Skip to main content
European Commission logo print header

New markers of immunological stress and welfare in animals: porcine acute phase proteins in the production of healthy pigs

Deliverables

In a previous study investigating the pig APP response in a model of inflammation induced by turpentine injection, a protein with an alpha- electrophoretic mobility was detected as a major negative APP in pigs and was preliminary characterised as alpha-lipoprotein (Lampreave et al, 1994). In this work, the major protein component of alpha-lipoprotein or high density lipoprotein (HDL) has been purified from normal pig sera by sequential ultracentrifugation (density range 1.080-1.160g/mL) followed by size exclusion chromatography on Sephadex G-150. Sequence analysis of the amino terminal part of the protein allowed its identification as apolipoprotein A-I (apo A-I). The isolated protein, was used for the immunization of rabbits, as well as for the primary standard in the immunological determinations. The antisera obtained, once adsorbed, showed by Western blotting a unique band specific for apo A-I, and it was subsequently used for the determination of apo A-I in pig serum by radial immunodiffusion. This assay has been used in other workpackages of the shared cost project to study the acute phase response of apo A-I compared to a number of positive and negative acute phase protein (in the sera of animals undergoing different experimental animal models and in animals from farms. Decreases in the concentration of apo A-I of around 50% were obtained after experimental inflammation. A higher response was observed in a model of bacterial infection (Actinobacillus pleuropneumoniae), where apo A-I showed minimum values, 2 to 5 times lower than the initial values, 2-4 days after initiation of the infection. The magnitude of the apo A-I response correlated with the magnitude of the response of the positive APP studied. It is concluded that apo A-I is a new negative acute phase protein in pigs. Further studies performed elsewhere in the project seem to confirm the reliability of Apo A-I as a parameter of health status or pig welfare. The results of these studies are described under individual result headings. The development of an ELISA for Apo A-I is a prioritary objective of our future research. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
An increase in haptoglobin and CRP serum concentrations were observed following a relatively short transport (1.3 h) and lairage of pigs. Whether this was due to sub-clinical tissue damage or haptoglobin and CRP production without tissue damage due to transport stress or other causes is unknown. Chronic lesions, which are commonly observed at slaughter without consequences for meat inspection, did not increase haptoglobin serum concentration significantly in the present investigation. This may be due to the small sample size. The study showed that blood samples for ante-mortem control in pig herds should preferentially be obtained before handling and transport to abattoir. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
A cross-sectional study was performed in 98 commercial finisher pig herds. Clinical signs were recorded for up to 1000 pigs in each herd and haptoglobin concentration in serum was determined in 30 pigs per herd. Lame pigs and pigs from herds with high prevalences of clinical signs of respiratory disease and diarrhea had high serum haptoglobin concentration. Increasing levels of antibodies against A. pleuropneumoniae serotype 2 and M. hyopneumoniae were associated with increasing serum haptoglobin concentration. Finishing pigs between 25 and 50 kg had high haptoglobin concentrations. Regional differences in serum haptoglobin concentration possibly influenced by different observers were found. Herds with up to 12 hours quarantine for visitors and with high stocking density had high haptoglobin concentrations. Herds with continuous production had higher haptoglobin concentrations than herds with batch production without all-in/all-out. Serum haptoglobin concentration was found to be a promising indicator of clinical and subclinical disease in finishing pigs. However, before the use in herd health programmes, further studies are needed. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
