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The International Colloquium On Animal Acute Phase Proteins
The 5th International Colloquium On Animal Acute Phase Proteins Dublin, Ireland March 14th – 15th 2005 Abstracts and Proceedings The 5th International Colloquium On Animal Acute Phase Proteins Dublin, Ireland March 14th – 15th 2005 Abstracts and Proceedings Book Scientific Committee Chairman: David Eckersall, United Kingdom Erik Gruys, The Netherlands Adrian Molenaar, New Zealand Brigit Petersen, Germany Fermin Lampreave, Spain Carlos Piniero, Spain Peter Heegaard, Denmark Satu Sankari, Finland Jean-Marie Godeau, Belgium Manfred Fuerll, Germany Jose Ceron, Spain Saverio Paltrinieri, Italy Gareth Evans, United Kingdom Tom McDonald, United States Brent Hoff, Canada Peter O'Brien, United Kingdom Paul Torgerson, Switzerland Local Organiser: Martin Gallagher, Ireland The Committee gratefully acknowledges the support of the sponsors of APP 5: Tridelta Development Limited PigCHAMP Pro Europa for the publication of this book of abstracts. Enterprise Ireland Biotechnology Directorate The Committee would also like to express it's gratitude to Laura Bence of Glasgow University for her invaluable work on the preparation of the abstracts for publication. Welcome From Chairman David Eckersall It gives me great pleasure to be able to welcome you to the 5th International Colloquium on Animal Acute Phase Proteins here in Dublin. Research in acute phase proteins in animals has been expanding rapidly and has been the focus of growing interest from veterinary medicine, biological science and comparative research in recent years. This has been reflected in the evolution of these regular meetings that have been held in locations around Europe over the last five years. Previous meetings have benefited from funding from the European Commission but the interest shown in this first independent meeting indicates that the research community establishing the fundamental pathophysiology of the acute phase proteins in animals and also the interpretation and use of diagnostic information from their assays has developed to a critical mass. The focus of the meeting has been altered for this occasion with less emphasis on regulatory application in food safety to provide greater opportunity to examine the acute phase proteins in companion and laboratory animals which is complementing the progress made in production animals. The role of the proteins in diagnosis of disease as markers of inflammation and infectious disease is established but there is much more to be determined on the correct interpretation of elevated concentrations in the different species studied. This meeting should be a valuable forum for exchange of information on diagnostic uses of the acute phase protein assays as well as the exchange of ideas on their biochemistry, function and other uses. The Scientific Committee is especially pleased to welcome Professor Mark Pepys to give the Plenary Lecture. As well as being the world authority on acute phase proteins in human medicine, Prof Pepys has published numerous papers on the acute phase response in animals, which have inspired many of us here at this Colloquium. We can hope that further cross fertilisation of ideas may develop in the future. The previous meeting in Glasgow, Bonn, Doorn and Segovia have all been memorable and have stimulated great collaboration between delegates. Hopefully this will continue with further links and collaborative projects being developed during the next two days of science and socialising. Ireland and Dublin are renowned for their hospitality and I am sure that the local organisers would wish your stay in their fair city to be exceedingly stimulating and enjoyable. Best Wishes David Eckersall Chairman of the Scientific Committee Abstracts INVITED PRESENTATIONS 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 THE USE OF ACUTE PHASE PROTEINS IN PIG PRODUCTION Petersen B1, Gymnich S1, Nielsen J-P2, Pineiro C3 1 Department Preventive Health Management, University of Bonn, Bonn, Germany 2Department of Swine Medicine, The Royal Veterinary and Agricultural University of Copenhagen, Copenhagen, Denmark 3 PigCHAMP PRO EUROPA SA, Segovia, Spain Email: firstname.lastname@example.com Objectives Acute phase proteins (APP) are proteins present in blood which respond to disease or other stressors by increasing or decreasing in concentration. The objective of Work Package 3 in the EU project was to study whether APP determination during production will be a valuable tool to indicate periods of reduced performance and clinical or subclinical disease prevalence with the aim that specific diagnostic methods and therapeutic approach would follow only if necessary. Furthermore to prove if APPs could be established as screening parameters for the health status of pigs during the production chain. Materials and Methods As variations exist in the processing of pig production in Europe studies in different European countries were necessary in order to find out an appropriate production process oriented strategy for the implementation of APP in the pig production chain in Europe. Different farms and production systems were included in the study in order to find out an appropriate production process oriented strategy for the implementation of APP in the pig production chain in Europe. In Germany three studies were performed. In the first study five variants of customer-supplier contacts were tested consisting of 20 piglet rearing farms involving the collection of about 1100 blood samples. In addition seven piglet breeding farms and one fattener farm were available for samples. 