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Influence of rearing conditions and respiratory disease on haptoglobin levels in the pig at slaughter
Research in Veterinary Science 83 (2007) 428–435 www.elsevier.com/locate/rvsc
Influence of rearing conditions and respiratory disease on haptoglobin levels in the pig at slaughter J.R. Amory b
a,*
, A.M. Mackenzie a, P.D. Eckersall b, M.J. Stear b, G.P. Pearce
a,1
a Animal Science Research Centre, Harper Adams University College, Newport, Shropshire TF10 8NB, UK Division of Animal Production and Public Health, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK
Accepted 30 January 2007
Abstract Associations between serum concentrations of haptoglobin, pathological lung lesions indicative of Mycoplasma hyopneumoniae (EP) or Actinobacillus pleuropneumoniae (PL) infection at slaughter and previous rearing environment were investigated in 510 pigs (90–100 kg live weight) from 17 farms in England. Haptoglobin concentrations were significantly higher in pigs showing pathological signs of EP infection compared to those without signs of this disease (EP positive median 0.43 mg ml 1 vs. EP negative median 0.26 mg ml 1, p < 0.01). However, there were no significant associations between serum haptoglobin concentrations and pathological signs of PL. The presence of solid partitions compared with barred or similar open partitions was associated with a decrease of 0.44 mg ml 1 farm mean haptoglobin concentration, whilst an increase in pen size of 10 m2 was associated with a decrease of 0.08 mg ml 1 farm mean haptoglobin concentration. The findings indicate that pathological signs of EP were associated with increased serum haptoglobin at slaughter, which in turn was influenced by components of the farm environment. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Pigs; Respiratory disease; Haptoglobin; Environment
1. Introduction The response of a pig to its environment may be monitored by using physiological markers, or ‘‘biomarkers’’ (Eckersall, 2000). Acute phase proteins (APPs) represent an important group of biomarkers with potential use as indicators of tissue injury associated with changes in health status in commercially reared pigs (Gymnich and Petersen, 2004). APPs are produced by the liver in response to cytokines associated with inflammation, infection or tissue injury and function to restore homeostasis in the body following injury or infection (Eckersall, 2000; Colditz, 2002; * Corresponding author. Present Address: Department of Science, Agriculture and Technology, Writtle College, Chelmsford, Essex CM1
E-mail address: [email protected] (J.R. Amory). Present address: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. 1
0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.01.012
Petersen et al., 2004) and the acute phase protein Haptoglobin, has been identified as a sensitive indicator of respiratory infection and its concentration in blood serum has been suggested as a means of non-specific surveillance of pig health status (Heegaard et al., 1998). There is an increasing number of scientific publications describing the acute phase response in the pig and the effects of infection (Heegaard et al., 1998), inflammatory response to an injection of turpentine (Eckersall et al., 1996), specific husbandry practices (Francisco et al., 1996a,b), transport (Saco et al., 2003), clinical signs of disease (Petersen et al., 2002a), histopathological signs of disease (Chen et al., 2003), and presence of clinical diseases such as Postweaning Multisystemic Wasting Syndrome (Segales et al., 2004) and Porcine Intestinal Adenomatosis (Geers et al., 2003). Haptoglobin levels have been reported to be higher in pigs from organic compared with non-organic pig herds (Millet et al., 2005), in indoor compared with outdoor herds (Franek and Bilkei,
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2004) and in herds with poor levels of hygiene (Gymnich and Petersen, 2004). However, there is little information describing the effects of specific aspects of the rearing environment on mediating the variability of acute phase proteins in the slaughter pig. Knowledge of such variation may be important in identifying factors within husbandry systems associated with increased acute phase response and also in the development of their use in ante- and post-mortem inspection (Saini and Webert, 1991) and production chain orientated health management systems (Gymnich and Petersen, 2004). This study was designed to investigate the relationship between pathological signs of two major respiratory diseases of pigs, enzootic pneumonia and pleuropneumonia, and serum haptoglobin concentrations at slaughter. The study also aimed to identify husbandry and management procedures important in influencing the acute phase response, as determined by levels of serum haptoglobin in slaughtered pigs reared under commercial conditions. 2. Materials and methods Seventeen farms were involved in the study that delivered pigs to one of three participating abattoirs in the midlands region of England. Each farmer was given a questionnaire detailing various aspects of the farm environment, husbandry system and pig health based on a previous study investigating risk factors for respiratory diseases in New Zealand (Stark et al., 1998). For the purpose of this study grower pigs were classified as 20–45 kg and finishers 45 kg – slaughter weight. Blood samples and lungs were collected from 30 randomly selected pigs from a single delivery. Following stunning, blood samples were collected in 30 ml universal tubes from the throat as they were exsanguinated. The collected blood was allowed to clot overnight at 4 °C and serum was then obtained by centrifugation for 15 min at 1000g and stored at 20 °C until subsequent analysis. The serum haptoglobin concentration was determined according to the haemoglobin binding activity method of Eckersall (2000) that allows measurement of haptoglobin from all species. A bovine standard of known haptoglobin concentration was used as standard (Horadagoda et al., 1994). Each farm was assessed for their pneumonia status by calculating the prevalence of affected lungs at slaughter and their degree of consolidation. In order to determine tissue damage due to enzootic pneumonia (EP score) the degree of consolidation was assessed by estimating the percentage of the surface of each lobe of the lung showing signs of consolidation. This percentage was multiplied by a weighting factor for each lobe based on their relative volumes (0.25 for the caudal lobes, 0.1 for the cranial and accessory lobes) and totalled to give a score out of 100 for each pig based on the methodology of Straw et al. (1986a). In order to determine tissue damage due to pleuropneumonia (PL score) the percentage of each lobe estimated to be missing due to adhesion of pleural
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membranes was scored pneumonias a measure of grade 2 pleurisy based on the methodology of Straw et al. (1986b). Although pleuritis can be attributed to a number of causal organisms, infection with Actinobacillus pleuropneumoniae is by far the most common in the UK (Done, 1999). Continuous variables were quantified by calculating median, minimum and maximum figures. Differences in serum haptoglobin concentration between pigs identified as having or not having pathological signs of enzootic pneumonia were calculated using the Kruskal–Wallis test (Petrie and Watson, 1999, p. 146). This was repeated for those identified as having or not having pathological signs of pleuropneumonia. In addition, both types of pneumonia were considered together by creating four groups one negative for both types, one positive for EP only, one positive for PL only and one positive for both types.. Regression equations were calculated to determine whether either EP or PL score could be predicted by the actual serum acute phase protein concentration in individual pigs. In order to determine which terms were to be considered for the construction of a multiple regression model, univariable analyses of variance were carried out between individual farm factors and the dependent variables. Those terms giving an F-probability p < 0.25 were put forward for the multiple regression model as suggested by Hosmer and Lemeshow (1989). Similarly for the continuous independent variables, those giving correlations of r > 0.23 (equivalent to p < 0.25 with 16 degrees of freedom based on 17 farms) were also put forward for the multiple regression model. This was carried out using forwards and backwards stepwise selection procedures to ascertain the best model. Finally all original terms were added individually to the model to check significance. Descriptive statistics, analysis of variance, Pearson correlation coefficients and the stepwise regression procedure were performed using S-Plus (version 6.2) for Windows statistics package. Non-parametric statistics were performed using Minitab (version 11) for Windows statistics package. 3. Results Of the 17 farms that took part in this study, eight were breeder-finisher units and 9 were finishing-only units, all slaughtering finished pigs between 90 and 100 kg live weight. Nine farms provided bedding for their grower pigs, while eight provided bedding for the finishers. Nine farms used a slatted method of slurry disposal for their growers, while 10 used slatted floors for their finishers. Five farms vaccinated against enzootic pneumonia, one farm vaccinated for pleuropneumonia, four farms injected individual grower pigs for treatment of respiratory disease and one farm injected finisher pigs for treatment of respiratory disease. However, the effects of vaccination and medication status were not investigated in detail in this study. Ten farms gave in-feed/water medication to their growers and five farms gave in-feed/water medication to their finishers.
