old pigs.

Procedures—Pigs were allocated to 2 groups (4 pigs/group). One group was inoculated with the CSF virus Alfort 187 strain, whereas the other groupwas inoculated with the ASF virus Spain 70 isolate. Blood samples were collected at various time points. At the end of the study, pigs were euthanized and a complete necropsy was performed, including histologic and immunohistochemical analyses.

Results—Serum concentrations of APPs increased in pigs inoculated with CSF and ASF viruses, which suggested an acute-phase response in the course of both diseases. The most noticeable increase in concentration was recorded for SAA in both groups (up to a 300-fold increase for CSF virus and an approx 40-fold increase for ASF virus), followed by CRP and then haptoglobin, which each had only 3- to 4-fold increases.

Conclusions and Clinical Relevance—Serum concentrations of APPs increased significantly in pigs inoculated with CSF and ASF viruses. However, differences were evident in serum concentrations of the proteins evaluated in this study.

Abstract

Objective—To determine serum concentrations of the selected acute-phase proteins (APPs) haptoglobin, serum amyloid A (SAA), and C-reactive protein (CRP) in pigs experimentally inoculated with classical swine fever (CSF) and African swine fever (ASF) viruses.

Animals—8 crossbred (Large White × Landrace) 10-week-old pigs.

Procedures—Pigs were allocated to 2 groups (4 pigs/group). One group was inoculated with the CSF virus Alfort 187 strain, whereas the other groupwas inoculated with the ASF virus Spain 70 isolate. Blood samples were collected at various time points. At the end of the study, pigs were euthanized and a complete necropsy was performed, including histologic and immunohistochemical analyses.

Results—Serum concentrations of APPs increased in pigs inoculated with CSF and ASF viruses, which suggested an acute-phase response in the course of both diseases. The most noticeable increase in concentration was recorded for SAA in both groups (up to a 300-fold increase for CSF virus and an approx 40-fold increase for ASF virus), followed by CRP and then haptoglobin, which each had only 3- to 4-fold increases.

Conclusions and Clinical Relevance—Serum concentrations of APPs increased significantly in pigs inoculated with CSF and ASF viruses. However, differences were evident in serum concentrations of the proteins evaluated in this study.

The acute-phase response is an early nonspecific defense mechanism in animals to local or general disturbances in homeostasis attributable to infection, inflammation, tissue injury, neoplastic growth, or immunologic disorders before specific immunity is achieved.1–3 The response includes changes in concentrations of some plasma proteins, primarily synthesized in the liver, which are referred to as APPs. During the acute-phase response, there is an increase in the serum concentration of some of these APPs (such as haptoglobin, SAA, and CRP), whereas there is a decrease in the serum concentration of other APPs (such as albumin).4,5

Monitoring the health and welfare of pigs during the production process and tracing pork products in general are concerns of consumers, producers, and regulators. Measurement of APPs is a valuable tool for evaluating health status in pigs; APPs can be used as nonspecific markers of clinical and subclinical disease as well as to distinguish between acute and chronic disease processes.2,6,7 Although APPs have been evaluated for numerous bacterial diseases in pigs,8–11 researchers have barely addressed APP concentrations in swine with viral diseases12,13 or examined in depth the relationship between serum APP concentrations and the number of cells expressing proinflammatory cytokines in the liver or elsewhere.

Cytokines, which are low–molecular-weight peptide mediators secreted primarily by leukocytes as well as by other cell types,14–16 stimulate the synthesis of APPs by hepatocytes.17,18 The major cytokines (TNF-α, IL-1, and IL-6), which have proinflammatory properties,16,19 exert their effect rapidly and jointly, inducing the activation of hepatic receptors that may lead to the formation of various APPs.15,17,20 Interleukin-6 plays an important role in the release of most APPs by hepatocytes,15,17,21,22 and Kupffer cells can play an intermediate role in APP release23 because stimulation by proinflammatory cytokines results in synthesis of IL-6 and transmission to hepatocytes.