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 which was obtained from the farmers or the veterinarians respectively. In aim the study 2 to evaluate if the serum concentration of haptoglobin respectively pig MAP is correlated to the concentration of these APPs 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. Study 3 was a short study where the acute phase protein haptoglobin was used as a screening parameter in five fattening farms. The aim of this work was to examine if haptoglobin could be used for single point investigations on the farms instead of time- course analyses. The four Danish studies are presented, too. In study 1, the design was 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. In study 2, 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. In study 3, 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. In study 4, paired blood samples were obtained at the farm before transport and at the abattoir to investigate the effect of routine transport and lairage and chronic lesions ante mortem in APP serum concentration. Four studies were conducted in Spain, two of them to assess the effect of stressors commonly present in pig production, such as mixing or an inadequate feeding pattern. In study 1 the effect of mixing animals, from different pens in the nursery, at entry to the fattening barn, on the levels of APP (PigMAP and haptoglobin) was studied. The possible interactions between different number of animals per pen (8 or 12 pigs per pen) and mixing were also evaluated. In study 2, the effect of a changeable pattern of food administration in the growth performance and APP levels (PigMAP, haptoglobin, Apo-AI, SAA, CRP and TT) of growing pigs was evaluated in the growing phase (74-116 d of age). Two hundred and forty pigs (26.3 ± 0.4 kg BW) were distributed into two treatments: fed ad libitum (AL) or disorderly (DIS), consisting in the administration of feed following a disorderly pattern, alternating periods of AL administration with periods of no feeding. Total feed intake (FI) was kept constant in both groups. In 1 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 study 3, the APP response (PigMAP, haptoglobin, ApoAI, SAA, CRP and TT) after a long transport (12 h) under commercial conditions was evaluated. Twenty pigs were bled in the farm the day before transport, at the arrival to the slaughterhouse and at the slaughter-line (about 24 h after the beginning of the transport). The fourth study included the determination of the serum concentration of the APP pig- MAP, haptoglobin, CRP and ApoA-I in pigs from twelve commercial farms located in Segovia, Spain. For this, two blood samplings were performed 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. Results APP in the rearing and fattening period (Germany, Study 1) 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 status of the breeding farms and the four APPs. Furthermore pigs causing costs of medical treatment above € 1.15 in the rearing period had significant higher Hp, PigMap and CRP serum concentration respectively and 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 are the results for CRP and ApoA1. These two parameters behaved vice versa. APP in meat juice (Germany, Study 2) A significant correlation between slaughter blood and meat juice of the pars costalis diaphragmatis was found for haptoglobin (p<0.001, r=0.7) respectively PigMAP (p<0.001, r=0.86). Haptoglobin in the fattening period (Germany, Study 3) Due to the average increases of daily weight gain as well as the average losses of all farms the pigs were divided into two classes. It was obvious that pigs living in farms where higher performance (daily weight gain > 715 g) and lower losses (< 3,14%) could be realized showed significantly lower haptoglobin concentrations. Samples 2 and 3, which were taken in the middle and at the end of the fattening, should give information on the health status of the animals in the farm. Again it turned out that pigs with a daily gain above 715g had significantly lower haptoglobin concentrations, compared with animals with lower performance. 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 time-course analysis, but also for a single point investigation in the farms. APP and clinical signs (Denmark, Study 1) 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 diarrhoea (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 1.1 mg/mL. When using a cut-off value of 1.8 mg/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. Health status & slaughter (Denmark, Study 2) 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. Risk factors in finishing pigs (Denmark, Study 3) Pigs from herds with high prevalences of clinical signs of respiratory disease and diarrhoea and lame pigs had high serum haptoglobin concentration. Increasing levels of antibodies against A. pleuropneumoniae serotype 2 and M. hyopneumoniae were associated with increasing serum haptoglobin concentration. 2 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 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. Handling/ transport & slaughter (Denmark, Study 4) 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 due to transport stress or other causes could not be concluded. 