The Veterinary Journal

Available online at www.sciencedirect.com The Veterinary Journal 177 (2008) 369 373 The Veterinary Journal www.elsevier.com/locate/tvjl Relationship between serum acute phase protein concentrations and lesions in finishing pigs Francisco J. Pallarés a, *, Silvia Martínez-Subiela b, Juan Seva a, Guillermo Ramis c, Pablo Fuentes b, Antonio Bernabé a, Antonio Muñoz c, José J. Cerón b a Departamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain b Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain c Departamento de Producción Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain Accepted 28 April 2007 Abstract To establish the relationship between serum levels of three acute phase proteins, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA), and the occurrence and severity of lesions at slaughter, a study was carried out using 70 fattening pigs from a finishing unit. Pigs were divided into three groups: Group 1 (25 pigs with clinical signs of disease), Group 2 (25 apparently healthy pigs with lesions at slaughter) and Group 3 (20 apparently healthy pigs with no lesions at slaughter). Serum levels of CRP, SAA and Hp were significantly higher in pigs with clinical signs of disease than in apparently healthy animals. Additionally, in apparently healthy pigs, serum levels of Hp and CRP were significantly higher in animals with lesions than those without lesions. The extent and severity of lung lesions were related to serum levels of Hp. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Pig; Acute phase proteins; Lesions; Lung; Slaughter Introduction The acute phase proteins (APPs) are a group of blood proteins that change in concentration in animals subjected to external or internal challenges, such as infection, inflammation, surgical trauma or stress (Murata et al., 2004). The serum concentrations of positive APPs, such as haptoglobin (Hp), serum amyloid A (SAA) and C-reactive protein (CRP), increase during the acute phase response, whereas the serum concentrations of negative APPs, such as albumin, decrease (Murata et al., 2004). Increased serum levels of APPs have been reported in pigs experimentally infected with porcine reproductive and respiratory syndrome (PRRS, Lelystad) virus, porcine circovirus type 2 (PCV2), Actinobacillus pleuropneumoniae * Corresponding author. Tel.: +34 968 364336; fax: +34 968 364147. E-mail address: pallares@um.es (F.J. Pallarés). and Streptococcus suis (Hall et al., 1992; Heegaard et al., 1998; Asai et al., 1999; Baarsch et al., 2000; Knura-Deszczka et al., 2002; Hultén et al., 2003; Díaz et al., 2005; Sorensen et al., 2006; Stevenson et al., 2006). Serum APPs also increase in pigs naturally infected with A. pleuropneumoniae (Hall et al., 1992) and in field cases of postweaning multisystemic wasting syndrome and porcine respiratory disease complex (Segalés et al., 2004; Pallarés et al., 2005, 2006; Parra et al., 2006). It has been postulated that concentrations of serum APPs are related to the severity of underlying disease and therefore may act as markers for the presence and extent of disease processes (Eckersall, 2000). Concentrations of APPs, such as Hp, CRP and SAA, may be used as tools for meat inspection and predicting meat quality at slaughter (Visser et al., 1992). However, despite the potential value of APPs in detection of lesions in finishing pigs, there are few published studies (Amory et al., 2000; Chen et al., 1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.04.019

370 F.J. Pallarés et al. / The Veterinary Journal 177 (2008) 369 373 2003). The aim of the present work was to establish the relationships between serum levels of three APPs (Hp, CRP and SAA) and the appearance and severity of lesions in pigs at slaughter. The study was designed to determine whether serum APP concentrations could be used as markers for the presence of lesions and as a tool to detect subclinical disease in fattening pigs. Materials and methods Animals and samples The study used 70 conventional Duroc x (Landrace x Large White) pigs from a finishing unit of 1,800 animals. The farm was seropositive to PRRS virus, PCV2, Mycoplasma hyopneumoniae and A. pleuropneumoniae. Pigs were vaccinated twice against M. hyopneumoniae (in the first week of life and 3 weeks later) and twice against Aujeszky s disease virus (ge negative modified live vaccine with oil in water adjuvant; at 11 and 14 weeks of age). Animals were introduced into the finishing unit at an average of 70 days of age and 25 kg weight and sent to the abattoir at an average of 190 days of age and 102 kg weight. All animals were reared and transported under welfare conditions described in the European Union Council Directive 91/630/EEC, Council Directive 2001/88/EC and Commission Directive 2001/93/EC. Group 1 was composed of 25 pigs with clinical signs of disease and poor body condition that were not suitable to be sent to the slaughterhouse (Table 1). Blood samples were collected