In swine, CSF and ASF are hemorrhagic viral diseases caused by viruses of different families.24 The main clinical and gross findings are loss of appetite; pyrexia; conjunctivitis; focal cyanosis of the skin and bleeding at various sites; tonsillar necrosis; and respiratory tract, gastrointestinal tract, and nervous system disorders accompanied by lymphopenia and thrombocytopenia. Major microscopic findings include lymphoid depletion in primary and secondary lymphoid tissues, multifocal hemorrhages, and inflammatory processes at a range of sites. It has been suggested that the major role played by inflammation in this pathologic process, which has been confirmed by measurement of proinflammatory cytokine concentrations,25–29,a is an alteration of APP patterns. Thus, the study reported here was conducted to determine serum concentrations of APPs in pigs experimentally infected with CSF and ASF viruses.

Materials and Methods

Animals—Eight crossbred (Large White × Landrace) pigs (4 males and 4 females) were used in the study. Pigs were 10 weeks old and weighed 30 kg. All pigs were healthy and seronegative for CSF, ASF, porcine reproductive and respiratory syndrome, and pseudorabies viruses. The pigs were allocated to 2 groups (4 pigs/group; 2 males and 2 females/group). Groups were housed separately in box pens at the Centro de Investigación en Sanidad Animal in Valdeolmos, Madrid, Spain. The study was performed in accordance with the Code of Practice for Housing and Care of Animals used in Scientific Procedures, which was approved by the European Economic Community Union in 1986 (86/609/ EEC amended by the directive 2003/65/EC).

Study design—One group of pigs was inoculated IM with 105 TCID50 of the virulent CSF virus strain Alfort 187.30 The other group of pigs was inoculated IM with 105 median hemagglutinating dose of the ASF virus Spain 70 isolate, which has been described as highly virulent.26 Day of inoculation was designated as day 0. After inoculation, clinical signs and rectal temperatures were monitored daily.

Collection of blood samples—Blood samples were collected from all pigs before inoculation to obtain baseline values. Blood samples from pigs inoculated with CSF virus were collected on days 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, 17, 21, and 24 after inoculation, whereas blood samples were collected from pigs inoculated with ASF virus on days 1, 2, 3, 4, 5, and 6 after inoculation. Considerable care was used in the collection of blood samples to prevent hemolysis and tissue contamination. Blood samples were obtained from a jugular vein by use of plastic syringes and disposable needles; samples were collected into evacuated tubes.b Samples were allowed to clot and then were centrifuged, and serum was harvested and frozen at −80°C until testing. Stability of APPs in frozen-thawed serum samples obtained from pigs and other species has been reported elsewhere.31,32

Processing of specimens for structural study and immunohistochemical analysis—Pigs were sedated with azaperonec and euthanized by administration of an overdose of thiopental sodiumd at the end of the study. A complete necropsy was performed on each pig. Samples of the liver, spleen, thymus, tonsils, lungs, kidneys, ileum, and lymph nodes (mediastinal, retropharyngeal, mesenteric, and mandibular for pigs inoculated with CSF virus and mesenteric, renal, and gastrohepatic for pigs inoculated with ASF virus) were collected and fixed via immersion in neutral-buffered 10% formalin solution (0.01M; pH, 7.2) and Bouin's solution for structural and immunohistochemical studies. After incubation in fixative, samples were dehydrated through a graded series of alcohol solutions, washed with xylol, and embedded in paraffin by use of routine techniques. For histologic examination, paraffin-embedded sections were cut at a thickness of 4 μm and stained with H&E.

The avidin-biotin-peroxidase complex technique has been used for the immunohistochemical detection of viral antigens on serial sections (thickness of 3 μm) obtained from various organs.33 To detect CSF viral glycoprotein E2 (glycoprotein 55), a monoclonal antibodye was used in accordance with an immunohistochemical technique described elsewhere.28 Viral protein 73 of ASF virus was detected by use of the viral protein 73 monoclonal antibodyf via an immunohistochemical technique described elsewhere.26 Tissue sections in which PBS solution or mouse nonimmune serum was substituted for the specific primary antibodies were used as negative control samples. Samples from healthy pigs were also used as control samples.