Stress represents the reaction of the body to stimuli that disturb its normal physiological equilibrium. As a consequence of these alterations to the homeostasis, a set of physiological changes that are part of the complex innate defence mechanism known as acute phase response are induced. In the productive systems, animals are exposed to multiple stressors that can affect their normal behaviour and growth, leading to production losses. In this trial the effect of a changeable pattern of food administration in the growth performance and APP (acute phase proteins) levels of growing pigs was evaluated. 240 pigs (LW x LR), of 74 days of life, from the same origin, with and initial weight of 26.3 + 0.39Kg, half entire males and half females, were used in the study. The experimental treatments consisted of pigs fed ad libitum (AL) or disorderly (DIS). Disorderly feeding consisted in an administration of food in a disorderly pattern, alternating in a day periods of ad libitum administration with periods of no feeding. The alteration in the feed pattern resulted in a lost of weight gain in males, whereas the growth of females was not significantly different between treatments. The behaviour of the APP was in good agreement with the growth performance parameters determined. Elevated levels of the positive APP Pig-MAP, Haptoglobin, SAA and CRP, and decreases in the concentration of the negative APP ApoAI, were observed in DIS males when compared with controls (AL), whereas females showed similar concentration in both treatments. The results of this trial confirms a good correlation between elevated APP levels and decreased performance, as well as the potential of this markers in the detection of situations causing stress for the animal. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
Thanks to the results of the studies conducted under the project, suggestions for proper sampling and better understanding of APP´s (acute phase proteins) on pig production could be made: Do's and don'ts when sampling for APP´s: Things you should do: - Record carefully the age, sex and physiological status. Important differences due to these factors can be found. - Take a big enough number of samples for every factor you want to study. Depending on the method you use, not less than 10 samples should be taken. - Comparisons with the typical pattern for your situation should be done. You will know what is usual or more commonly found. - Take your samples randomly using animals of different pens. - Understand variability. Use average with standard deviation or standard error always. Same average can mean very different things. - Use the APP´s in combination with serum profile against infection diseases. This can provide a better understanding of the situation. - Check suspected as critical or hazardous points in your production system. Subclinical situations with low performance can be detected more easily. Once you have taken measures to correct it, check again the levels to confirm the efficancy. Things you should not do: - Do not forget that the acute phase reaction has a biological cost which means not only higher mortality but losses of performance are also very frequent and are well described. - Do not try to understand everything from a single analysis of APP´s. Very often you are going to need other tools. - Do not take samples up to 10 days after a vaccination. Vaccination leeds to increased APP-levels. - Do not forget your other tools in the analysis for the situation. Clinical symptoms, necropsies, serum profiles or others will lead you to a better understanding of the situation. - Do not take samples on runts or chronically sick animals. Your results could be biased. - Do not forget to ask your vet or consultant. Only he can provide a good understanding and the right approach. Furthermore a recommendation for sampling ages respectively weights were performed. Suggested sampling ages respectively weights when sampling pigs for APP´s: - Age (days) 28 60 80 116; - Weight (kg) 7 20 35 60 Age. - Age (days) Slaughter (-3); Weight (kg) 100 +. Finally a test strategy for APP Screening was developed. In this model, APP concentrations are measured at the time of each inspection. Preconditions for the blood analysis, known from literature, were defined. If the pigs were not weaned or if the last vaccination was done within the last 10 days, then the APP levels could be influenced by these factors and blood testing should not be done. If the APP concentrations are below the physiological norm then the group can be assessed as a group without deviation. But if the APP levels were above the norm then the pigs will be ranked as a group with deviations, and adverse effects on the production will be expected. Hence, a weak-point analysis should be done, as well as testing for infectious diseases like porcine reproductive and respiratory syndrome virus infection etc. If there are no disease indications the weak-point analysis should be continued. Any disease indications should be investigated and their successful control monitored. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