from the jugular vein using single-use blood collection tubes without any additive (Vacutainer, Becton Dickinson). Afterwards, the animals were killed by an intravenous overdose of sodium pentobarbital (Dolethal, Vétoquinol) and complete postmortem examinations were performed. Gross lung lesions were scored according to the estimated percentage of lung affected by pneumonia (Halbur et al., 1995). Samples of lung, heart, kidney, ileum, spleen, tonsil, brain, liver and tracheobronchial, mediastinal, inguinal and mesenteric lymph nodes were collected in 10% neutral buffered formalin for histopathology. Pigs of Group 1 were divided in two subgroups according to the presence of lesions in the lungs only (Subgroup 1a; 12 pigs) or in the lungs and one or more other organs (Subgroup 1b; 13 pigs). Groups 2 and 3 comprised 45 apparently healthy pigs from the same fattening unit sent to the abattoir (Table 1). These animals were transported for no longer than 1.5 h and were rested for 4 h before slaughter. They were killed by cutting the throat after being stunned in a chamber containing carbon dioxide, and were bled out with a Rotastick pump (Anitec). Blood samples were collected directly from the neck wounds. Carcasses and organs were carefully examined as for Group 1 and samples of lesions were collected into 10% neutral buffered formalin for histopathology. Lesions in the oesophagogastric mucosa, ranging from parakeratosis to erosions, were present in almost 100% of stomachs studied previously at the abattoir (Ramis et al., 2004) and were not used to differentiate groups of pigs. Pigs with any other gross lesions (25 animals) Table 1 Groups of animals included in the study Groups n Animals Group 1 25 Pigs with clinical signs Subgroup 1a 12 Pigs with lesions only in the lungs Subgroup 1b 13 Pigs with lesions in the lungs and one or more other organs Group 2 25 Apparently healthy pigs showing any gross lesions at slaughter Group 3 20 Apparently healthy pigs showing no gross lesions at slaughter were included in Group 2; pigs with no other gross lesions (20 animals) were included in Group 3. All blood samples obtained were allowed to clot at 4 C for 1 h; the serum was separated by centrifugation (2000 g for 15 min) and then frozen at 20 C until testing (1 week later). Determination of acute phase proteins All methods used for the determination of serum concentrations of all three APPs had been previously validated (Kjelgaard-Hansen et al., 2007; Tecles et al., 2006). Serum concentrations of CRP were determined using a commercial immunoturbidimetric assay (Randox Laboratories) and results were expressed in lg/ml. Serum concentrations of Hp were quantified by a spectrophotometric method using a commercial kit (Phase Range Haptoglobin Assay, Tridelta Development) and results were expressed in mg/ml. These assays were performed according to the manufacturers instructions on an automated analyser (Cobas Mira Plus, ABX Diagnostics). Serum concentrations of SAA were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Phase Tridelta Development) and the results expressed in lg/ml. All samples were analysed in the same analytical run to avoid high between-run imprecision as had been previously reported for SAA (Tecles et al., 2006). Statistical analysis The values of each protein concentration were evaluated for approximate normality of distribution using the Kolmogorov Smirnov statistic. Limits of confidence intervals were calculated as the 25th and 75th percentiles. To evaluate differences in the serum concentrations of CRP, SAA and Hp between groups, the non-parametric test of Mann Whitney was performed. A difference was considered significant at P < 0.05. The data were analysed using the statistical analysis program SPSS v.11 (SPSS Inc.). Results Lesions All animals in Group 1 had lung lesions (Table 2), including cranioventral suppurative bronchopneumonia (20/25; 80%), chronic pleurisy (13/25; 52%), embolic pneumonia (7/25; 28%), enzootic (mycoplasmal) pneumonia (4/25; 16%) and fibrinous bronchopneumonia (2/25; 8%). The percentage of lung surface affected in pigs of Group 1 varied from 6 85%, with an average of 45%. Lesions in other organs are listed in Table 2. Table 2 Main lesions observed in pigs at slaughter Lesion Group 1 (n = 25) Suppurative bronchopneumonia Group 2 (n = 25) 20 0 0 Chronic pleurisy 13 5 0 Embolic pneumonia 7 0 0 Enzootic (mycoplasmal) 4 20 0 pneumonia Fibrinous bronchopneumonia 2 0 0 Chronic ileitis 8 0 0 Chronic arthritis 6 0 0 Tail (bite) wounds 4 0 0 Polyserositis 1 0 0 Group 3 (n = 20)