Determination of APP concentrations in serum— Concentrations of haptoglobin, CRP, and SAA were measured in all serum samples. All determinations were performed in duplicate. A commercial automated spectrophotometric assayg based on the peroxidase activity of haptoglobin-haemoglobin complexes was used to determine haptoglobin concentrations; assays were performed in an automated analyzer.h A commercial solid-phase sandwich immunoassayi was used to determine concentrations of CRP. The final absorbance of the samples was measured in a microtiter plate reader at 450 nm. A commercial solid-phase sandwich ELISAj was used to determine concentrations of SAA. Final absorbance of the samples was also measured in a microtiter plate reader at 450 nm. All of these methods have been validated for use in measuring APP concentrations in porcine serum samples and have had good within-assay precision and accuracy. However, CRP and SAA assays can have high between-day variation34; thus, in the study reported here, all samples were assayed for APP concentrations on the same day.

Statistical analysis—Data were analyzed by use of a statistical analysis program.k Serum concentrations of the various APPs were assessed to calculate mean and SD values. Differences between the values of APP serum concentrations of pigs before inoculation and at various time points after inoculation with CSF or ASF viruses were analyzed by use of the Mann-Whitney U test for nonparametric distributions. Values were considered significant at P < 0.05.

Results

Pigs inoculated with CSF virus—Pigs inoculated with CSF virus had nonspecific clinical signs (loss of appetite, varying degrees of anorexia, constipation, and pyrexia [40.5° to 41.5°C]) beginning 2 days after inoculation. Beginning on day 4 or 5 after inoculation, pigs had ocular discharge associated with conjunctivitis; they also had mild diarrhea. In addition, rectal temperature increased to 42°C beginning on day 6 after inoculation. Pigs had decreased activity and semiliquid, yellowish-gray diarrheic feces beginning on day 8 after inoculation.

Necropsy revealed gross lesions typical of acute CSF. Hyperemia and petechiae were evident in submandibular, mesenteric, and ileocecal lymph nodes and the tonsils; petechial and ecchymotic hemorrhages were evident in the lungs and kidneys; and necrosis was detected in the tonsils. Congestion of the ileum, a reduction in the size of the thymus, and small splenic infarcts characteristic of CSF were also detected.

Histologically, a severe decrease in lymphocyte numbers was detected in the B and T areas of lymphoid organs (spleen, thymus, lymph nodes, and gastrointestinal tract–associated lymphoid tissues). Depletion of lymphocytes was accompanied by pyknosis and karyorrhexis indicative of apoptosis, and this coincided with an increase in the size and number of monocytes or macrophages. Acute hemorrhage was evident in the capsule and medulla of lymph nodes, whereas hyperemia, edema, and microhemorrhage were detected in the ileum and lungs. A moderate periportal infiltrate and foci of lymphocyte-like cells were found in the liver in combination with enlarged septa in the lungs and moderate bronchopneumonia. Necrotic foci were found in the tonsils, and moderate tubular nephrosis, microhemorrhages, and mild infiltrates of lymphocyte-like cells were found in the kidneys.

Virus antigen was detected in all organs evaluated. The monocyte or macrophage population was identified as the main target cells; this included Kupffer cells, interstitial macrophages, and circulating monocytes of the liver.

Serum concentrations of APPs after inoculation with CSF virus were determined (Figure 1). Pigs inoculated with CSF virus had a significant increase in all APPs during the study period. Mean preinoculation concentration of SAA was 1.14 mg/L, and a significant increase was detected on days 4 to 6. The maximum concentration was 359.9 mg/L, which was approximately 300 times the baseline value. Serum concentrations of SAA decreased from days 4 to 6 until day 9, when a second increase was detected. Mean preinoculation concentration of CRP was 56.25 mg/L; the CRP concentration significantly increased by day 3 and remained increased until the end of the study period. The maximum CRP concentration reached was 178.9 mg/L, which was approximately 3 times the preinoculation value. Mean preinoculation concentration of haptoglobin was 1,650 mg/L. The mean haptoglobin concentration increased significantly by day 14 after inoculation. The maximum concentration reached was 4,190 mg/L, which was approximately 3 times the preinoculation value.