The aim of the present work was to examine the influence of different rearing systems on the practicality of including acute phase proteins (APP) as screening. Five variants of customer-supplier contacts were tested consisting 20 piglet rearing farms involving the collection of about 1100 blood samples. In addition, seven piglet breeding farms and one fattener farm were available. Out of these farms five piglet breeding farms, 12 piglet rearing farms and one fattener farm were selected with a total of 680 blood samples. The selection was based on the amount of serum we got from the farmers vets respectively. A significant influence of the origin could be observed for Haptoglobin (Hp), Pig Major Protein (pigMap), C - reactive protein (CRP) and Alpha Lipoprotein (ApoA1). There was a strong significant relationship between the hygiene statuses of the breeding farm and the four acute phase proteins. Furthermore, pigs causing cost of medical treatment above 1,15 Euros in the rearing period had significant higher Hp, pigMap and CRP serum concentration respectively lower ApoA1 concentrations. Concerning the daily weight gain lower haptoglobin and pigMap serum concentration at final inspection in the rearing phase could be observed in the group with the higher daily weight gain. Nevertheless, the correlation was not significant. In contrast to these results we have to see the results for CRP and ApoA1. These two parameters behaved vice versa. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
A case-control study including 340 finishing pigs in 15 commercial Danish pig herds was carried out in order to study haptoglobin concentration in serum as an objective marker of different clinical signs. Rectal temperature and haptoglobin concentration in serum was compared as markers of clinical disease. Finishing pigs aged 10 to 25 weeks with different clinical signs were matched to control pigs without clinical signs with respect to herd, pen, estimated weight and gender. Each pig was subjected to a standard clinical examination and a serum sample was obtained. In 86 of the case-control pairs, the rectal temperature was also recorded. A substantial and significantly elevated mean haptoglobin concentration in serum was found in pigs with lameness (p < 0.0001), respiratory disease (p = 0.0002), tail or ear bite (p < 0.0001) and diarrhea (p = 0.02). Similarly, a higher mean rectal temperature was found in pigs with lameness (p < 0.0001), respiratory disease (p = 0.002) and tail or ear bite (p = 0.0003) when compared to the controls. A significant but low correlation between rectal temperature and haptoglobin concentration in serum was observed (p = 0.003, r = 0.20). Maximum simultaneously sensitivity (0.61 - 0.71) and specificity (0.61 - 0.77) of serum haptoglobin for the different clinical signs was found at a cut-off value of 1.1mg/mL. When using a cut-off value of 1.8mg/mL, the sensitivity decreased (0.31 - 0.60) and the specificity increased (0.82 - 0.86). The area under the ROC-curve was found to be 0.67 - 0.78 for the different clinical signs. Defining a cut-off value which classified individual pigs according to clinical signs was not possible. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
A cross-sectional study was conducted with 617 finishing pigs aged 10 to 25 weeks in 11 commercial herds of different health status as defined by the Danish monitoring program for specific pathogen free (SPF) herds. A standard clinical examination was performed and a blood sample was obtained from each pig for determination of haptoglobin concentration in serum. A significant difference in haptoglobin concentration between herds was observed. This difference was not only related to the health status declarations. Likewise, SPF-status combined with age was found to influence the haptoglobin concentration. Pigs aged 10 to 14, 15 to 19 and 20 to 25 weeks in conventional herds had a significantly higher haptoglobin concentration compared to SPF-x pigs of the same age (P = 0.01, <0.001 and <0.001, respectively). No difference between SPF-x pigs of different age was observed. Conventional pigs aged 15 to 19 and 20 to 25 weeks were found to have a higher haptoglobin concentration than conventional pigs aged 10 to 14 weeks (P = 0.005 and 0.01, respectively). Lame pigs and pigs with tail or ear bite were found to have an elevated haptoglobin concentration in serum (P < 0.001). No significant effect of respiratory symptoms or umbilical hernia was found. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