C-reactive protein(ug/ml) F.J. Pallarés et al. / The Veterinary Journal 177 (2008) 369 373 371 a b c 3.5 350 300 100 3.0 2.5 250 75 200 2.0 150 50 1.5 Serum AmyloidA(ug/mL) Haptoglobin(mg/mL) 100 50 25 1.0 0.5 0 0 0.0 Fig. 1. Serum concentrations of (a) C-reactive protein, (b) serum amyloid A and (c) haptoglobin in pigs in Groups 1, 2 and 3. The median is marked with a line. The box shows the 25th 75th percentiles. The whiskers show maximum and minimum In Subgroup 1a (12 pigs with lesions only in the lungs), only one type of lung lesion was found in three pigs and two or more different types of lung lesions in nine pigs. The most frequent combination of lung lesions was suppurative bronchopneumonia and chronic pleurisy, observed in six pigs. The percentage of lung surface affected ranged from 8 85%, with an average of 51%. In Subgroup 1b (13 pigs with lesions in the lungs and one or more organs), only one type of lung lesion was observed in three pigs and two or more different types of lung lesions in ten pigs. The most frequent combination of lung lesions in Subgroup 1b was suppurative bronchopneumonia and chronic pleurisy, found in five pigs. The percentage of lung surface affected ranged from 6 70%, with an average of 40%. In Group 2, enzootic (mycoplasmal) pneumonia was detected in 20/25 (80%) pigs and chronic pleurisy was seen in 5/25 (20%) (Table 2). The percentage of lung surface affected ranged from 5 50%, with an average of 15%. Only one type of lung lesion was observed in each pig in this group. No significant lesions were detected in other organs. In Group 3, no significant lesions were detected in the lungs or other organs. All pigs in Groups 1 3 had parakeratosis and/or erosions in the oesophagogastric mucosa. Serum concentrations of acute phase proteins Serum concentrations of CRP, SAA and Hp in all groups are shown in Fig. 1a c, respectively. The median concentration of CRP in Group 1 (pigs with clinical disease) was 131.3 lg/ml, which is 2.1-fold higher than Group 2 (apparently healthy pigs with gross lesions at slaughter; 62.7 lg/ml) and 2.6-fold higher than Group 3 (apparently healthy pigs without gross lesions at slaughter; 50.2 lg/ml). There were significant differences between Groups 1 and 2 (P < 0.01), Groups 1 and 3 (P < 0.0001) and Groups 2 and 3 (P < 0.05). The median concentrations of SAA were 18.6 lg/ml in Group 1 and 0.0 lg/ml in Groups 2 and 3. There were significant differences between Groups 1 and 2 (P < 0.01) and Groups 1 and 3 (P < 0.0001). There were no significant differences between Groups 2 and 3 (P = 0.109). The median concentration of Hp in Group 1 was 2.4 mg/ml and this value was 2.4-fold higher than Group 2 (1.0 mg/ml) and 4.8-fold higher than Group 3 (0.5 mg/ ml). There were significant differences between Groups 1 and 2 (P < 0.0001), Groups 1 and 3 (P < 0.0001) and Groups 2 and 3 (P < 0.05). Serum concentrations of CRP, SAA and Hp in pigs in Subgroups 1a (Group 1 pigs showing lesions only in the lungs) and 1b (Group 1 pigs showing lesions in the lungs and one or more other organs) are shown in Table 3. The median serum concentration of CRP in Subgroup 1b was 1.7-fold higher than Subgroup 1a and was significantly different (P < 0.05). The median serum concentrations of SAA and Hp in Subgroups 1a and 1b were not significantly different (P = 0.168 and 0.979, respectively). Table 4 shows the concentrations of serum APPs in Groups 1 and 2 showing only lung lesions (37 pigs) grouped according to the percentage of lung surface affected: 1 30% (25 pigs) and >30% (12 pigs). Significant differences were found between these two categories for SAA (P < 0.05) and for Hp (P < 0.0001). No significant Table 3 (Hp) concentrations in pigs in Subgroups 1a and 1b Subgroup 1a (Pigs in Group 1 with lesions only in the lungs) Subgroup 1b (Pigs in Group 1 with lesions in the lungs and one or more other organs) n CRP (lg/ml) 12 90.6 a (49.9 143.8) 13 154.2 b (117.7 283.0) SAA (lg/ml) 1.4 a (0.0 39.1) 39.9 a (0.2 166.9) Hp (mg/ml) 2.4 a (2.1 2.6) 2.6 a (1.8 2.8)

372 F.J. Pallarés et al. / The Veterinary Journal 177 (2008) 369 373 Table 4 (Hp) concentrations of pigs in Groups 1 and 2 showing only lung lesions and grouped according to the percentages of lung surface affected n CRP (lg/ml) SAA (lg/ml) Hp (mg/ml) 1 30% 25 61.8 a (50.0 114.3) 0.0 a (0.0 4.7) 1.1 a (0.5 1.6) >30% 12 90.6 a (57.6 143.8) 9.3 b (0.0 39.1) 2.4 b (1.9 2.6) Table 5 (Hp) concentrations in pigs in Groups 1 and 2 showing only lung lesions grouped according with the presence of one or more than one different types of lung lesions n CRP (lg/ml) SAA (lg/ml) Hp (mg/ml) One type of 29 70.8 a (55.2 123.6) 0.0 a (0.0 9.8) 1.1 a (0.5 1.7) lung lesion More than one type of lung lesion 8 90.6 a (49.5 119.7) 0.0 a (0.0 26.1) 2.5 b (2.1 2.8) differences were found for the serum concentrations of CRP (P = 0.261). Serum APP concentrations in Groups 1 and 2 showing only lung lesions (37 pigs) grouped according to the presence of one or more than one different types of lung lesions are shown in Table 5. Significant differences were found between these two categories only for the serum concentration of Hp (P < 0.0001). No significant differences were found for the serum concentrations of CRP (P = 0.985) or SAA (P = 0.814). Discussion Our results indicate that serum concentrations of CRP, SAA and Hp can be used as