Figure 1—
Figure 1—

Mean ± SD serum concentrations of haptoglobin (diamonds), SAA (squares), and CRP (triangles) in 4 pigs before and after inoculation with the CSF virus Alfort 187 strain. Day of inoculation was designated as day 0. 'Within an APP, value differs significantly (P < 0.05; Mann-Whitney U test), compared with the preinoculation (PI) value.

Citation: American Journal of Veterinary Research 68, 7; 10.2460/ajvr.68.7.772

Pigs inoculated with ASF virus—Pigs inoculated with ASF virus had clinical signs beginning 2 days after inoculation. Clinical signs consisted of pyrexia (39.5° to 41°C), loss of appetite, lethargy, dyspnea, and diarrhea (often with bloody content).

During necropsy, macroscopic lesions were typical of ASF, with hemorrhagic splenomegaly, necrosis in the tonsils, and hemorrhagic lymphadenitis (particularly evident in the gastrohepatic and renal lymph nodes but also evident in the mesenteric lymph nodes) also typical. Septal edema in the lungs, hydrothorax, hydropericardium, and petechiae in the kidneys were also detected.

Histologically, pigs had hemorrhagic splenomegaly and acute, severe hemorrhages in the lymph nodes (particularly evident in the gastrohepatic and renal lymph nodes) and kidneys. Severe lymphoid depletion was evident in the thymus, B and T areas of the spleen, and lymph nodes. This was accompanied by moderate periportal infiltrates with foci of dead cells, tubular nephrosis, and necrosis of the tonsils. Alveolar and interstitial edemas, as well as bronchopneumonia and hyperemia, were detected in the lungs.

African swine fever viral protein 73 was detected in all tissues examined in the study; it was especially evident in the monocytes or macrophages. In the liver, Kupffer cells, circulating monocytes, and hepatocytes were immunolabelled for ASF antigen.

Pigs inoculated with ASF virus had increased concentrations of all APPs during the study (Figure 2). Mean preinoculation concentration of SAA was 2.75 mg/L; there was a significant increase detected on days 3 and 4 after inoculation. Maximum concentration reached was 112.18 mg/L, which was 41 times the baseline value. After days 3 and 4, concentrations of SAA decreased to preinoculation values. Mean preinoculation concentration of CRP was 16.76 mg/L, which increased significantly by day 1 after inoculation. The highest concentration detected was 167.48 mg/L, which was approximately a 10-fold increase. Concentrations of CRP remained increased until the end of the study. Mean preinoculation concentration of haptoglobin was 1,340 mg/L, which increased significantly by day 3 and remained increased until the end of the study. The highest haptoglobin concentration reached was 4,890 mg/L, which represented a 4-fold increase.

Figure 2—
Figure 2—

Mean ± SD serum concentrations of haptoglobin (diamonds), SAA (squares), and CRP (triangles) in 4 pigs before and after inoculation with the ASF virus Spain 70 isolate. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 68, 7; 10.2460/ajvr.68.7.772

Discussion

The increase in serum concentrations of APPs in pigs experimentally inoculated with CSF or ASF viruses, compared with baseline concentrations, suggested an acute-phase response in the course of both viral diseases. The response pattern of APPs is a species-specific event. In pigs, haptoglobin and CRP are considered the major APPs,9,35,36 whereas SAA is considered an APP with moderate increases.9,37 An increase in serum haptoglobin concentrations has been reported in swine during the course of viral12,13 and bacterial8–10 diseases, and an increase in CRP concentrations has also been described in pigs experimentally infected with Actinobacillus pleuropneumoniae9 and in pigs injected with turpentine to stimulate development of an inflammatory lesion.35,36