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. Effect of bad management. Mixing. (Spain, Study 1) The day following placement at the fattening barn, mixed pigs had higher PigMAP values (2.07 vs 1.36 mg/mL; P=0.0006) than non-mixed pigs and were still higher 5 d after placement (1.18 vs 0.90 mg/mL; P=0.04), and the difference disappeared thereafter. No consistent pattern was observed for haptoglobin, but the concentration was always higher for mixed pigs being significant at d 14 (1.83 vs 1.31 mg/mL; P=0.05). Males were more sensitive to both stressors than females and showed higher PigMAP concentration (P<0.05) and lower growth (P<0.05). As conclusion, mixing of pigs at the start of the fattening period induces stress independently of pen size and serum concentration of APP's can be used to detect the intensity of stress. Effect of bad management. Feeding. (Spain, Study 2) Average daily gain (ADG) was higher in AL than in DIS group (592 vs 548 g/d; P<0.01). Differences in ADG between AL and DIS animals in the 74-88 d subperiod were due to the males (523 vs 398 g/d), having the females of both groups similar ADG. Differences in the behaviour of the APP were found between DIS males and females. In females, no differences were found between feeding patterns. However, in DIS males, PigMAP and haptoglobin remained elevated respect the AL ones at 88 and 102 d of age (P<0.05 and P<0.01 for PigMAP and P=0.11 and P<0.01 for haptoglobin). SAA was also numerically higher in DIS males at 88 d of age and CRP was higher (P=0.07) at 102 d. The concentration of negative APP ApoAI, increased slightly with time in AL males, whereas in DIS males the concentration remained lower than in the AL. No differences were observed in TT. The acute phase index also showed differences at 88 d of age and DIS males had a higher index than AL ones (58.7 vs 5.7; P<0.05), with no differences in females. These results confirm a good correlation between daily gain and APP serum levels. Hence, APP can be proposed as an interesting parameter to be used in the detection of situations that cause stress in the pig. Long distance transport (Spain, Study 3 ) All the positive APP determined showed a significant increase of concentration after transport (P=0.0001 for PigMAP and CRP, P=0.001 for SAA and P=0.02 for haptoglobin), being values obtained in slaughter line the highest. The magnitude of the change of concentration observed after transport varied from one protein to another. The concentration of ApoAI decreased about 30% in the day after transport (P=0.0001), whereas transthyretin did not show significant changes respect the values obtained in the farm the day before transport. These results suggest that APP may be interesting parameters to evaluate welfare during transport, and useful tools in the evaluation of transport conditions. It is interesting to remark that the use of the acute phase index (PigMAP x CRP/ApoAI) improved the sensitivity in the detection of the APP response that follows transport. APP in commercial farms (Spain, Study 4) In the first study, 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, being the rest of the values similar. In fattening pigs, PigMAP concentration tended to decrease slightly with age, whereas haptoglobin showed a slight slope at 12 weeks of life. From this study, a cut off value for normal state based on the mean plus 1.96 sd was established for reproductive sows and boars and for fattening pigs. In the second study, the acute phase index (PigMAP x CRP/ApoAI) was compared in two farms of different health status. The APP concentration pattern obtained in the high-health status farm showed constant APP levels in all the growing period, whereas the presence of disease or stress due to bad managing conditions would alter this APP pattern in the low-health status farm, that could be used in the 3 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 detection of problems in the farm. As conclusion, APP may become an useful tool when evaluating the general health status of farms, and the APP index increases the sensitivity. Conclusions The project, divided in three work packages (WP) has come to a successful conclusion achieving its milestones and producing the deliverables expected. From the WP1, the assessment APP as markers of immunological stress, nutrition and the acute phase was positively validated. From the WP2 the identification of the best combinations that can be employed to identify animals with infectious/inflammatory processes was obtained. The establishment of the acute phase index (CRP x PigMAP) / (AporA1) increase the robustness of analysis, whereas the combination of Hp, ApoA1 and PigMAP was also validated. The studies performed in WP3 at differing stages of the pig production process and in different countries with differing production systems were able to demonstrate the usefulness of APP analysis in the production chain. There are numerous examples from the studies undertaken that demonstrate the value of APP analysis. However, the full potential of their measurement will need much further investigation as this project is undoubtedly only a starting point for our full understanding of how APP assay will be used to enhance the identification of immunological stress in pigs during production. The full range of APP was not measured in every study due to the interactions of time and resource availability. Nevertheless there were a number of fascinating discoveries made in the course of the project some of which were predictable from prior knowledge of the acute phase response in other species while others could suggest an even wider role for their analysis. In the former category of result would be the investigations confirming the APP are