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Median and range figures for the continuous variables are shown in Table 1. Signs of enzootic pneumonia were the most prevalent lesion type in this study with 147 pigs out of the 510 examined showing these signs (varying from 0% to 26% of total lung volume affected). Only one of the 17 farms did not show gross lesions associated with enzootic pneumonia. Pathological lesions associated with pleuropneumonia were found in 34 of the 510 pigs examined (signs of pleurisy varying from 0% to 48% of total lung volume affected in all pigs). Four out of the 17 farms did not show the lung tissue damage attributed to pleuropneumonia in this study. Distributions of lesion scores are presented in Table 2. Eleven out of the 510 pigs showed signs of both diseases. Mean individual serum haptoglobin was 0.64 mg ml 1 (range 0–8.55 mg ml 1), whilst the farm mean haptoglobin concentration varied from 0.27 to 1.26 mg ml 1 (Table 3). There was a significant increase in haptoglobin concentra-
Table 1 Median, minimum and maximum figures for continuous management variables from the respiratory disease and acute phase protein study Median
Minimum
Maximum
No. of sows on unit
78
491
Distance to nearest pig farm (miles) Weaning age (days) No. of stages after weaning Grower pen size (m2) No. of grower pigs per pen Grower space allowance (m2/ pig) No. of grower pigs per room Finisher pen size (m2) No. of finisher pigs per pen Finisher space allowance (m2/ pig) No. of finisher pigs per room
2
0 (finishers only) 0.25
5
24 2 12.5 25 0.5
21 1 5 14 0.36
28 4 100 200 1.25
140 13.3 20 0.68
20 6.75 10 0.47
290 100 200 0.93
149
100
220
Table 2 Distribution of the severity of lesion scores for enzootic pneumonia and pleuropneumonia Pneumonia scorea (%)
Enzootic pneumonia (% of pigs, n = 510)
Pleuropneumonia (% of pigs, n = 510)
0 >0–1 >1–2 >2–5 >5–10 >10–20 >20–30 >30–40 >40–50 >50
71.2 5.9 7.1 9.0 3.3 2.9 0.6 0 0 0
93.3 0.0 0.8 1.6 1.8 1.8 0.2 0.4 0.2 0
a Score was ascribed by estimating the percentage of the surface of each lobe of the lung showing signs of consolidation multiplied by a weighting factor for each lobe based on their relative volumes (0.25 for the caudal lobes, 0.1 for the cranial and accessory lobes) and totalled to give a score out of 100 for each pig.
Table 3 Descriptive statistics regarding serum haptoglobin
Mean Minimum First quartile Median Third quartile Maximum
Individual pig serum haptoglobin (mg ml 1, n = 455)
Farm mean serum haptoglobin (mg ml 1, n = 17)
0.64 0.00 0.04
0.64 0.27 0.43
0.33 0.96
0.51 0.82
8.55
1.26
tion in those pigs showing pathological signs of EP compared to those without pathological signs of this disease (EP positive median 0.43 vs. EP negative median 0.26, p < 0.01). However, there were no significant differences in serum haptoglobin concentration between pigs negative or positive for pathological signs of pleuropneumonia. Histograms showing the population profiles for animals identified as having pathological signs of EP or PL are presented in Figs. 1 and 2, respectively. When considering both types of pneumonia together, analysis showed significant differences for haptoglobin (p < 0.05, see Fig. 3). Nine factors were found to be associated (p < 0.25) with an increase in farm mean haptoglobin concentration (categorical variables are shown in Table 4). There was a high level of correlation between these factors (Table 5). From a relatively large number of input variables only two remained in the final multiple regression model for serum haptoglobin concentration based on the farm mean values. Solid pen divisions in the finisher accommodation and finisher pen were associated with a decrease of 0.44 mg ml 1 mean serum haptoglobin concentration compared with a lack of solid partitions. An increase in finisher pen size of 10 m2 was associated with a decrease of 0.08 mg ml 1 mean serum haptoglobin concentration. Table 6 shows the predictive model for farm mean serum haptoglobin concentration. 4. Discussion and conclusions In addition to being used for monitoring infectious disease progression it has been suggested that acute phase protein measurements can be used for the prognosis and diagnosis of disease and the evaluation of general health status (Heegaard et al., 1998; Eckersall, 2000; Petersen et al., 2002a). In the present study, haptoglobin concentrations were significantly higher (0.26–0.43 mg ml 1, p < 0.01) in pigs that showed pathological signs of enzootic pneumonia compared to those that did not. Although reported normal levels of serum haptoglobin concentrations vary (probably due to the lack of a recognised reference preparation for harmonised calibration of assays) concentrations are generally quoted as being less than 0.5 mg ml 1 (Heegaard et al., 1998). In the animals in this study a number in all groups (EP and PL positive and
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431
45 40
Proportion of population
35 30 25 20 15 10 5
4
10
3.8
3.6
3.4
3
3.2
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 >4
Serum Haptoglobin concentration (mg/ml) EP free (341 pigs)
EP positive (147 pigs)
Fig. 1. Distribution of serum haptoglobin concentration for pigs with or without pathological signs of enzootic pneumonia (EP).
60
Proportion of population
50
40
30
20
10
10
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0
0.2
0 >4
Serum Haptoglobin concentration (mg/ml) PL free (455 pigs)
PL positive (33 pigs)
Fig. 2. Distribution of serum haptoglobin concentration for pigs with or without pathological signs of pleuropneumonia (PL).
negative) had haptoglobin concentrations greater than 3 mg ml 1 and without the possibility of identifying the cause of these elevated levels these outliers were retained in the analysis. In a study of a conventional herd, (Eurell et al., 1992) found levels varying between 0.1 and 0.6 mg ml 1compared to the range of 0–8.55 mg ml 1 found in the current study, however the 3rd quartile of 0.96 mg ml 1 is a more equivalent comparator. Others have reported normal levels as low as 0.06 mg ml 1, a mean of 0.19 mg ml 1 in a herd chronically infected with pleuropneumonia and a mean of 0.24 mg ml 1 in a herd undergoing acute Actinobacillus infection (Hall et al., 1992). The
higher mean levels found in the current study may be due to the standard used for calibration (bovine haptoglobin) instead of the human haptoglobin used by Hall et al. (1992) as previously suggested by Heegaard et al. (1998). Respiratory lesions in pigs at slaughter are common in the UK. Done (1999) reported that the prevalence of enzootic pneumonia in the UK can be as high as 90%, although the average remains about 50% (Done, 1999). This average figure is higher than the 29% prevalence of enzootic pneumonia found in the current study, which was carried out during the months of April and May and the prevalence of pneumonia is known to be reduced during the summer
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J.R. Amory et al. / Research in Veterinary Science 83 (2007) 428–435 Table 4 Farm factors associated with differences in serum haptoglobin concentration Farm factor
Fig. 3. Serum haptoglobin concentrations of pigs identified as negative for EP and PL, having either just EP or just PL or both conditions.
