The Veterinary Journal

Available online at www.sciencedirect.com The Veterinary Journal 177 (2008) 110 115 The Veterinary Journal www.elsevier.com/locate/tvjl Changes in haptoglobin, C-reactive protein and pig-map during a housing period following long distance transport in swine Germana Salamano a, Elisabetta Mellia a, Denise Candiani b, Francesco Ingravalle a, Renato Bruno b, Giuseppe Ru a, Luca Doglione a, * a IZS-State Veterinary Institute of Piedmont, Liguria and Aosta Valley, Turin, Italy b Department of Veterinary Morphophysiology, Faculty of Veterinary Medicine, University of Turin, Turin, Italy Accepted 18 March 2007 Abstract The aim of this study was to investigate the effects of a housing period following long distance transport on haptoglobin (Hp), C- reactive protein (CRP) and pig major acute phase protein (pig-map) in swine. After transportation, 80 gilts were allotted to group A, B, C, or D. Blood samples were collected on arrival and 28 days later; additional samples were collected from Group C on day 14, and from Group D on days 3, 5 and 14. Acute phase proteins (APPs) in Group A were significantly lower on day 28 than on day 1; the opposite occurred in Group B because of a tail biting episode. In Group C, values remained elevated on day 14 and showed a reduction on day 28; in Group D elevated levels detected on day 14 were preceded by a decrease from days 1 to 5. The results indicate that stressors associated with transportation and new accommodation can cause an increase in APPs that could be useful indicators of welfare during transport and routine management. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Haptoglobin; C-reactive protein; Pig-MAP; Transport; Acute phase proteins Introduction During production and marketing, pigs are exposed to many environmental stressors (including thermal extremes, restraint, weaning, mixing, handling and shipping) that increase disease susceptibility and impair immune function (Kelley, 1985; Sheridan et al., 1994). An animal s response to a stressor involves a variety of adaptive physiological mechanisms designed to restore homeostasis. Among these, the acute phase reaction (APR) is a non-specific immune response that has been defined as the entire array of metabolic and physiological changes which occur in response to many different stimuli such as infections, tissue damage, neoplastic growth, * Corresponding author. Tel.: +39 011 2686269; fax: +39 011 2475933. E-mail address: luca.doglione@izsto.it (L. Doglione). immunological disorders or stress (Baumann and Gauldie, 1994; Gabay and Kushner, 1999; Murata et al., 2004). The APR is a complex reaction mediated by proinflammatory cytokines, mainly interleukin (IL)-6, and involving both local and systemic effects (Heinrich et al., 1990; Sehgal et al., 1989). One of these effects results in changes in the concentration of some plasma proteins, mainly synthesised in the liver, which are called acute phase proteins (APPs). The APPs have been grouped according to the direction of change (positive if their synthesis increases, negative if it decreases), and according to the extent to which their concentrations change (minor, intermediate and major) during an APR (Kushner and Mackiewicz, 1987; Steel and Whitehead, 1994). The response pattern of APPs is species-specific: haptoglobin (Hp), C-reactive protein (CRP) and pig major acute phase protein (pig-map) belong to the major positive APPs in swine (Eckersall et al., 1996; González- Ramón et al., 1995; Gruys et al., 1994). Recently it has 1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.03.015

G. Salamano et al. / The Veterinary Journal 177 (2008) 110 115 111 been suggested that APPs may be useful in the assessment of animal welfare (Eckersall, 2000; Murata et al., 2004). The aim of the present field study was to investigate the effect of long distance transport on Hp, CRP and pig-map serum concentrations and the changes of these APPs over a 28-day housing period following transportation. Materials and methods Eighty Landrace Large White 4-month-old gilts were transported from Denmark to Italy by truck in 48 h and then monitored for 28 days. The animals were randomly allotted to four different groups (A, B, C, D) each of 20 pigs. Each group was located in a 6 3.5 m pen. Food and water were provided ad libitum throughout the study. Blood samples were collected via jugular venepuncture immediately upon arrival (T1) and 28 days later (T28), for each pig using one tube containing EDTA and one without anticoagulant. The animals in Group C were additionally bled on day 14 (T14) while those in Group D were