raised in animals with clinical conditions such as respiratory lesions, lameness or diarrhoea and that mean levels can vary between (especially rearing) farms with differing levels of hygiene. Economic implications of the results are also evident from the association between high positive APP (low negative APP) and the future cost of medical treatment. Important issues of welfare are relevant to the findings that stress of moving to new accommodation can cause an increase in APP and also the interesting observation that there is a sex difference in the response to disorderly feeding with males giving an APP response absent in females. In conclusion the project has delivered a range of methods to monitor positive and negative acute phase protein and has utilised them to establish a core group of proteins to be assessed for their value in quantification of the acute phase reaction and immunological stress in pigs. These tests are already showing their ability to monitor health and disease in pigs through the production chain but there is a need for further extensive national and international studies to fully understand and exploit their diagnostic potential. Acknowledgements: Support from the European Commission for these studies is gratefully acknowledged (Shared Cost Project QLK5-2001-02219) 4 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 PORCINE ACUTE PHASE PROTEINS IN EXPERIMENTAL MODELS OF INFECTIOUS DISEASES Heegaard PMH, Stockmarr A, Sorensen NS Danish Institute for Food and Veterinary Research, Department of Veterinary Diagnostics and Research, Copenhagen, Denmark. E-mail: firstname.lastname@example.com Introduction We have studied the porcine acute phase protein (APP) response in a number of infection models for bacterial and viral infections through collaboration with Danish and European groups, especially in the EU funded Shared Cost project Acute Phase Proteins in Pigs (QLK5-2001-02219). This has led to the identification and characterisation of a number of new negative pig acute phase proteins (some of which are being presented at this meeting) and the description of concentration changes of a selection of positive and negative pig APPs during various experimental infections. Materials & Methods A typical infection experiment entailed a group of pigs being infected at the same time by well-described infection methods aimed at reproducing the corresponding natural infection. The pigs were then followed daily to inspect for clnical signs of disease. Blood samples were obtained before infection and at regular intervals after infection, sampling several times from the same animal. At the end of the experiment animals were euthanised and autopsied to record pathological changes and to recover tissue for demonstration of the presence of the infective agent and other microorganisms, if present. Serum samples were analysed by immunochemical methods and calibrated whenever possible with porcine acute phase protein standards from the EU concerted action "Animal Acute Phase Proteins" (QLK5-CT-1999-153). Data from a number of such experiments will be presented together with the methods used for statistical treatment of the data. Results Strongly reacting positive pig APPs include serum amyloid A (SAA), C-reactive protein (CRP), pig major acute phase protein (pig MAP) and haptoglobin; SAA is often undetectable in normal pig serum but its big incremental change during the acute phase reaction makes it one of the most prominent of the acute phase proteins. Hemopexin and complement C3 are weakly reacting positive acute phase proteins and transthyretin (TTR) and alpha-1 apolipopoprotein (apo A1) are strong negative acute phase proteins, while albumin, although negatively reacting is quite inconsistent in doing so. Surprisingly, alpha-1-acid glycoprotein is nonresponsive in pigs while in most other species it is a prominent positive APP. Comparing different infections, in many cases SAA was not increased and albumin was not decreased, while generally, CRP, haptoglobin, pig MAP and transthyretin and apoA1 reacted strongly to the infections studied. Using the most sensitive and specific of these APPs a good reflection of the severity of infection could generally be obtained. Discussion It can be concluded that certain APPs are rather insensitive to infection, either by not reacting (SAA, albumin) or by showing a pronounced baseline variation that lowered the sensitivity for detecting an acute phase condition (haptoglobin, TTR). Furthermore, the specificities of some APPs may be hard to establish (SAA) because pre-infection levels are undetectable. Using a selection of APPs with rapid and prolonged AP changes and low prechallenge variations a composite parameter could be constructed that was investigated statistically for general responsiveness to infections. In conclusion, it is possible by using such a parameter to gain precise information on the AP status of an animal. A number of basic questions might be investigated by the use of such a combination of measurements, including the possibility of detecting subclinical infections by APP reactions, the differentiation if possible between acute vs. chronic infection and the influence of physical and psychological stress on APP reactions. This will then lay the foundation for the practical use of APP measurements as a clinical and surveillance aid. Acknowledgements: Support from the European Commission for these studies is gratefully acknowledged (Shared Cost Project QLK5-2001-02219) 5 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 