months (Stark et al., 1998). Prevalence figures are similar for other pig producing countries with climates similar to the UK (52% in New Zealand (Stark et al., 1998); 84% in Sweden (Wallgren et al., 1994a); 70% in Norway (Lium and Falk, 1991); 75% in Ont., Canada (Wilson et al., 1986)). There is little information as to actual prevalence of Actinobacillus pleuropneumoniae infection in the UK, more often the presence of pleuritis (the formation of pleural membranes) is recorded and has been reported as being less than 20% in the UK (Done, 1999). This is similar to the seven per cent prevalence for pigs showing pleuritis in the current study, pleuritis also being expected to be reduced in the summer months (Stark et al., 1998). Prevalence figures are rare for other countries for actual pleuropneumonia (2.7% in New Zealand (Stark et al., 1998)) but are more common for pleuritis, which was measured in the current study (19.1% in New Zealand (Stark et al., 1998); 6% in Sweden (Wallgren et al., 1994a); 29% in Norway (Lium and Falk, 1991); 11% in Ont., Canada (Wilson et al., 1986)). It is recommended that to develop this research, future studies would utilise histological and serological techniques to confirm the causes of infection.
No. farms
Mean
Pooled standard deviation
pValue
Solid partitions between finisher pens Yes 8 0.45 No 9 0.80
0.250
0.012
Fan-assisted ventilation for finishers Yes 5 0.90 No 12 0.53
0.253
0.014
Solid partitions between grower pens Yes 10 0.54 No 7 0.78
0.286
0.115
Fan-assisted ventilation for growers Yes 4 0.87 No 13 0.57
0.279
0.076
Finishers kept on an all-in all-out policy Yes 7 0.51 No 10 0.73
0.290
0.146
Finishers treated by injection for respectively disease Yes 1 1.12 0.284 No 16 0.61 Growers’ effluent Removed daily Removed less than daily
6 11
Coughing observed in finishers Yes 12 No 5
0.103
0.46 0.74
0.278
0.070
0.70 0.50
0.296
0.228
Pigs > 12 weeks old in room with pigs 5+ weeks younger Yes 3 0.85 0.294 No 14 0.59
0.195
Type of unit Breeder-finisher Finishers only
0.242
8 9
0.73 0.56
0.297
This is the first report finding a relationship between pathological lesions indicative of M. hyopneumonia infection and serum haptoglobin concentrations. This is consistent with report of Franek and Bilkei (2004), where serum haptoglobin concentration was highly correlated to serum antibody titres to Mycoplasma hyopneumonia in outdoor
Table 5 Correlations between dependent variables related to farm mean haptoglobin concentration Dependent variables
1
1 Solid partitions between finisher pens 2 Fan-assisted ventilation for finishers 3 Solid partitions between grower pens 4 Fan-assisted ventilation for growers 5 Finishers kept on an all-in all-out policy 6 Finishers treated by injection for respectively disease 7 Growers’ effluent removed daily 8 Coughing observed in finishers 9 Pigs >12 weeks old in room with pigs 5+ weeks younger 10 Breeder/finisher unit (vs. finisher only)
–
2
3
4
5
6
7
8
9
– 0.55
– 0.86
0.41
– 0.46
– 0.45
0.54 0.43
– 0.63
– – 0.45
(N.B., Pearson’s coefficient ‘r’ for p = 0.1 and p = 0.05 are 0.40 and 0.47, respectively. For clarity only relationships of p < 0.1 are shown).
– 0.49
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Table 6 The multiple regression model for the serum haptoglobin concentration for pigs at slaughter Dependent variable
Fitted terms in regression model
Haptoglobin (mg ml 1)
Constant +Finisher pens separated by solid partitions +10 m2 increase in finisher pen size
Regression coefficient 1.06 0.44** 0.08*
Standard error 0.11 0.03
Lower confidence interval (95%) 0.66 1.34
Upper confidence interval (95%) 0.22 0.18
Model r2 = 0.58. * and ** indicate p < 0.05 and p < 0.01, respectively.
reared pigs. Also infection with Mycoplasma hyorhinis has been shown to be associated with an increase in serum haptoglobin concentration in pigs (Magnusson et al., 1999). The degree of pathological lesions was not significantly associated with differences in serum haptoglobin in the current study. This may be due to the fact that these lesions are likely to be residual from a previous infection rather than reflecting a current acute condition although elevated haptoglobin does persist for a number of weeks post-infection (Hulten et al., 2003). Other work in cattle (Horadagoda et al., 1999) has shown that in cases where inflammation had been identified, the acute phase proteins serum amyloid A and haptoglobin were associated with acute rather than chronic infection. In addition, it is of note that the highest recorded values for haptoglobin were not associated with either type of pneumonia. The results are also consistent with the report of (Chen et al., 2003) who found moderate but significantly increased haptoglobin concentration in normal pigs with lesions at post-mortem compared to those without lesions, though when subdivided by disease the differences were not significant. It is probable that disease conditions other than those indicated by the respiratory lesions examined in this study were responsible for raised levels of the acute phase protein in these pigs. Apart from infection with A. pleuropneumoniae (Hall et al., 1992) and pathological signs of enzootic pneumonia (findings of the current study), other infections reported to increase serum haptoglobin concentration in the pig include Bordetella bronchiseptica and Pasteurella multocida (Van Miert, 1996), M. hyorhinis (Magnusson et al., 1999), Toxoplasma gondii (Jungerson et al., 1999) Lawsonia intracellularis (Geers et al., 2003) and PMWS (Segales et al., 2004). Although it is possible that some of the pigs may have been suffering from these conditions it is more likely that there are diseases other than those already reported, such as septicaemia arising from severe tail biting, that would cause the high levels of serum haptoglobin concentration (over 8.5 mg ml 1) observed in this study. Clinical signs of disease in finishing pigs including lameness, respiratory disease, tail or ear bites, diarrhoea and increased rectal temperatures have all been associated with increased serum haptoglobin concentration (Petersen et al., 2002a,b) but were not investigated in detail in the present study. This suggests that haptoglobin should be regarded more of a general rather than a specific disease health marker before further clinical or pathological
inspection is carried out on any given herd as suggested by Gymnich and Petersen (2004). The presence of free haemoglobin in a sample, for example through haemolysis, may result in a false negative result in the estimation of serum haptoglobin concentration (Eckersall et al., 1996). It is of note that very little haemolysis was detected in any of the samples collected in the current study, following centrifugation. This shows that there was very little damage to the red blood cells within the sample. This may suggest that a suitable point for the collection of blood to be used in this particular assay is at the point of exsanguination during the slaughter process. Haptoglobin can also be measured in meat juice, which could make its application more practical (Hiss et al., 2003). Two farm level environmental factors were shown to be significantly associated with lower levels of serum Haptoglobin in this study. The use of solid partitions between individual pens of finisher pigs was associated with reduced mean serum haptoglobin concentration compared with pigs that were allowed physical contact between pens. It has been demonstrated that solid pen walls reduce the risk of infection between pens (Morris et al., 1995) and that they are associated with improved respiratory health at the farm level (Flesja et al., 1982; Hurnik et al., 1994). The current findings reflect the reduced risk of infection of diseases transmissible through physical contact that would result in an acute phase response, although this term showed some colinearity with an all-in all-out policy and may point towards the importance of general hygiene. An increase in finisher pen size was also associated with a reduction in mean serum haptoglobin concentration. The importance of adequate pen size on a pig’s health and welfare is well known (Edwards et al., 1988). A reduced pen area to below 0.7 m2 per pig has been reported to greatly increase the risk of respiratory disease (Lindquist, 1974), although pen size and not space allocation was the significant term in this model. As the minimum pen size in this study was 13.3 m2 and the maximum 100.0 m2 this translates, according to the regression equation presented in Table 6, to a significant difference of 0.51 mg ml 1 serum haptoglobin concentration. Although not a large difference, this level of increase is now considered to be a positive acute phase response (Heegaard et al., 1998). There are a number of mechanisms by which increased space might reduce the acute phase response. The increased space and corresponding air volume will reduce the concentra-