also bled on days 3, 5 14 (T3, T5, T14). On day 25 (T25), pigs in Group B developed aggressive behaviour resulting in tail biting. Complete haematology was carried out on all samples using an automatic blood counter calibrated for swine (HemaVet 3500, CDC Technologies). In addition, the serum from each blood sample was separated by centrifugation (1560 g, 10 min) and frozen at 80 C. Serum haptoglobin and CRP concentrations were determined in duplicate using commercial assay kits (Tridelta Development Ltd.). Serum pig-map concentrations were assayed in duplicate with a sandwich ELISA test (PigCHAMP Pro Europa S.A.). Hp and pig-map results were expressed as mg/ml while CRP results as lg/ml. The experimental procedures used in this study conformed to the regulations of Decreto Legislativo no. 116/92, concerning the protection of animals used for scientific research. The distribution of APPs was described using boxplots. Statistical analysis was performed using STATA9 software package (Stata Corporation). After checking normality and homogeneity of variances for all data, the Friedman test was applied to the values of Hp, CRP and pig- MAP for each group of pigs in order to perform a non parametric ANOVA for repeated measures. To evaluate the comparability of the different groups on the same day of sampling, the Kruskal Wallis test was calculated from the values of Hp, CRP and pig-map on the first day of blood sampling. The Wilcoxon and the Friedman nonparametric tests were used to compare within each group the APP concentrations at different times of sampling. Results Haematology profiles were within the normal range of reference values in all gilts. However, higher values in the neutrophil count accompanied by lower values in the lymphocyte count were detected immediately after transport (T1) in comparison with the same counts at the end of the trial (T28). On day T1, the groups showed APP concentrations that were similar and comparable, with the exception of Hp in Group A where concentrations were almost doubled. APP concentrations observed in Group A on day T28 were significantly lower than those on day T1 whereas the opposite phenomenon occurred in Group B. In this box a tail biting episode occurred on day T25 and on T28 the injured pigs had the highest APP levels observed in this study. In Group C, values remained elevated on T14 and showed a reduction on T28, while in Group D high levels detected on T14 reducing on T28 were preceded by a gradual decrease from days T1 to T5 (Figs. 1 3). Discussion In the present study changes in Hp, CRP and pig-map levels were evaluated as an indicator of stress in pigs. The results suggest that APPs can be affected by transport stress and by other stressors commonly present in pig husbandry practices (Tables 1 and 2). During inflammation or infection, concentrations of serum CRP, Hp and pig-map increase (often substantially) soon after the insult and remain elevated for several days. For instance, it has been reported that local inflammation induced in pigs by turpentine injection increased Hp and CRP concentrations 2 8-fold in 48 72 h although values returned to normal around day 7 (Eckersall et al., 1996); pig-map concentrations increased 12-fold (González-Ramón et al., 1995). Experimental infection by aerosol inoculation with Actinobacillus pleuropneumoniae increased Hp levels more than 26-fold and concentrations were significantly elevated from days 1 to 15; CRP increased approximately seven times prechallenge levels and were significantly elevated on days 1 8; pig-map increased approximately 12-fold and was significantly elevated from days 1 to 15 (Heegaard et al., 1998). Recently, it has been suggested that APPs could be useful not only for monitoring the inflammatory process for diagnostic and prognostic purposes, but also for analysing various non-inflammatory conditions, which it was previously thought did not affect APP values (Murata et al., 2004). The pigs in the present study were clinically normal and apparently healthy. The fact that their haematology profiles were within the normal range and the clinical observations made seem to support the idea that changes in APP concentrations were not related to infection or disease. We could not exclude the presence of sub-clinical infections, but it is not unlikely that the variations were caused by other stressors. As it was impossible to obtain serum samples before shipment, as has been done in other studies (Piñeiro et al., 2006), it was assumed that about 1 month was sufficient time for the gilts to recover from the stress of transport and to establish a normal state