THE ACUTE PHASE RESPONSE IN CLINICAL PRACTICE: THE FIRST 75 YEARS Pepys MB Royal Free and University College Medical School, University College London, Department of Medicine, London, UK. Email:firstname.lastname@example.com Study of the acute phase response started with the discovery of C-reactive protein (CRP) 75 years ago. Clinical interest in and use of acute phase proteins in human medicine has waxed and waned over the years since then for technical, cultural and educational reasons. Although CRP is overwhelmingly the best systemic marker of the acute phase response in humans, clinicians in different specialties and in different countries have shown, and still show, remarkable differences in their capacity to understand and apply the profound clinical utility of measuring this exquisitely sensitive, precise, and quantitative but non-specific, non-diagnostic, marker of disease. Nevertheless, routine use of CRP assays is now firmly embedded as an established part of investigation and management in most parts of the world, and properly standardised on the WHO International Reference Standard that I produced 20 years ago. The only other human acute phase protein that merits routine measurement as a marker of inflammation is serum amyloid A protein (SAA), monitoring of which is essential in management of reactive systemic, AA, amyloidosis, in which the aim is reduction, to normal if possible, of the circulating SAA concentration, in order to arrest amyloid deposition and allow amyloid regression with associated clinical benefit. We also made the WHO standard for this analyte. Following our original demonstration in 1994 that high sensitivity measurements of CRP provide significant prognostic information in patients with acute coronary syndromes, there has been an explosion of interest in CRP and cardiovascular disease, greatly enhanced by the finding that baseline CRP values in the general population also predict future atherothrombotic events. The avalanche of publicity and hype about this has generated a bandwagon of enthusiasm for such measurements in risk assessment that is not well supported by the epidemiological evidence. There have also been enormously exaggerated claims, based on very poor experimental evidence, that CRP is pro-atherogenic. In fact no rigorous studies to date support these claims. In marked contrast, after ischaemic necrosis of tissue has occurred, we have robustly demonstrated that human CRP can be pro-inflammatory and enhance tissue damage in vivo. CRP is thus a valid therapeutic target and we are designing and developing novel CRP-inhibitory drugs that may have cardioprotective and neuroprotective effects after myocardial infarction and stroke. In the past my laboratory has also studied various acute phase proteins in different animals, identifying and/or characterising them in detail for the first time, in particular mouse serum amyloid P component (SAP), rat CRP, dog CRP and horse SAA. Although both the pentraxin proteins, CRP and SAP, and the SAA family are stably conserved in evolution, there are notable differences between species in their circulating concentrations and behaviour as acute phase reactants. Mouse SAP is an exquisitely sensitive acute phase protein with important and, as yet, largely unrealised potential for use in toxicological screening. The accelerating recent progress of clinical applications of animal acute phase protein measurement in veterinary medicine and in food production is both timely and appropriate, and will surely continue to yield wide ranging benefits 6 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 MAMMARY SERUM AMYLOID A3, PROGRESS TOWARD DETERMINING STRUCTURE AND FUNCTION. A. Molenaar, P. Harris, J. Green, M. Callaghan, K. Kim, R. Wieliczko, K. Johannessen, R. Dines, R. Watson, V. Arkus, J. Dalziel, K. Singh, G. Rajan, M. Pearson, V. Farr, M. Miles, K. Oden, L. Good, R. McLaren, C. Prosser, M. Grigor, S. Davis, K. Stelwagen. AgResearch, Ruakura Research Centre, Hamilton, New Zealand. Email: email@example.com The serum amyloid A protein (SAA) family are one of the major reactants in the acute-phase response (Sellar 1993, Eckersall 2004). We found the mRNA encoding for the SAA protein (M-SAA) to be present in bovine mammary tissue by representational difference analysis in a comparison of RNA from normal and involuting quarters of a dairy cow udder (Molenaar, 1999). SAA3 proteins have also been detected by in situ hybridisation in a range of human tissues including; some epithelial cells of the intestine, breast and pancreas (Urieli-Shoval, 1998). It is thought that local serum amyloid production may fulfill a short term requirement (Sellar, 1993), or that the locally expressed proteins may play a role related to the site of expression and be produced under conditions that do not initiate the systemic acute phase response (Urieli-Shoval, 1998). The mRNA was found by in situ hybridisation to be localised to restricted populations of mammary epithelial cells. In-situ hybridization, northern analysis and real-time PCR revealed that it was expressed at a moderate level in late pregnancy, at a low level throughout lactation, was induced during early milk stasis, and expressed at a high level during mid to late involution and inflammation/mastitis. The expression patterns of the mRNA and the location of the protein in the udder were consistent with an involvement of M-SAA in mammary remodelling during pregnancy and involution, and response to stress caused by cellular engorgement and