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tion of infectious agents found as airborne or non-airborne particles (Stark, 2000). The associated reduction in pig contact may also reduce cross-infection and possible stress due to social interaction. Stress resulting in the production of cortisol that has been demonstrated to influence the ability of a pig to cope with pathogenic infection (Johnson et al., 1994; Wallgren et al., 1994b) affecting the risk of an acute phase response occurring and has also been shown to be correlated to the haptoglobin response to exposure to respiratory pathogens such as M. hyopneumoniae (Franek and Bilkei, 2004). Notable were the absence of other factors, such as medication and vaccination against respiratory disease. However, the records of this were variable and may have reflected actual practice meaning that variation was too high to find a significant effect. In conclusion, this study has demonstrated that the presence of pathological signs of enzootic pneumonia was associated with variation in levels of serum haptoglobin concentration measured at slaughter, which in turn was influenced by components of the farm environment. Although there has not been a previous study assessing the levels of acute phase proteins in commercial pig herds in the UK, the levels measured in the present study are consistent with those previously reported in controlled trials with A. pleuropneumoniae infection in this species (Heegaard et al., 1998). It is suggested that although haptoglobin is a useful indicator of enzootic pneumonia (infection with M. hyopneumoniae), the level of variation, presumably from other inflammatory conditions, suggests that this acute phase protein may be more useful as a general indicator of pig health status rather than specific respiratory infection. This is in agreement with the findings of other authors (Petersen et al., 2002b). References Chen, H.H., Lin, J.H., Fung, H.P., Ho, L.L., Yang, P.C., Lee, W.C., Lee, Y.P., Chu, R.M., 2003. Serum acute phase proteins and swine health status. Can. J. Vet. Res. 67, 283–290. Colditz, I.G., 2002. Effects of the immune system on metabolism: implications for production and disease resistance in livestock. Livestock Product. Sci. 75, 257–268. Done, S.H., 1999. Respiratory infections. Pig Int. 29 (5), 35. Eckersall, P.D., 2000. Acute phase proteins as markers of infection and inflammation: monitoring animal health, animal welfare and food safety. Irish Vet. J. 53, 307–311. 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. Vet. Immunol. Immunopathol. 51, 377–385. Edwards, S.A., Armsby, A.W., Large, J.W., 1988. Effects of floor area allowance on performance of growing pigs kept on fully slatted floors. Animal Product. 46, 453–459. Eurell, T.E., Bane, D.P., Hall, W.F., Schaeffer, D.J., 1992. Serum haptoglobin concentration as an indicator of weight gain in pigs. Can. J. Vet. Res. 56, 6–9. Flesja, K.I., Forus, I.B., Solberg, I., 1982. Pathological lesions in swine at slaughter. V. Pathological lesions in relation to some environmental factors in the herds. Acta Vet. Scand. 23, 169–183. Francisco, C.J., Bane, D.P., Unverzagt, J., 1996a. The effects of enrofloxacin and tiamulin on serum haptoglobin and a-1-acid glyco-
protein concentrations in modified medicated-early-weaned pigs. Swine Health Prod. 4, 113–117. Francisco, C.J., Bane, D.P., Wiegel, R.M., Unverzagt, J., 1996b. The influence of pen density, weaning age, and feeder space on serum haptoglobin concentration in young growing swine. Swine Health Prod. 4, 67–71. Franek, S.P., Bilkei, G., 2004. Influence of non-confinement rearing under high infectious pressure from Mycoplasma hyopneumoniae: pig performance, acute phase proteins and cortisol assessment. Acta Veterinaria Brno 73, 335–340. Geers, R., Petersen, B., Huysmans, K., Knura-Deszczka, S., De Becker, M., Gymnich, S., Henot, D., Hiss, S., Sauerwein, H., 2003. On-farm monitoring of pig welfare by assessment of housing, management, health records and plasma haptoglobin. Animal Welfare 12, 643–647. Gymnich, S., Petersen, B., 2004. Haptoglobin as a screening parameter in health management systems in piglet rearing. Pig News Info. 15, 111– 118. Hall, W.F., Thomas, E.E., Hansen, R.D., Herr, L.G., 1992. Serum haptoglobin concentration in swine naturally or experimentally infected with Actinobacillus pleuropneumoniae. JAVMA 201, 1730–1733. Heegaard, P.M., Klausen, J., Nielsen, J.P., Gonzalez-Ramon, N., Pineiro, 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. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 119, 365–373. Hiss, S., Knura-Deszcka, S., Regula, G., Hennies, M., Gymnich, S., Petersen, B., Sauerwein, H., 2003. Development of an enzyme immuno assay for the determination of porcine haptoglobin in various body fluids: testing the significance of meat juice measurements for quality monitoring programs. Vet. Immunol. Immunopathol. 96, 73–82. Horadagoda, A., Eckersall, P.D., Hodgson, J.C., Gibbs, H.A., Moon, G.M., 1994. Immediate responses in serum TNF-alpha and acutephase protein concentrations to infection with Pasteurella haemolytica A1 in calves. Res. Vet. Sci. 57, 129–132. Horadagoda, N.U., Knox, K.M.G., Gibbs, H.A., Reid, S.W.J., Horadagoda, A., Edwards, S.E.R., Eckersall, P.D., 1999. Acute phase proteins in cattle: discrimination between acute and chronic inflammation. Vet. Rec. 144, 437–441. Hosmer, D.W., Lemeshow, S., 1989. Model-building strategies and methods for logistic regression. Appl. Logistic Reg.. Wiley, New York, pp. 82–134. Hulten, C., Johansson, E., Fossum, C., Wallgren, P., 2003. Interleukin 6, serum amyloid A and haptoglobin as markers of treatment efficacy in pigs experimentally infected with Actinobacillus pleuropneumoniae. Vet. Microbiol. 95, 75–89. Hurnik, D., Dohoo, I.R., Bate, L.A., 1994. Types of farm management as risk factors for swine respiratory disease. Prev. Vet. Med. 20, 147–157. Johnson, R.W., von Borell, E.H., Anderson, L.L., Kojic, L.D., Cunnick, J.E., 1994. Intracerebroventricular injection of corticotrophin releasing hormone in the pig: acute effects on behaviour, adrenocorticotrophic secretion and immune suppression. Endocrinology 135, 642–648. Jungerson, G., Jensen, L., Riber, U., Heegaard, P.M.H., Petersen, E., Poulsen, J.S.D., Billehansen, V., Lind, P., 1999. Pathogenicity of selected Toxoplasma gondii isolates in young pigs. Int. J. Parasitol. 29, 1307–1319. Lindquist, J.O., 1974. Animal health and environment in the production of fattening pigs. Acta Veter. Scand. 51 (Suppl.), 1–78. Lium, B.M., Falk, K., 1991. An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. Acta Veter. Scand. 32, 55–65. Magnusson, U., Wilkie, B., Artursson, K., Mallard, B., 1999. Interferonalpha and haptoglobin in pigs selectively bred for high and low immune response and infected with Mycoplasma hyorhinis. Vet. Immunol. Immunopathol. 68, 131–137. Millet, S., Cox, E., Buyse, J., Goddeeris, B.M., Janssens, G.P.J., 2005. Immunocompetence of fattening pigs fed organic versus conventional diets in organic versus conventional housing. Vet. J. 169, 293–299.