A sandwich type ELISA for the quantification of Pig-MAP has been developed and validated. The assay is based in two monoclonal antibodies, specific for pig-MAP and showing no crossreactivity with other plasma proteins when tested by western-blot. To increase the probability of obtaining antibodies adequate for sandwich ELISA, intact protein and a fragment of 30K corresponding to the carboxy-terminal end where used during the selection of hybridomas secreting antibodies that reacted with Pig-MAP. This 30K fragment belongs to the region of Pig-MAP showing less homology with the ITI family, and probably containing the most inmunogenic part of the protein. The performance of the ELISA was good, the assay kept linearity under dilution, and precision was adequate, with coefficients of variation lowest of 8% for both, inter and intra assay. The test was validated by comparing the pig-MAP concentration values obtained with this technique with values determined by radial immunodiffusion, the technique routinely used in our laboratory for the quantification of Pig-MAP. Although radial immunodiffusion is a very precise technique, it has the inconvenience of being time- and reaction consuming method, not adequate for automatisation. The concentration of Pig-MAP was measured with both techniques in 80 serum samples with Pig-MAP values ranging from 0.33 to 4.5mg/mL. A high degree of correlation between the two immunochemical methods was found (R=0,978, b=1,02), confirming the accuracy of the assay. The assay described here appears as an adequate alternative to radial immunodifussion for the quantification of Pig-MAP, and inceases the set of specific assays availables for pig APP (acute phase proteins). Further studies have been performed elsewhere in the project to confirm the reliability of Pig-MAP as a parameter of health status or pig welfare. The results of these studies are described under individual result headings. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
In the context of a short project, the acute phase protein (APP) haptoglobin was used as a screeningparameter in five fattening farms. The aim of the present work was to examine if the parameter haptoglobin could be used for punctual investigation in the enterprises instead of a course analyses. It turned out that this method of blood sampling of pig groups of different ages on one day showed that the parameter haptoglobin is suitable not only for a course analyses, but also for a punctual investigation in the enterprises. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
Mixing of animals from different origin is a well-known stressful situation for pigs. There are different moments in the productive life of pigs in which mixing is unavoidable, as after weaning or at the entry to the fattening barn. After mixing, pigs must establish again the dominance hierarchy’s relationships through aggressive interactions. Previous works have shown that those initial aggressions, and probably stress, that follow mixing can reduce growth rate. In this trial the effect of mixing animals from different pens than in nursery, at the entry to the fattening barn, on the levels of APP was studied. 240 pigs (Large White x Landrace-Large White), half entire males and half females, of 60 d of age, with and initial body weight of 20.2 + 0.9 Kg were randomly distributed in 24 pens. The experimental design consisted in a 2X2 factorial arrangement, with the following treatments: mixed or unmixed animals and two different number of animals per pen (8 or 12). Serum samples were obtained from 12 animals of each treatment, at experimental days 1, 5, 14 and 28. Elevated levels of the positive APP (acute phase proteins) and a decrease in the concentration of negative APP were observed the day before moving the pigs to the experimental installations. Thus the stress caused by the change of place resulted in a significant APP response. The APP response was higher in the mixed animals than in the unmixed. The effect was especially significant for Pig-MAP (p=0.0007). No interactions between the number of animals per pen and mixing were found, indicating that in this trial crowding has no effect on the APP response caused by mixing. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
In this work the concentration of the APP (acute phase proteins) Pig-MAP, haptoglobin, CRP and ApoA-I has been determined in pigs from commercial farms located in Segovia, Spain. The aim of the study was to establish a range of concentration of these proteins in normal state, as well as to analyse the potential of these markers to evaluate the general health status of farms. Serum samples from reproductive sows (Large White x Landrace) having none to five parturitions were collected in 9 commercial farms (5 animals per age and farm, randomly selected). The mean values of Pig-MAP and haptoglobin in the total of sows studied were respectively of 0.81+0.39 and 1.16+0.59mg/ml. There were no differences in the concentration of Pig-MAP or haptoglobin depending on the number of parturitions of the sows. In the case of the negative APP ApoA-I higher levels (p<0.0001) were found in sows having none parturition (2,45 mg/ml), being the rest of the values similar (1,59mg/ml). The mean values of Pig-MAP and haptoglobin concentration in reproductiveboars (Large White) were respectively 1.26mg/ml and 0.75mg/ml. Thus, a sex effect was found, being the opposite for haptoglobin and Pig-MAP. The mean concentration of the APP studied was not modified during the period of collection