consequent inhibition of milk protein mRNA transcription, and, as it was elevated during mastitis, during inflammation and infection. The association of the M-SAA with the vesicle or fat globule membranes was demonstrated by immunohistochemistry and this association is also consistent with a role in the removal of accumulated lipid which becomes trapped in the alveoli during milk stasis (Molenaar, 1995). An extra-mammary role has been suggested in that M-SAA, and in particular its TFLK motif, has been shown to be protective in neonates and possibly adults against gastrointestinal infections by inducing mucin production (McDonald 2003). In order to attempt to clarify the function of the M-SAA we cloned the cDNA, expressed the mature peptide in E. coli, confirmed the reading frame using LCMS, developed a sensitive antibody and attempted the isolation of the recombinant and native form for functional testing. Difficulties were encountered such as poor solubility and aggregation, nevertheless these give some clues about the properties of the molecule. Various extracts have been tested in direct and indirect assays and the work is ongoing. Our experiences, the results of bioinformatic and theoretical structural studies, and future plans will be presented. References Eckersall PD. The time is right for acute phase protein assays. Veterinary Journal. Jul;168(1):3-5. 2004. Mack D.R., McDonald T.L., Larson M.A., Wei S., and Weber A. The conserved TFLK motif of mammary-associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in vitro. Pediatric Research. Jan;53(1):137-42. 2003 Molenaar A. J., Davis S. R., Jack L. J. W., and Wilkins R. J. Expression of the butyrophilin gene, a milk fat globule membrane protein, is associated with the expression of the αs1casein gene. Histochemical Journal. Vol 27 pages 389-395. 1995. Molenaar A., Rajan G., Stelwagen K., and Grigor M. A. Serum Amyloid Protein homologue is expressed by the Mammary Gland. Research Seminars of the Waikato Academic division of the University of Auckland. 17 March 1999. Urieli-Shoval S., Cohen P., Eisenberg S., and Matzner Y. Widespread expression of serum amyloid A in histologically normal human tissues: predominant localization to the epithelium. The Journal of Histochemistry and Cytochemistry 46 (12):1377-1384. 1998 Sellar G.C. and Whitehead A.S. The Acute Phase Response and Major Acute Phase Proteins in Early Host Defence. Journal of Biomedical Science 4, 1-9. 1993. 7 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 THE GLYCOSYLATION OF ALPHA-1-ACID GLYCOPROTEIN – STRUCTURALLY COMPLEX, FUNCTIONALLY IMPORTANT? Smith, K Department of Bioscience, University of Strathclyde, Glasgow G1 1XW Alpha-1-acid glycoprotein (AGP) is a human plasma protein that belongs to the group of positive acute- phase proteins that are produced by the liver. It is also has the ability to bind and carry numerous basic and neutral lipophilic drugs from endogenous (steroid hormones) and exogenous origin. During several physiological and pathological conditions, the total concentration of AGP increases and has been shown to be an early indicator of a change in condition, disruption to homeostasis or background illness since it occurs before the production of antibodies by the immune system or before clinical symptoms are apparent. AGP levels are also valuable for prognosis and monitoring of treatment and are particularly useful as a marker for the detection of early stage disease, to judge the extent of progression of a malady and to assess the effectiveness of treatments or changes related to attempts to improve management or environment. Often overlooked is the fact that AGP, like many APP, is glycosylated with oligosaccharide chains attached to the protein sequence. Oligosaccharide chains are ordered structures composed of various monosaccharides and charged molecules (sialic acids) in a specific sequences that are important determinants of biological function mainly because they contribute significantly to the hydrodynamic mass of individual glycoconjugates. The degree of AGP glycosylation is variable between species but normally accounts for between 23.5% and 43.5% of the molecular weight (typically 42,000 to 50,000). Interestingly, during the course of an acute phase reaction the glycosylation of AGP is capable of becoming uniquely altered in the presence of specific individual diseases. For example our research to date, has proved that it is possible to distinguish individual liver diseases on the basis of their AGP glycosylation e.g. hepatitis C the oligosaccharide chains have the highest amount of fucose in comparison to the other liver diseases and also contain the rare monosaccharide N-acetylgalactosamine (GalNAc). A further complexity of AGP glycosylation is the fact that, in normal serum, the glycoprotein does not exist in a single form but as a heterogeneous population of glycosylated variants (glycoforms) owing to differing occupancy of the five glycosylation sites. Heterogeneity arises through subtle structural differences in monosaccharide sequence and linkages, degree of branching (bi-, tri-, tetra- antennary) and extent of sialylation. The relative proportions of these "normal" AGP glycoforms have been found to change and abnormal glycoforms are expressed during disease. Our ongoing research interest is not only interested in the diagnostic potential of AGP glycosylation in various disease states but also the functional significance of these modifications. In rheumatoid arthritis (RA), our research has demonstrated hyperfucosylation, hypersialylation and increased chain branching (from an increased content of biantennary chains in the early acute stages to a reduced content in the chronic patient) in AGP. Moreover fucosylation is present as part of the tetrasaccharide antigen sialyl Lewis X which is known to be the simplest structure recognised by the selectin family of cell adhesion molecules thus suggesting a role for rheumatoid AGP in the restriction of leukocyte extravasation to a centre of inflammatory activity. Conversely, the increased fucosylation of AGP from the plasma of burns' patients has been directly correlated with the atypical collagen deposition associated with hypertrophic scarring. 