J.R. Amory et al. / Research in Veterinary Science 83 (2007) 428–435 Morris, C.R., Gardner, I.A., Hietala, S.K., Carpenter, T.E., Anderson, R.J., Parker, K.M., 1995. Seroepidemiologic study of natural transmission of Mycoplasma hyopneumoniae in a swine herd. Prev. Vet. Med. 21, 323–337. Petersen, H.H., Dideriksen, D., Christiansen, B.M., Nielsen, J.P., 2002a. Serum haptoglobin concentration as a marker of clinical signs in finishing pigs. Vet. Rec. 151, 85–89. Petersen, H.H., Ersboll, A.K., Jensen, C.S., Nielsen, J.P., 2002b. Serumhaptoglobin concentration in Danish slaughter pigs of different health status. Prev. Vet. Med. 54, 325–335. Petersen, H.H., Nielsen, J.P., Heegaard, P.M.H., 2004. Application of acute phase protein measurement in veterinary clinical chemistry. Vet. Res. 35, 163–187. Petrie, A., Watson, P., 1999. Statistics for Veterinary and Animal Science. Blackwell Science. Saco, Y., Docampo, M.J., Fa´brega, E., Manteca, X., Diestre, A., Lampreave, F., Bassols, A., 2003. Effect of transport stress on serum haptoglobin and pig-MAP in pigs. Animal Welfare 12, 403–409. Saini, P.K., Webert, D.W., 1991. Application of acute phase reactants during antemortem and postmortem meat inspection. J. Am. Vet. Assoc. 198, 1898–1901. Segales, J., Pineiro, C., Lampreave, F., Nofrarias, M., Mateu, E., Calsamiglia, M., Andres, M., Morales, J., Pineiro, M., Domingo, M., 2004. Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS). Vet. Res. 35, 275–282.
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Stark, K.D.C., 2000. Epidemiological investigation of the influence of environmental risk factors on respiratory diseases in swine – a literature review. Vet. J. 159, 37–56. Stark, K.D.C., Pfeiffer, D.U., Morris, R.S., 1998. Risk factors for respiratory diseases in New Zealand pig herds. New Zeal. Vet. J. 46, 3– 10. Straw, B.E., Backstrom, L., Leman, A.D., 1986a. Examination of swine at slaughter. Part II. Findings at slaughter and their significance. Comp. Cont. Edu. Pract. Veter. 8, S106–S112. Straw, B.E., Backstrom, L., Leman, A.D., 1986b. Evaluation of swine at slaughter. Part I. The mechanics of examination, and epidemiologic considerations. Comp. Cont. Edu. Pract. Veter. 8, S41–S48. Van Miert, A.S., 1996. Serum haptoglobin concentration in growing swine after intrenasal challenge with Bordetella bronchiseptica and toxigenic Pasteurella multocida type D. Can. J. Vet. Res. 60, 222–227. Wallgren, P., Beskow, P., Fellstrom, C., Renstrom, L.H.M., 1994a. Porcine lung lesions at slaughter and their correlation to the incidence of infections by Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae during the rearing period. J. Vet. Med. B 41, 441–452. Wallgren, P., Wilen, I., Fossum, C., 1994b. Influence of experimentally induced endogenous production of cortisol on the immune capacity in swine. Vet. Immunol. Immunopathol. 42, 301–316. Wilson, M.R., Takov, R., Friendship, R.M., Martin, S.W., McMillan, I., Hacker, R.R., Swaminathan, S., 1986. Prevalence of respiratory diseases and their association with growth rate and space in randomly selected swine herds. Can. J. Vet. Res. 50, 209–216.
Influence of rearing conditions and respiratory disease on haptoglobin levels in the pig at slaughter J.R. Amory b
a,*
, A.M. Mackenzie a, P.D. Eckersall b, M.J. Stear b, G.P. Pearce
a,1
a Animal Science Research Centre, Harper Adams University College, Newport, Shropshire TF10 8NB, UK Division of Animal Production and Public Health, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK
Accepted 30 January 2007
Abstract Associations between serum concentrations of haptoglobin, pathological lung lesions indicative of Mycoplasma hyopneumoniae (EP) or Actinobacillus pleuropneumoniae (PL) infection at slaughter and previous rearing environment were investigated in 510 pigs (90–100 kg live weight) from 17 farms in England. Haptoglobin concentrations were significantly higher in pigs showing pathological signs of EP infection compared to those without signs of this disease (EP positive median 0.43 mg ml 1 vs. EP negative median 0.26 mg ml 1, p < 0.01). However, there were no significant associations between serum haptoglobin concentrations and pathological signs of PL. The presence of solid partitions compared with barred or similar open partitions was associated with a decrease of 0.44 mg ml 1 farm mean haptoglobin concentration, whilst an increase in pen size of 10 m2 was associated with a decrease of 0.08 mg ml 1 farm mean haptoglobin concentration. The findings indicate that pathological signs of EP were associated with increased serum haptoglobin at slaughter, which in turn was influenced by components of the farm environment. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Pigs; Respiratory disease; Haptoglobin; Environment
1. Introduction The response of a pig to its environment may be monitored by using physiological markers, or ‘‘biomarkers’’ (Eckersall, 2000). Acute phase proteins (APPs) represent an important group of biomarkers with potential use as indicators of tissue injury associated with changes in health status in commercially reared pigs (Gymnich and Petersen, 2004). APPs are produced by the liver in response to cytokines associated with inflammation, infection or tissue injury and function to restore homeostasis in the body following injury or infection (Eckersall, 2000; Colditz, 2002; * Corresponding author. Present Address: Department of Science, Agriculture and Technology, Writtle College, Chelmsford, Essex CM1
E-mail address: [email protected] (J.R. Amory). Present address: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. 1
0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.01.012
Petersen et al., 2004) and the acute phase protein Haptoglobin, has been identified as a sensitive indicator of respiratory infection and its concentration in blood serum has been suggested as a means of non-specific surveillance of pig health status (Heegaard et al., 1998). There is an increasing number of scientific publications describing the acute phase response in the pig and the effects of infection (Heegaard et al., 1998), inflammatory response to an injection of turpentine (Eckersall et al., 1996), specific husbandry practices (Francisco et al., 1996a,b), transport (Saco et al., 2003), clinical signs of disease (Petersen et al., 2002a), histopathological signs of disease (Chen et al., 2003), and presence of clinical diseases such as Postweaning Multisystemic Wasting Syndrome (Segales et al., 2004) and Porcine Intestinal Adenomatosis (Geers et al., 2003). Haptoglobin levels have been reported to be higher in pigs from organic compared with non-organic pig herds (Millet et al., 2005), in indoor compared with outdoor herds (Franek and Bilkei,
J.R. Amory et al. / Research in Veterinary Science 83 (2007) 428–435