of the samples (1 year, at 4 months intervals), and there were no significant differences between the two insemination centres included in the study. To determine the concentration of APP in the growing finishing period, serum samples were obtained in 6 farms from animals of 28, 60, 90, 120, 150 days age. The concentration of Pig-MAP tended to decrease slightly with time. Mean values for each age ranged from 1.16 to 0.73mg/ml, and from 1.10 to 0.72mg/ml for haptoglobin (no significant differences between ages). In a second study, the APP levels were compared in two farms of different health status and managing conditions. One of the farms was a high health status farm with excellent performance, free of A. pleuropneumoniae, PRRSv, S. suis and mange, and with low mortality (<2%). Farm B was a low health status farm, with high prevalence of actinobacilosis due to A. pleuropneumoniae, PRSSv and mycoplasmosis due to M. hyopneumoniae, and a mortality >12% during the period of analysis. The concentration of the positive APP in the farm of high health status remained constant during the period of analysis. In the farm of low health status significantly higher levels of the positive APP, and a decrease of ApoA-I were found in animals of 3, 9 and 13 weeks of live. Results obtained in this study indicate that the determination of APP may become a useful tool when evaluating the general health status of farms. The use of an APP index increased the differences observed between farms. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
Meat juice is increasingly used for pork quality assessments in surveillance and control programs. Hereby the determination of pathogen-specific antibodies, in particular for salmonella (Quirke et al., 2001; Alban et al., 2002) but also for other pathogens (Kapel et al., 1998; Mortensen et al., 2001) currently accounts for the major task. The aim of this study was to evaluate if the serum concentration of haptoglobin respectively pigMap is correlated to the concentration of these APPs (acute phase proteins) in meat juice which is routinely collected at slaughterhouse for salmonella testing. Slaughter blood and muscle samples for extraction of meat juices from 299 slaughter pigs were collected. For haptoglobin, it turned out that a significant correlation of 0.7 between slaughter blood and meat juice of the pars costalis diaphragmatis existed respectively 0.6 for pigMap. The relation of Hp and pigMap concentrations in meat juice with those in blood serum established in our study suggests that meat juice may also be applied for general assessments of animal health and welfare. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
For the first time, a method to measure the levels of transthyretin in porcine serum has been developed. It shows that TTR is a negative acute phase reactant in pig. Immunoblotting and mass spectrometry demonstrated that commercially available sheep polyclonal antiserum raised to human transthyretin cross reacted with porcine TTR. This cross reactivity is probably due to the 85% amino acid sequence homology between porcine and human transthyretin protein sequences and the lack of glycosylation of TTR. The specific antibody was used to develop an assay for measuring transthyretin in pig serum. Using this assay TTR in porcine sera can be measured in a consistent and reproducible manner with acceptable precision. Following Strep.suis infection transthyretin showed a negative acute phase response with serum levels falling significantly two days following treatment then returning to pretreatment values after 5 days. The fall in levels of TTR during the acute phase response was relatively small compared to the increases found in other acute phase proteins such as SAA. However, it is consistent with drops in human transthyretin levels following infection reported previously (Ferard et al. 2002). The assay we have developed for TTR may have the disadvantage of relying on passively adsorbing the antigen to a surface in competition with all the other serum proteins. This, on the other hand may be less of a drawback considering that TTR concentrations vary only slightly. However, the detection limit might be lowered significantly by using a sandwich ELISA with antibodies raised to purified porcine transthyretin to trap the TTR. We are presently developing such an ELISA. In conclusion, we have developed a means to measure TTR in pig serum and demonstrated that the concentration of this protein in serum falls in response to infection. This assay was used to measure TTR levels in the porcine serum samples provided by other project partners. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