8 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 RELATIONSHIP BETWEEN HAPTOGLOBIN, SERUM AMYLOID A AND CLINICAL STATUS IN THE SURVEY OF DAIRY HERDS DURING A SIX MONTHS HOUSING PERIOD Humblet M1, Guyot H2, Boudry B2, Mbayahi F1, Hanzen C2, Rollin F2, Godeau J1 1 Department of Functional Science 2Department of Clinical Sciences, University of Liège, Liege, Belgium. Email:email@example.com Introduction Haptoglobin (Hp) and serum amyloid A (SAA) are considered as two of the major acute phase proteins (APPs) in cattle (Conner et al.,. Res Vet Sci., 1986; 41: 126-128; Skinner et al., Vet Rec. 1991; 128: 147- 149). They were measured in dairy herds and compared to clinical examination in order to assess their capacity to identify the animals with acute inflammation. Then a comparison was made between these two APPs to check if they were correlated, and if they could help the farmer to identify the animals undergoing an acute inflammatory process. Material and Methods Two hundred and sixteen pregnant dairy cows, from two to nine years old, were included in the study. They were randomly chosen from four commercial farms of the Eastern part of Belgium. Blood was sampled every fifteen days during a six months housing period. A complete clinical and gynaecological exam was performed at blood sampling, which allowed the classification of the animals in two categories: healthy and diseased cows. Serum Hp determination was carried on by quantitative measurement of haemoglobin binding capacity as reported by Skinner et al. (Vet. Rec., 1991, 128:7, 147-149). SAA was determined by mean of an ELISA kit (Tridelta Development Ltd, Ireland). Hp and SAA status were allocated to the samples according to the Cut-off point (COP) for medical decision (Table I). Samples were gathered into three different periods: P1 (prepartum), P2 (week 1 postpartum) and P3 (postpartum beyond week 1). APP prevalence was assimilated to the percentage of samples with APP values above the COP (positive APP test). APP sensitivity was assimilated to the incidence of a positive APP test among diseased cows and specificity, to the frequency of a negative test in the population of healthy cows. The assignment of the health status based on clinical and gynaecological examinations was considered to be the gold-standard. Hp and SAA concentrations were related to clinical observations using standard unpaired and paired t tests of the Statview program (SAS Dole Institute Inc.). Differences were considered as significant (S) for P ≤ 0.05. APP Periods Multiparous cows Primiparous cows Hp– :< 30 mg/L P1 + P3 Hp +: 30 - 100 mg/L Hp ++: > 100 mg/L Hp Hp– :< 150 mg/L Hp– :< 150 mg/L P2 Hp +: 150 - 200 mg/L Hp +: 150 - 250 mg/L Hp ++: > 200 mg/L Hp ++: > 250 mg/L P1 + P3 SAA + : > 25 000 µg/L SAA P2 SAA +: > 60 000 µg/L Table I: reference values for Hp and SAA were established for dairy cows in a preliminary study (not published). Hp–: absence of inflammation; Hp+: mild inflammation and Hp++: severe inflammation. Results The profiles of mean Hp and SAA concentrations throughout the whole study were similar in the four herds: they significantly peaked during the first week after calving both in diseased and healthy cows. Hp and SAA mean concentrations were significantly higher in the samples from diseased cows. A total of 9.7% of samples from healthy cows presented a Hp value above the COP. Hp sensitivity and specificity reached 38.0% and 90.3% respectively. If only P2 was considered, 35.8% of samples from healthy cows presented a Hp value above the COP; Hp sensitivity reached then 85.0%, with a 91.6% specificity. The results were similar in the four herds. In a different approach, 37.0% of Hp+/++ samples were identified as diseased by the clinical exam. This proportion reached a 85.0% value if P2 was considered alone. SAA sensitivity and specificity were similar to Hp (38.7% and 89.2% respectively) and 10.8% of samples from healthy cows presented a SAA value above the COP. SAA prevalence reached 40.0% and its sensitivity increased to reach a 79.2% value in P2. Only 56.1% of samples SAA+ came from cows clinically diseased. SAA was then investigated on the basis of the samples status allocated by Hp (Hp–, Hp+ or Hp++), and it appeared that 93.2% of samples Hp– were also SAA–, and 76.2% of samples Hp++ were also SAA+. SAA profiles showed that cows with a Hp++ status presented also SAA concentrations corresponding to an acute inflammation, corroborating thus the classification of samples according to Hp. More than 95% of samples from healthy cows were Hp– and SAA–: they identified well animals that were not under an inflammatory status. On the other hand, 76.0% of samples from diseased cows were 9 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Hp+/++ and SAA+. Among samples from healthy cows, 48.6% were SAA+ and Hp–, but only 4.2% were SAA– and Hp+/++. Hp was not correlated with SAA (r2= 49.5%). Discussion Neither Hp nor SAA should be used as inflammation markers in the week following parturition, as they physiologically rise at that time. The ability of Hp and SAA to identify diseased animals was quite low, but they both presented a better specificity. SAA confirmed the inflammatory status defined on the basis of Hp. An inflammatory status was also detected by the acute phase proteins in about 10% of samples from healthy cows. These acute phase proteins could be useful to the farmer: the animals with pathological serum concentrations could be submitted to a deeper clinical investigation. Both APPs were not correlated. 