comparable to that prior to transportation. A large biological variation between individual pigs was found (Table 1) but interestingly it was always same the pigs that showed a raised or reduced APP response during the study. Moreover, the higher the peak value of APPs, the faster the value decreased closer to the mean values for the other pigs. The physiological stress response depends on individual psychological perception and emotional involvement (Von Borell, 1995), so it is possible that among animals experiencing the same stressor(s), one animal may be more sensitive than another. One study showed that in pigs inoculated with the same dose of Streptococcus suis there were varying individual responses although a significant increase in Hp

112 G. Salamano et al. / The Veterinary Journal 177 (2008) 110 115 Fig. 1. Pig-MAP concentration over the time and by group (n = 20 in each group). The line within the box marks the median; the boundaries of the box represent the 25th and 75th percentiles; whiskers above and below the box indicate the 10th and 90th percentiles and the points outside the end of the whiskers are outliers. Fig. 2. CRP concentration over the time and by group (n = 20 in each group). concentration was recorded in all infected animals until 10 days after infection (Knura-Deszczka et al., 2002). Similarly, in our study, in spite of differences in the magnitude of the response between individuals, a clear time course of changes in the concentration of APPs could be defined. Loading, transport and unloading of pigs and new accommodation all cause stress: the animals are compelled to move, the structure of the animal group changes, and there is physical and emotional stress owing to the unfamiliar environment. Tail biting may be a sign of disharmony between animals and the environment. Despite the research undertaken so far, it has been impossible to induce tail biting experimentally and no external factors have been shown to play a specific role in triggering this behaviour, which is considered a multifactorial syndrome with the involvement of environmental features (Schrøder-Petersen and Simonsen, 2001). In the present study, all of the gilts were in the same herd but only Group B showed this abnormal behaviour 3 days before the last sampling. For this reason injured gilts in Group B T28 had the highest APP concentrations recorded throughout the study. Unexpectedly in Groups C and D we saw no constant trend toward lower concentrations of APP from days T1 T28. Furthermore in these groups the maximum response was reached at day T14 for all three proteins and not immediately after transport or on day T3. Lower serum concentrations on day T5 in Group D suggest that there was an initial attempt to recover from the stress of

G. Salamano et al. / The Veterinary Journal 177 (2008) 110 115 113 Fig. 3. Hp concentration over the time and by group (n = 20 in each group). Table 1 Summary values of haptoglobin (Hp), C-reactive protein (CRP) and pig major acute phase protein (pig-map) by group (A, B, C, D) and by time of blood collection (days T1, T3, T5, T14, T28) Pig-MAP (mg/ml) CRP (lg/ml) Hp (mg/ml) T1 T3 T5 T14 T28 T1 T3 T5 T14 T28 T1 T3 T5 T14 T28 Group A Min 0.24 0.19 17.04 13.42 0.06 0.04 Max 2.35 0.88 964.58 35.00 2.64 1.84 Median 0.76 0.41 53.58 19.14 1.17 0.59 Mean 0.91 0.45 161.02 21.19 1.33 0.67 SEM 0.13 0.04 56.79 1.55 0.17 0.11 Group B Min 0.22 0.67 19.52 17.61 0.22 1.40 Max 1.43 5.40 241.60 883.80 1.73 3.88 Median 0.61 3.27 41.76 340.19 0.52 3.07 Mean 0.70 3.06 65.65 372.27 0.60 2.88 SEM 0.08 0.31 12.18 63.83 0.08 0.15 Group C Min 0.14 0.13 0.06 15.63 15.73 13.58 0.15 0.32 0.05 Max 2.29 1.61 0.78 368.87 608.27 336.32 2.31 2.26 1.54 Median 0.54 0.56 0.26 41.70 46.44 19.42 0.70 1.22 0.70 Mean 0.75 0.62 0.30 94.07 147.75 35.53 0.85 1.19 0.76 SEM 0.12 0.08 0.04 23.42 40.78 15.86 0.13 0.10 0.09 Group D Min 0.18 0.35 0.10 0.36 0.22 10.06 13.80 14.66 15.43 14.24 0.02 0.18 0.05 0.74 1.00 Max 2.25 2.53 2.75 3.58 2.46 453.06 1023.54 784.40 1047.83 555.03 1.44 3.30 4.00 3.55 3.34 Median 0.84 0.76 0.42 1.15 0.98 75.04 66.65 54.01 201.55 27.57 0.64 1.13 0.57 2.36 1.66 Mean 0.96 1.02 0.68 1.33 1.09 110.73 127.20 102.11 266.85 67.55 0.68 1.25 0.89 2.27 1.82 SEM 0.12 0.15 0.16 0.18 0.11 28.27 49.69 38.31 56.65 27.25 0.09 0.19 0.21 0.19 0.14 The number of pigs sampled (n) is 20 in each group. transportation but additional stressors such as handling procedures, mixing and adaptation to a new environment, occurred during the housing period. This may explain the increase in APP sera concentrations in both Groups C and D on day T14, and it is reasonable to think that also in Groups A and B (bled only on days T1 and T28) something similar may have happened. Group D on day T14 showed concentrations for pig-map and Hp that had almost doubled, and the values were about four times greater for CRP compared to those obtained in Group C on day T14 (Table 1). Repeated exposure to a stressor can result in alteration in the response that can increase in intensity, a process that has been called sensitization (Ladewig, 2000). As Group D was the most repeatedly