Retinol Binding Protein (RBP) is a small molecular weight protein (MW 21,000 Da). It is the exclusive protein for transport of vitamin A (retinol) in the body. Almost all the vitamin A in blood is bound to RBP. RBP is synthesized mainly in the liver by parenchymal hepatocytes (Yamada et al 1987). In the endoplasmic reticulum in the liver RBP picks up one molecule of retinol. The synthesis of RBP and its secretion from hepatocytes is mainly controlled by retinol. RBP in the plasma is bound to another, larger protein, transthyretin (TTR). TTR is a tetrameric protein with a molecular mass of approximately 55,000 Da. Monoclonal antibodies were prepared against porcine RBP. Development of an ELISA is in progress. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
A number of combinations of acute phase proteins (APP) were found to work well in detecting infectious/inflammatory processes, the most sensitive combinations in general being (Hp, apoA1, pigMAP) and (CRP, apoA1, pigMAP). These are as good as or only very slightly lower than four protein combination in all cases. An acute phase index based on such a combination of APPs (for example (CRPxpigMAP) / ApoA1)), thus including a negative APP, would give an increased robustness of conclusions based on these measurements and would correct for possible dilution and evaporation artefacts. It could be argued that the combination with Hp is less usable than the other one, as pre-infection levels of Hp vary considerably making it a more difficult task to determine a cut-off value for this protein as compared to the others. It also seems worthwhile to consider some of the two-protein combinations (e.g. (Hp, pig MAP); (Hp, ApoA1) and (CRP, apoA1)), as these two-protein combinations perform almost as well as the three-protein combinations. Since the situation may be different with real samples, all promising combinations of proteins should be tested on real herd derived blood samples before a final decision is made. It furthermore remains to define operational, real-life cut-off values for the APPs to be tested in an acute phase index and for the index value based on these APPs. Thus, in conclusion, the combinations of (Hp, apoA1, pigMAP) or (CRP, ApoA1, pigMAP) give the most sensitive detection of inflammation/infection, but two-protein combinations like (Hp, ApoA1) should also be considered, especially when a precise cut-off level for Hp is at hand. The aim of an acute phase index based on such a combination of APPs will be a high specificity (few false positives). Such an index will enable us to detect with great certainty an ongoing infection/inflammatory situation, but will not enable us to declare an animal healthy in the absence of an elevated APP-level. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/
Interest is being increasingly focused on the welfare of transported animals, because of the consequences of the stress generated during transport in meat quality and the value of the final product, as well as the apparent awareness of consumers over the ethical quality of the meat they eat. Transport is an inherent stressful procedure, and circumstances as vibration, restricted space, mixing, lack of ventilation, or deprivation of food and water can compromise pig welfare. Different parameters have been used in the evaluation of animal welfare during transport, including behavioural observations, or physiological parameters as glucocorticoids or heart rate, but it does not exist a consensus about which of them is the most adequate. Recently it has been suggested that APP (acute phase proteins) could be used in the evaluation of transport conditions. In this study the APP response after a 700 km transport by road in commercial conditions was evaluated. Serum samples were obtained at farm the day before transport, immediately after arrival to the abattoir (12 hours of trip )and the day before, in the slaughter line, after 12 hours of lairage. An increase of the positive APP pig-MAP (2 times, p=0.0001), haptoglobin (1.5 times p=0.0227), CRP (5 times, p=0.0001) and SAA (14 times p=0.0014) and a decrease of the negative APP ApoA-I (30%, p=0.0001) was observed the day after transport. Different factors concur during transport, including stress caused by coping with new environments, mixing, fight between animals as well as spread of pathogens that could be the cause of APP elevation. However it is clear that the increase of APP levels reflex the presence of situations that compromise animal welfare. Studies have shown that APP can experiment higher increases in these transport conditions that represent higher levels of stress for the animal. Thus APP appears interesting parameters to determine welfare during transport, and to evaluate transport conditions. In the other hand, if the APP assay is going to be integrated in the quality systems, it is necessary to consider this effect. More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/

Searching for OpenAIRE data...

There was an error trying to search data from OpenAIRE

No results available