markers of clinical disease in pigs. These findings agree with those of Chen et al. (2003), in which serum Hp and CRP concentrations were significantly higher in culled pigs than in clinically normal pigs. Tourlomoussis et al. (2004) also observed a significant increase in serum concentrations of Hp and SAA in cows with pathological conditions compared to healthy animals. Serum Hp values in healthy pigs without any lesions (Group 3) in this study were similar to levels (0.76 mg/ ml) found in healthy pigs by Sepponen and Pösö (2006). According to our results, only serum Hp and CRP could be used as markers of the presence of lesions at slaughter, since they appear to differentiate apparently healthy pigs with lesions from pigs with no lesions. The presence of elevated serum Hp and CRP concentrations in apparently healthy pigs at slaughter could provide important information to a veterinary inspector about the presence of subclinical lesions that could lead to condemnations or a decrease in the quality of carcasses. In order to progress towards a wider use of APPs at slaughter in the future, strategies such as the use of whole blood or meat juice, or the development of on-line testing systems, should be explored to make these assays faster and easier to perform. Serum concentrations of APPs are affected by age (Petersen et al., 2002; Tourlomoussis et al., 2004), breed (Clapperton et al., 2005), housing and management conditions (Geers et al., 2003), and prolonged transportation by road and rest periods (Piñeiro et al., 2007). All pigs included in this study were of the same age and genetic crossbreed and had been reared in the same fattening unit under the same housing and management conditions. Pigs in Groups 2 and 3 were transported for 1.5 h to the abattoir, with a rest period of approximately 4 h, whereas pigs in Group 1 were killed on the farm. Therefore, we postulate that differences in serum concentrations of APPs in our study were influenced mainly by the pathological conditions affecting the animals. Lesions in the oesophagogastric mucosa were detected in all pigs included in the study and therefore were unlikely to account for differences in APPs between groups. Parakeratosis and erosions of the oesophagogastric mucosa are common in pigs at slaughter and are probably associated with diet and intensive production systems (Ramis et al., 2004). High serum concentrations of APPs can be used to differentiate pigs with signs of disease from clinically normal pigs and are correlated with the presence and degree of lesions within clinically normal pigs (Chen et al., 2003). To our knowledge, there are no data in the literature about the possible use of serum levels of APPs to differentiate the degree of lesions among pigs with clinical signs. Our results show that serum levels of CRP in pigs with clinical signs are significantly lower in animals showing only lung lesions than when lesions were seen in the lungs and one or more other organs. Previously, the presence of enzootic pneumonia has been associated with increased serum Hp levels (Amory et al., 2000; Geers et al., 2003). A weak correlation was found between the severity of these lesions and Hp concentrations, but there was no relationship between Hp and the extent of pneumonia (Amory et al., 2000); other types of lung lesions, including pleurisy and bronchopneumonia, were not examined. In our study, all types of lung lesions were recorded and we demonstrated a relationship between serum Hp values and extent (% of lung surface affected) and severity (presence of one or more types of lesions) of lung lesions, independent of the type of lesions. Since several different types of lung lesions often occurred concurrently, it was not possible to study the correlation between serum APPs and the extent and severity of every type of lung lesion individually. Conclusions From the results obtained in this study, serum values of CRP and Hp could be used to differentiate healthy finishing

F.J. Pallarés et al. / The Veterinary Journal 177 (2008) 369 373 373 pigs without any lesions from pigs with subclinical lesions. In addition, serum concentrations of Hp could be used as an indicator of the extent and severity of lung lesions of pigs at slaughter. The possibility of measuring serum concentrations of APPs in finishing pigs just before slaughter could provide information for veterinary inspectors about the possible occurrence of lesions in these pigs and could serve as a tool for the meat industry to differentiate between pigs with a different health status and carcass quality. Acknowledgements This study was founded by Project AGL2003-01325 from the Spanish Ministry of Science and Technology and by Project PB/13/FS/02 from the Fundación Seneca of the Comunidad Autónoma de la Región de Murcia (Spain). References Amory, J.R., Mackenzie, A.M., Pearce, G.P., Eckersall, P.D., Lampreave, F., Alava, M.A., 2000. 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