2004) and in herds with poor levels of hygiene (Gymnich and Petersen, 2004). However, there is little information describing the effects of specific aspects of the rearing environment on mediating the variability of acute phase proteins in the slaughter pig. Knowledge of such variation may be important in identifying factors within husbandry systems associated with increased acute phase response and also in the development of their use in ante- and post-mortem inspection (Saini and Webert, 1991) and production chain orientated health management systems (Gymnich and Petersen, 2004). This study was designed to investigate the relationship between pathological signs of two major respiratory diseases of pigs, enzootic pneumonia and pleuropneumonia, and serum haptoglobin concentrations at slaughter. The study also aimed to identify husbandry and management procedures important in influencing the acute phase response, as determined by levels of serum haptoglobin in slaughtered pigs reared under commercial conditions. 2. Materials and methods Seventeen farms were involved in the study that delivered pigs to one of three participating abattoirs in the midlands region of England. Each farmer was given a questionnaire detailing various aspects of the farm environment, husbandry system and pig health based on a previous study investigating risk factors for respiratory diseases in New Zealand (Stark et al., 1998). For the purpose of this study grower pigs were classified as 20–45 kg and finishers 45 kg – slaughter weight. Blood samples and lungs were collected from 30 randomly selected pigs from a single delivery. Following stunning, blood samples were collected in 30 ml universal tubes from the throat as they were exsanguinated. The collected blood was allowed to clot overnight at 4 °C and serum was then obtained by centrifugation for 15 min at 1000g and stored at 20 °C until subsequent analysis. The serum haptoglobin concentration was determined according to the haemoglobin binding activity method of Eckersall (2000) that allows measurement of haptoglobin from all species. A bovine standard of known haptoglobin concentration was used as standard (Horadagoda et al., 1994). Each farm was assessed for their pneumonia status by calculating the prevalence of affected lungs at slaughter and their degree of consolidation. In order to determine tissue damage due to enzootic pneumonia (EP score) the degree of consolidation was assessed by estimating the percentage of the surface of each lobe of the lung showing signs of consolidation. This percentage was multiplied by a weighting factor for each lobe based on their relative volumes (0.25 for the caudal lobes, 0.1 for the cranial and accessory lobes) and totalled to give a score out of 100 for each pig based on the methodology of Straw et al. (1986a). In order to determine tissue damage due to pleuropneumonia (PL score) the percentage of each lobe estimated to be missing due to adhesion of pleural
429
membranes was scored pneumonias a measure of grade 2 pleurisy based on the methodology of Straw et al. (1986b). Although pleuritis can be attributed to a number of causal organisms, infection with Actinobacillus pleuropneumoniae is by far the most common in the UK (Done, 1999). Continuous variables were quantified by calculating median, minimum and maximum figures. Differences in serum haptoglobin concentration between pigs identified as having or not having pathological signs of enzootic pneumonia were calculated using the Kruskal–Wallis test (Petrie and Watson, 1999, p. 146). This was repeated for those identified as having or not having pathological signs of pleuropneumonia. In addition, both types of pneumonia were considered together by creating four groups one negative for both types, one positive for EP only, one positive for PL only and one positive for both types.. Regression equations were calculated to determine whether either EP or PL score could be predicted by the actual serum acute phase protein concentration in individual pigs. In order to determine which terms were to be considered for the construction of a multiple regression model, univariable analyses of variance were carried out between individual farm factors and the dependent variables. Those terms giving an F-probability p < 0.25 were put forward for the multiple regression model as suggested by Hosmer and Lemeshow (1989). Similarly for the continuous independent variables, those giving correlations of r > 0.23 (equivalent to p < 0.25 with 16 degrees of freedom based on 17 farms) were also put forward for the multiple regression model. This was carried out using forwards and backwards stepwise selection procedures to ascertain the best model. Finally all original terms were added individually to the model to check significance. Descriptive statistics, analysis of variance, Pearson correlation coefficients and the stepwise regression procedure were performed using S-Plus (version 6.2) for Windows statistics package. Non-parametric statistics were performed using Minitab (version 11) for Windows statistics package. 3. Results Of the 17 farms that took part in this study, eight were breeder-finisher units and 9 were finishing-only units, all slaughtering finished pigs between 90 and 100 kg live weight. Nine farms provided bedding for their grower pigs, while eight provided bedding for the finishers. Nine farms used a slatted method of slurry disposal for their growers, while 10 used slatted floors for their finishers. Five farms vaccinated against enzootic pneumonia, one farm vaccinated for pleuropneumonia, four farms injected individual grower pigs for treatment of respiratory disease and one farm injected finisher pigs for treatment of respiratory disease. However, the effects of vaccination and medication status were not investigated in detail in this study. Ten farms gave in-feed/water medication to their growers and five farms gave in-feed/water medication to their finishers.
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Median and range figures for the continuous variables are shown in Table 1. Signs of enzootic pneumonia were the most prevalent lesion type in this study with 147 pigs out of the 510 examined showing these signs (varying from 0% to 26% of total lung volume affected). Only one of the 17 farms did not show gross lesions associated with enzootic pneumonia. Pathological lesions associated with pleuropneumonia were found in 34 of the 510 pigs examined (signs of pleurisy varying from 0% to 48% of total lung volume affected in all pigs). Four out of the 17 farms did not show the lung tissue damage attributed to pleuropneumonia in this study. Distributions of lesion scores are presented in Table 2. Eleven out of the 510 pigs showed signs of both diseases. Mean individual serum haptoglobin was 0.64 mg ml 1 (range 0–8.55 mg ml 1), whilst the farm mean haptoglobin concentration varied from 0.27 to 1.26 mg ml 1 (Table 3). There was a significant increase in haptoglobin concentra-
Table 1 Median, minimum and maximum figures for continuous management variables from the respiratory disease and acute phase protein study Median
Minimum
Maximum
No. of sows on unit
78
491
Distance to nearest pig farm (miles) Weaning age (days) No. of stages after weaning Grower pen size (m2) No. of grower pigs per pen Grower space allowance (m2/ pig) No. of grower pigs per room Finisher pen size (m2) No. of finisher pigs per pen Finisher space allowance (m2/ pig) No. of finisher pigs per room
2
0 (finishers only) 0.25
5
24 2 12.5 25 0.5
21 1 5 14 0.36
28 4 100 200 1.25
140 13.3 20 0.68
20 6.75 10 0.47
290 100 200 0.93
149
100
220
Table 2 Distribution of the severity of lesion scores for enzootic pneumonia and pleuropneumonia Pneumonia scorea (%)
Enzootic pneumonia (% of pigs, n = 510)
Pleuropneumonia (% of pigs, n = 510)
0 >0–1 >1–2 >2–5 >5–10 >10–20 >20–30 >30–40 >40–50 >50
71.2 5.9 7.1 9.0 3.3 2.9 0.6 0 0 0
93.3 0.0 0.8 1.6 1.8 1.8 0.2 0.4 0.2 0
a Score was ascribed by estimating the percentage of the surface of each lobe of the lung showing signs of consolidation multiplied by a weighting factor for each lobe based on their relative volumes (0.25 for the caudal lobes, 0.1 for the cranial and accessory lobes) and totalled to give a score out of 100 for each pig.