In the study reported here, patterns of APP concentrations (especially haptoglobin and CRP) for both diseases did not coincide with those for other inflammatory processes in which pigs had marked increases of APPs during initial stages, which were followed by a progressive decrease in the serum concentrations.9,35,36 However, our results are in agreement with the patterns for APPs in pigs after experimental infection with Streptococcus suis.38 Furthermore, there were differences between CSF and ASF in terms of the patterns for APPs, although the concentrations and patterns for haptoglobin and CRP were similar for both diseases. The maximum concentration of haptoglobin was detected during the delayed stages of ASF and CSF, with significantly increased haptoglobin concentrations (compared with preinocualtion values) evident only from 14 to 21 days after inoculation and decreased by 24 days after inoculation with CSF virus. Serum concentrations of CRP remained high during the entire experimental period in inoculated pigs (both viruses), whereas maximum SAA concentrations were reached earlier in pigs inoculated with ASF virus (4 days after inoculation) than in pigs inoculated with CSF virus (6 days after inoculation). Serum concentrations of APPs were higher at all stages of disease in pigs inoculated with CSF virus than in those inoculated with ASF virus. Moreover, the 2-phase pattern for SAA recorded in pigs inoculated with CSF virus was not evident in pigs inoculated with ASF virus, perhaps because the experiment and course of disease with this highly virulent strain of ASF were of a shorter duration.

With regard to the magnitude of the increase, the highest change in concentration was recorded for SAA in both inoculation groups (up to a 300-fold increase after inoculation of CSF virus and an approx 40-fold increase after inoculation of ASF virus), which was followed by CRP and then by haptoglobin (only a 3- to 4-fold increase for each). These results are in agreement with the belief that SAA and CRP responses are generally stronger than the response for haptoglobin in that the fold increases from baseline values are higher. However, the concentrations of CRP and SAA obtained during acute-phase responses are substantially lower than the concentration of haptoglobin.2,39

Differences in the amount of cytokines secreted by macrophage populations in the liver and other tissues during ASF and CSF infection may have a direct influence on serum APP concentrations and patterns because proinflammatory cytokines induce synthesis of these proteins by hepatocytes,39 a mechanism in which Kupffer cells play an intermediate role.23 It has been suggested1,2 that serum concentrations of SAA and CRP (classified as type 1 APPs) are predominantly induced by IL-1 and TNF-α (synergistically with IL-6), whereas type 2 APPs (such as haptoglobin) are mainly induced by IL-6.

The increase in serum concentrations of SAA and CRP in the study reported here coincides with increased serum concentrations of IL-1, beginning during the initial stages (1 day after inoculation) of CSF and ASF, and with increased expression of IL-1 by Kupffer cells, beginning during the initial stages of CSF (2 days after inoculation) and ASF (1 day after inoculation) described in other studies40,a,l that involved the use of blood, serum, and liver samples from the same group of pigs reported here. In agreement with these results, IL-1 could play a major role in the synthesis of type 1 APPs by hepatocytes during both diseases. Moreover, an increase in TNF-α expression by Kupffer cells and interstitial hepatic macrophages has been detected beginning at an early stage (1 day after inoculation) of ASF,a whereas the expression of IL-6 by Kupffer cells and interstitial hepatic macrophages has been shown to significantly increase from the initial stages of CSF (2 days after inoculation),40 thus inducing effects that may synergistically compliment the systemic response to IL-1.14,41

The decrease in serum concentrations of SAA in the study reported here coincided with a decrease in the number of Kupffer cells expressing IL-1 and TNF-α in other studies40,a and also with a decrease in the concentrations of these cytokines (particularly in pigs inoculated with CSF) in seruml and other tissues or fluids.26–29 Because SAA synthesis is not limited to the liver, it is likely to be regulated by inflammatory changes and cytokine concentrations (mainly IL-1 and TNF-α) at various sites.42