114 G. Salamano et al. / The Veterinary Journal 177 (2008) 110 115 Table 2 Q values (Friedman s test) for all possible pairwise comparisons by times of sampling in each group of animals and by each protein Pig-MAP CRP Hp T3 T5 T14 T28 T3 T5 T14 T28 T3 T5 T14 T28 Group A T1 5.69 6.33 4.43 2.77 Group B T1 5.06 5.06 6.33 2.77 Group C T1 0.78 5.59 0.45 5.14 3.13 0.45 T14 4.81 5.59 3.58 3.31 Group D T1 0.71 3.96 2.12 1.13 0.85 0.28 2.69 1.84 3.54 0.42 7.35 5.66 T3 4.67 1.41 0.42 1.13 1.84 2.69 3.11 3.82 2.12 T5 6.08 5.09 2.97 1.56 6.93 5.23 T14 0.99 4.53 1.70 3.86 A statistically significant result (marked in bold) is obtained when the values within the table are higher than the critical value for that particular group. bled, it is likely that successive exposure to that stimulus within a few days may have led to higher responses. Conclusions Our findings suggest that not only transportation but also the stress of adapting to new accommodation may cause an increase in APP concentrations. This confirms that pigs are affected by both physical and psychological stressors and not only by inflammation caused by tissue damage or infection (Murata et al., 2004; Piñeiro et al., 2006). Thus APPs may be useful parameters for the detection of stress in pigs and in the assessment of welfare during transport and herd management. Before using APP concentrations as objective and non-specific markers of animal health, however, it is important to take the possible influence of envinronmental factors, handling and other type of stress in the absence of disease into consideration (Petersen et al., 2004). Further research is needed to define the usefulness of APPs as potential markers of animal welfare and to develop a baseline for practical applications. Finally, the use of a standardized acute phase index, derived from a mathematical formula that uses both positive and negative reacting APPs, could be used to improve the sensitivity of the detection of the APP response (Skinner, 2001). References Baumann, H., Gauldie, J., 1994. The acute phase response. Immunology Today 15, 74 80. Eckersall, P.D., Saini, P.K., McComb, C., 1996. The acute phase response of acid soluble glycoprotein, a(1)-acid glycoprotein, ceruloplasmin, haptoglobin and C-reactive protein, in the pig. Veterinary Immunology Immunopathology 51, 377 385. Eckersall, P.D., 2000. Recent advances and future prospects for the use of acute phase proteins as markers of disease in animals. Revue de Médicine Vétérinaire 151, 577 584. Gabay, C., Kushner, I., 1999. Acute-phase proteins and other systemic responses to inflammation. New England Journal of Medicine 340, 448 454. González-Ramón, N., Alava, M.A., Sarsa, J.A., Piñeiro, M., Escartin, A., Garcia-Gil, A., Lampreave, F., Piñeiro, A., 1995. The major acute phase serum protein in pigs is homologous to human plasma kallikrein sensitive PK-120. FEBS Letters 371, 227 230. Gruys, E., Obwolo, M.J., Toussaint, M.J.M., 1994. Diagnostic significance of the major acute phase proteins in veterinary clinical chemistry: a review. Veterinary Bulletin 64, 1009 1018. Heegaard, P.M., Klausen, J., Nielsen, J.P., González-Ramón, N., Piñeiro, M., Lampreave, F., Alava, M.A., 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. Comparative Biochemistry and Physiology B 119, 365 373. Heinrich, P.C., Castell, J.V., Andus, T., 1990. Interleukin-6 and the acute phase response. Biochemical Journal 265, 621 636. Kelley, K.W., 1985. Immunological consequences of changing environmental stimuli. In: Moberg, G.P. (Ed.), Animal Stress. American Physiological Society, Bethesda, MD, pp. 193 223. Knura-Deszczka, S., Lipperheide, C., Petersen, B., Jobert, J.L., Berthelot- Herault, F., Kobish, M., 2002. Plasma haptoglobin concentration in swine after challenge with Streptococcus suis. Journal Veterinary Medicine B 49, 240 244. Kushner, I., Mackiewicz, A., 1987. Acute phase proteins as disease markers. Disease Markers 5, 1 11. Ladewig, J., 2000. Chronic intermittent stress: a model for the study of long term stressors. The biology of animal stress. Basic Principles and

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