Table 3 Descriptive statistics regarding serum haptoglobin
Mean Minimum First quartile Median Third quartile Maximum
Individual pig serum haptoglobin (mg ml 1, n = 455)
Farm mean serum haptoglobin (mg ml 1, n = 17)
0.64 0.00 0.04
0.64 0.27 0.43
0.33 0.96
0.51 0.82
8.55
1.26
tion in those pigs showing pathological signs of EP compared to those without pathological signs of this disease (EP positive median 0.43 vs. EP negative median 0.26, p < 0.01). However, there were no significant differences in serum haptoglobin concentration between pigs negative or positive for pathological signs of pleuropneumonia. Histograms showing the population profiles for animals identified as having pathological signs of EP or PL are presented in Figs. 1 and 2, respectively. When considering both types of pneumonia together, analysis showed significant differences for haptoglobin (p < 0.05, see Fig. 3). Nine factors were found to be associated (p < 0.25) with an increase in farm mean haptoglobin concentration (categorical variables are shown in Table 4). There was a high level of correlation between these factors (Table 5). From a relatively large number of input variables only two remained in the final multiple regression model for serum haptoglobin concentration based on the farm mean values. Solid pen divisions in the finisher accommodation and finisher pen were associated with a decrease of 0.44 mg ml 1 mean serum haptoglobin concentration compared with a lack of solid partitions. An increase in finisher pen size of 10 m2 was associated with a decrease of 0.08 mg ml 1 mean serum haptoglobin concentration. Table 6 shows the predictive model for farm mean serum haptoglobin concentration. 4. Discussion and conclusions In addition to being used for monitoring infectious disease progression it has been suggested that acute phase protein measurements can be used for the prognosis and diagnosis of disease and the evaluation of general health status (Heegaard et al., 1998; Eckersall, 2000; Petersen et al., 2002a). In the present study, haptoglobin concentrations were significantly higher (0.26–0.43 mg ml 1, p < 0.01) in pigs that showed pathological signs of enzootic pneumonia compared to those that did not. Although reported normal levels of serum haptoglobin concentrations vary (probably due to the lack of a recognised reference preparation for harmonised calibration of assays) concentrations are generally quoted as being less than 0.5 mg ml 1 (Heegaard et al., 1998). In the animals in this study a number in all groups (EP and PL positive and
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431
45 40
Proportion of population
35 30 25 20 15 10 5
4
10
3.8
3.6
3.4
3
3.2
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 >4
Serum Haptoglobin concentration (mg/ml) EP free (341 pigs)
EP positive (147 pigs)
Fig. 1. Distribution of serum haptoglobin concentration for pigs with or without pathological signs of enzootic pneumonia (EP).
60
Proportion of population
50
40
30
20
10
10
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0
0.2
0 >4
Serum Haptoglobin concentration (mg/ml) PL free (455 pigs)
PL positive (33 pigs)
Fig. 2. Distribution of serum haptoglobin concentration for pigs with or without pathological signs of pleuropneumonia (PL).
negative) had haptoglobin concentrations greater than 3 mg ml 1 and without the possibility of identifying the cause of these elevated levels these outliers were retained in the analysis. In a study of a conventional herd, (Eurell et al., 1992) found levels varying between 0.1 and 0.6 mg ml 1compared to the range of 0–8.55 mg ml 1 found in the current study, however the 3rd quartile of 0.96 mg ml 1 is a more equivalent comparator. Others have reported normal levels as low as 0.06 mg ml 1, a mean of 0.19 mg ml 1 in a herd chronically infected with pleuropneumonia and a mean of 0.24 mg ml 1 in a herd undergoing acute Actinobacillus infection (Hall et al., 1992). The
higher mean levels found in the current study may be due to the standard used for calibration (bovine haptoglobin) instead of the human haptoglobin used by Hall et al. (1992) as previously suggested by Heegaard et al. (1998). Respiratory lesions in pigs at slaughter are common in the UK. Done (1999) reported that the prevalence of enzootic pneumonia in the UK can be as high as 90%, although the average remains about 50% (Done, 1999). This average figure is higher than the 29% prevalence of enzootic pneumonia found in the current study, which was carried out during the months of April and May and the prevalence of pneumonia is known to be reduced during the summer
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J.R. Amory et al. / Research in Veterinary Science 83 (2007) 428–435 Table 4 Farm factors associated with differences in serum haptoglobin concentration Farm factor
Fig. 3. Serum haptoglobin concentrations of pigs identified as negative for EP and PL, having either just EP or just PL or both conditions.
months (Stark et al., 1998). Prevalence figures are similar for other pig producing countries with climates similar to the UK (52% in New Zealand (Stark et al., 1998); 84% in Sweden (Wallgren et al., 1994a); 70% in Norway (Lium and Falk, 1991); 75% in Ont., Canada (Wilson et al., 1986)). There is little information as to actual prevalence of Actinobacillus pleuropneumoniae infection in the UK, more often the presence of pleuritis (the formation of pleural membranes) is recorded and has been reported as being less than 20% in the UK (Done, 1999). This is similar to the seven per cent prevalence for pigs showing pleuritis in the current study, pleuritis also being expected to be reduced in the summer months (Stark et al., 1998). Prevalence figures are rare for other countries for actual pleuropneumonia (2.7% in New Zealand (Stark et al., 1998)) but are more common for pleuritis, which was measured in the current study (19.1% in New Zealand (Stark et al., 1998); 6% in Sweden (Wallgren et al., 1994a); 29% in Norway (Lium and Falk, 1991); 11% in Ont., Canada (Wilson et al., 1986)). It is recommended that to develop this research, future studies would utilise histological and serological techniques to confirm the causes of infection.
No. farms
Mean
Pooled standard deviation
pValue
Solid partitions between finisher pens Yes 8 0.45 No 9 0.80
0.250
0.012
Fan-assisted ventilation for finishers Yes 5 0.90 No 12 0.53
0.253
0.014
Solid partitions between grower pens Yes 10 0.54 No 7 0.78
0.286
0.115
Fan-assisted ventilation for growers Yes 4 0.87 No 13 0.57
0.279
0.076
Finishers kept on an all-in all-out policy Yes 7 0.51 No 10 0.73
0.290
0.146
Finishers treated by injection for respectively disease Yes 1 1.12 0.284 No 16 0.61 Growers’ effluent Removed daily Removed less than daily
6 11
Coughing observed in finishers Yes 12 No 5
0.103
0.46 0.74
0.278
0.070
0.70 0.50
0.296
0.228
Pigs > 12 weeks old in room with pigs 5+ weeks younger Yes 3 0.85 0.294 No 14 0.59
0.195
Type of unit Breeder-finisher Finishers only
0.242
8 9
0.73 0.56
0.297
This is the first report finding a relationship between pathological lesions indicative of M. hyopneumonia infection and serum haptoglobin concentrations. This is consistent with report of Franek and Bilkei (2004), where serum haptoglobin concentration was highly correlated to serum antibody titres to Mycoplasma hyopneumonia in outdoor
Table 5 Correlations between dependent variables related to farm mean haptoglobin concentration Dependent variables
1
1 Solid partitions between finisher pens 2 Fan-assisted ventilation for finishers 3 Solid partitions between grower pens 4 Fan-assisted ventilation for growers 5 Finishers kept on an all-in all-out policy 6 Finishers treated by injection for respectively disease 7 Growers’ effluent removed daily 8 Coughing observed in finishers 9 Pigs >12 weeks old in room with pigs 5+ weeks younger 10 Breeder/finisher unit (vs. finisher only)
–
2
3
4
5
6
7
8
9
– 0.55
– 0.86
0.41
– 0.46
– 0.45
0.54 0.43
– 0.63
– – 0.45
(N.B., Pearson’s coefficient ‘r’ for p = 0.1 and p = 0.05 are 0.40 and 0.47, respectively. For clarity only relationships of p < 0.1 are shown).