The maintenance (in pigs inoculated with CSF or ASF viruses) of CRP concentrations, a protein synthesized primarily by hepatocytes,42 may have been attributable to the role played by this protein in processes that modulate monocytes and macrophages.43,44 When there are large amounts of viral antigen in the liver or there is severe tissue damage, CRP concentrations may be maintained to favor monocytes and macrophages. This maintenance of serum CRP concentrations may be enhanced during the latter stages of both ASF and CSF by an increase in the number of Kupffer cells and other interstitial macrophages that secrete IL-6,40,a which is a cytokine that acts synergistically with IL-114,41 to support CRP production by hepatocytes.

Serum concentrations of haptoglobin increased mildly from the start of the experiment, reaching maximum values during the final stages of both diseases. Peak concentrations for the study reported here coincided with the greatest number of IL-6–secreting hepatic macrophages in pigs inoculated with CSF (14 to 17 days after inoculation)40 or ASF (5 to 6 days after inoculation)a viruses, thus highlighting the influence of these macrophages on serum concentrations, patterns, and maintenance of haptoglobin concentrations during CSF and ASF. This role has been reported for other diseases of animals.22,45 Haptoglobin in pigs appears to act as a type 2 APP induced by IL-6 and is characterized by a later increase and a response lasting for up to 2 weeks.46

An acute-phase response is evident after inoculation with ASF and CSF viruses. The characteristic tissue damage is prompted by the viruses involved. However, differences were evident in serum concentrations of the APPs evaluated in the study reported here. Differences in patterns of proinflammatory cytokine expression for each disease may ultimately be responsible for serum concentrations of APPs and patterns for the APPs. Thus, cytokines may play a major role in the various mechanisms that regulate the acute-phase response in pigs infected with CSF and ASF viruses.

ABBREVIATIONS

APP

Acute-phase protein

SAA

Serum amyloid A

CRP

C-reactive protein

TNF

Tumor necrosis factor

IL

Interleukin

CSF

Classical swine fever

ASF

African swine fever

a.

Gómez-Villamandos JC, Mekonnen T, Salguero F, et al. Immunohistochemical detection of monokines in the liver of pigs with acute African swine fever (abstr), in Proceedings. 17th Annu Meet Eur Soc Vet Pathol 1999;236.

b.

Venoject, Terumo Europe, Leuven, Belgium.

c.

Stresnil, Jannsen Animal Health, Beerese, Belgium.

d.

Thiovet, C-Vet Veterinary Products Ltd, Leyland, Lancashire, England.

e.

Monoclonal antibody WH303, Veterinary Laboratories Agency, Addlestone, Surrey, England.

f.

Ingenasa, Madrid, Spain.

g.

Phase haptoglobin assay, Tridelta Development Ltd, Bray, Ireland.

h.

Cobas Mira Plus analyzer, ABX diagnostic, Montpellier, France.

i.

Tridelta Phase Range-CRP canine assay, Tridelta Development Ltd, Bray, Ireland.

j.

Phase Serum Amyloid A assay, Tridelta Development Ltd, Bray, Ireland.

k.

GraphPad InStat, version 3.0, GraphPad Software Inc, San Diego, Calf.

l.

Fernández de Marco M, Salguero FJ, Sánchez-Cordón PJ, et al. A comparative study of the serum profile of cytokines in the pathogenesis of experimental African swine fever and classical swine fever (abstr), in Proceedings. 22nd Annu Meet Eur Soc Vet Pathol 2004;84.

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Contributor Notes

Supported by grants from the Ministry of Education and Science (PB98-1033, AGL 2003-00252, and AGL 2003-01325) and the Seneca Foundation (PB/13/FS/02).

Dr. Sánchez-Cordón was supported by a contract from the Juan de la Cierva program (Ministry of Education and Science, Spain).

The authors thank M. C. Camacho for technical assistance.

Address correspondence to Dr. Sánchez-Cordón.