– 0.49
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Table 6 The multiple regression model for the serum haptoglobin concentration for pigs at slaughter Dependent variable
Fitted terms in regression model
Haptoglobin (mg ml 1)
Constant +Finisher pens separated by solid partitions +10 m2 increase in finisher pen size
Regression coefficient 1.06 0.44** 0.08*
Standard error 0.11 0.03
Lower confidence interval (95%) 0.66 1.34
Upper confidence interval (95%) 0.22 0.18
Model r2 = 0.58. * and ** indicate p < 0.05 and p < 0.01, respectively.
reared pigs. Also infection with Mycoplasma hyorhinis has been shown to be associated with an increase in serum haptoglobin concentration in pigs (Magnusson et al., 1999). The degree of pathological lesions was not significantly associated with differences in serum haptoglobin in the current study. This may be due to the fact that these lesions are likely to be residual from a previous infection rather than reflecting a current acute condition although elevated haptoglobin does persist for a number of weeks post-infection (Hulten et al., 2003). Other work in cattle (Horadagoda et al., 1999) has shown that in cases where inflammation had been identified, the acute phase proteins serum amyloid A and haptoglobin were associated with acute rather than chronic infection. In addition, it is of note that the highest recorded values for haptoglobin were not associated with either type of pneumonia. The results are also consistent with the report of (Chen et al., 2003) who found moderate but significantly increased haptoglobin concentration in normal pigs with lesions at post-mortem compared to those without lesions, though when subdivided by disease the differences were not significant. It is probable that disease conditions other than those indicated by the respiratory lesions examined in this study were responsible for raised levels of the acute phase protein in these pigs. Apart from infection with A. pleuropneumoniae (Hall et al., 1992) and pathological signs of enzootic pneumonia (findings of the current study), other infections reported to increase serum haptoglobin concentration in the pig include Bordetella bronchiseptica and Pasteurella multocida (Van Miert, 1996), M. hyorhinis (Magnusson et al., 1999), Toxoplasma gondii (Jungerson et al., 1999) Lawsonia intracellularis (Geers et al., 2003) and PMWS (Segales et al., 2004). Although it is possible that some of the pigs may have been suffering from these conditions it is more likely that there are diseases other than those already reported, such as septicaemia arising from severe tail biting, that would cause the high levels of serum haptoglobin concentration (over 8.5 mg ml 1) observed in this study. Clinical signs of disease in finishing pigs including lameness, respiratory disease, tail or ear bites, diarrhoea and increased rectal temperatures have all been associated with increased serum haptoglobin concentration (Petersen et al., 2002a,b) but were not investigated in detail in the present study. This suggests that haptoglobin should be regarded more of a general rather than a specific disease health marker before further clinical or pathological
inspection is carried out on any given herd as suggested by Gymnich and Petersen (2004). The presence of free haemoglobin in a sample, for example through haemolysis, may result in a false negative result in the estimation of serum haptoglobin concentration (Eckersall et al., 1996). It is of note that very little haemolysis was detected in any of the samples collected in the current study, following centrifugation. This shows that there was very little damage to the red blood cells within the sample. This may suggest that a suitable point for the collection of blood to be used in this particular assay is at the point of exsanguination during the slaughter process. Haptoglobin can also be measured in meat juice, which could make its application more practical (Hiss et al., 2003). Two farm level environmental factors were shown to be significantly associated with lower levels of serum Haptoglobin in this study. The use of solid partitions between individual pens of finisher pigs was associated with reduced mean serum haptoglobin concentration compared with pigs that were allowed physical contact between pens. It has been demonstrated that solid pen walls reduce the risk of infection between pens (Morris et al., 1995) and that they are associated with improved respiratory health at the farm level (Flesja et al., 1982; Hurnik et al., 1994). The current findings reflect the reduced risk of infection of diseases transmissible through physical contact that would result in an acute phase response, although this term showed some colinearity with an all-in all-out policy and may point towards the importance of general hygiene. An increase in finisher pen size was also associated with a reduction in mean serum haptoglobin concentration. The importance of adequate pen size on a pig’s health and welfare is well known (Edwards et al., 1988). A reduced pen area to below 0.7 m2 per pig has been reported to greatly increase the risk of respiratory disease (Lindquist, 1974), although pen size and not space allocation was the significant term in this model. As the minimum pen size in this study was 13.3 m2 and the maximum 100.0 m2 this translates, according to the regression equation presented in Table 6, to a significant difference of 0.51 mg ml 1 serum haptoglobin concentration. Although not a large difference, this level of increase is now considered to be a positive acute phase response (Heegaard et al., 1998). There are a number of mechanisms by which increased space might reduce the acute phase response. The increased space and corresponding air volume will reduce the concentra-
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tion of infectious agents found as airborne or non-airborne particles (Stark, 2000). The associated reduction in pig contact may also reduce cross-infection and possible stress due to social interaction. Stress resulting in the production of cortisol that has been demonstrated to influence the ability of a pig to cope with pathogenic infection (Johnson et al., 1994; Wallgren et al., 1994b) affecting the risk of an acute phase response occurring and has also been shown to be correlated to the haptoglobin response to exposure to respiratory pathogens such as M. hyopneumoniae (Franek and Bilkei, 2004). Notable were the absence of other factors, such as medication and vaccination against respiratory disease. However, the records of this were variable and may have reflected actual practice meaning that variation was too high to find a significant effect. In conclusion, this study has demonstrated that the presence of pathological signs of enzootic pneumonia was associated with variation in levels of serum haptoglobin concentration measured at slaughter, which in turn was influenced by components of the farm environment. Although there has not been a previous study assessing the levels of acute phase proteins in commercial pig herds in the UK, the levels measured in the present study are consistent with those previously reported in controlled trials with A. pleuropneumoniae infection in this species (Heegaard et al., 1998). It is suggested that although haptoglobin is a useful indicator of enzootic pneumonia (infection with M. hyopneumoniae), the level of variation, presumably from other inflammatory conditions, suggests that this acute phase protein may be more useful as a general indicator of pig health status rather than specific respiratory infection. This is in agreement with the findings of other authors (Petersen et al., 2002b). References Chen, H.H., Lin, J.H., Fung, H.P., Ho, L.L., Yang, P.C., Lee, W.C., Lee, Y.P., Chu, R.M., 2003. Serum acute phase proteins and swine health status. Can. J. Vet. Res. 67, 283–290. Colditz, I.G., 2002. Effects of the immune system on metabolism: implications for production and disease resistance in livestock. Livestock Product. Sci. 75, 257–268. Done, S.H., 1999. Respiratory infections. Pig Int. 29 (5), 35. Eckersall, P.D., 2000. Acute phase proteins as markers of infection and inflammation: monitoring animal health, animal welfare and food safety. Irish Vet. J. 53, 307–311. 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. Vet. Immunol. Immunopathol. 51, 377–385. Edwards, S.A., Armsby, A.W., Large, J.W., 1988. Effects of floor area allowance on performance of growing pigs kept on fully slatted floors. Animal Product. 46, 453–459. Eurell, T.E., Bane, D.P., Hall, W.F., Schaeffer, D.J., 1992. Serum haptoglobin concentration as an indicator of weight gain in pigs. Can. J. Vet. Res. 56, 6–9. Flesja, K.I., Forus, I.B., Solberg, I., 1982. Pathological lesions in swine at slaughter. V. Pathological lesions in relation to some environmental factors in the herds. Acta Vet. Scand. 23, 169–183. Francisco, C.J., Bane, D.P., Unverzagt, J., 1996a. The effects of enrofloxacin and tiamulin on serum haptoglobin and a-1-acid glyco-
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