10 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 HAPTOGLOBIN IN ROUTINE ANALYSIS OF BOVINE SERUM Füerll M, Kirbach H, Pietsch H Large Animal Internal Clinic, Veterinary Faculty, Leipzig, Germany E-mail: firstname.lastname@example.com Background Haptoglobin (Hp) has proven to be very valuable for the analysis of the causes of important bovine diseases. Hp concentration, for example, is increased after parturition especially in those cows that developed retained placenta, mastitis or abomasal displacement (DA). DA may be considered to be a result of acute phase reaction. Hp increases during acute parturition stress. However, little is known about the usage and benefits of Hp in routine diagnostics in cattle. Objective The investigation aimed at checking the diagnostic, pathophysiologic and prognostic information of the Hp analysis during the routine operation of a ruminant clinic. Material and Methods Hp analyses were carried out a) in 116 healthy HF-cattle as well as 126 ill cattle in various farms and b) in 141 ill cows of our ruminant clinic. Determinations: Hp was determined using the test set by Tridelta (Dublin), metabolic parameters with the Hitachi 704 analysis machine (Bayer) and blood cells with the hematological system Technicon 1 (Bayer). Results a) The highest Hp concentrations were found in cows as well as calves with pneumonia (range 1.6 – 4.8 g/l), followed by cows with DA (range 1.0 – 3.5 g/l) as well as cows with mastitis and endometritis (range 0.5 – 2.8 g/l). b) The patients of the ruminant clinic, all ill with DA as well as additional troubles, had the following Hp concentrations (g/l, median): enteritis 1,10, bronchitis 0.95, nephritis 0.92, mastitis 0.45, endometritis 0.40, left side DA 0.46, right side DA 0.38 and laminitis 0.22. Hp correlated significantly with the following parameters (p< 0.05): Fe -0.40, AST -0.30 GLDH -0.30, AP -0.30, Ca -0.30, BHB -0.20, urea 0.20, albumin -0.20, CK -0.20, GGT -0.20, LDH -0.20, ALT -0.20, bilirubin II -0.20. Neither the number of leukocytes nor other blood cells correlated significantly with the Hp concentration. By comparing recovered cows and cows with exitus letalis, no significant differences occurred, that is the Hp analysis has no prognostic information. Hp concentration increases especially in diseases with acute inflammation (pneumonia, mastitis, enteritis, retained placenta/endometritis). Hence in extremely high Hp concentrations the mentioned organ diseases can be defined by differential diagnosis. Regarding the Fe-Bond to the APP transferrin, the negative correlation with Fe becomes clear. Because the clinic's patients were intensely medicated, the metabolic parameters mentioned above could be stabilised relatively quickly, which means that the increased concentrations or activities are normalised, thus the negative correlation with Hp is explained. In untreated cattle of practising farms, the Hp concentrations correlated in most diseases positively (p<0.05) with protein as well as negatively with albumin and AST-activity. The lack of prognostic information is surprising and could be interpreted as a consequence of the intensive medication that influenced the tested parameters. Conclusions In routine analysis in cattle, Hp indicates serious inflammation (pneumonias, mastitis, enteritis). Significant correlations with metabolic parameters exist mainly with Fe, less so with other metabolic parameters. Hp has no prognostic value with regards to the outgoing of the disease. 11 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 AN UPDATE ON ACUTE PHASE PROTEINS IN DOGS: ADVANCES IN MEASUREMENT ASSAYS AND CLINICAL APPLICATION Cerón J, Parra MD Department of Animal Medicine and Surgery, Veterinary School, University of Murcia, Murcia. Spain. E-mail: email@example.com Acute phase proteins are part of a non-specific inflammatory reaction of the host that occurs shortly after any tissue injury. Origin of the response can be attributed to infective, immunologic, neoplastic, traumatic, parasitic or other causes. Dogs have some particularities in the acute phase protein response compared with other species such as ruminants that should be taken in consideration; for example higher concentrations of haptoglobin in healthy animals which require larger sample dilutions with spectrophotometric methods or a very high response of CRP. In this presentation, recent advances in the application of acute phase proteins in dogs will be review and discussed with special emphasis in some topics such as: - Development of new assays. New sensitive assays have been specifically developed for canine CRP and haptoglobin based on Time Resolved Flourometry. This technique has allowed measurements of these proteins in canine effusions and the differentiation between exudates and transudates as well as the use of whole blood and saliva as alternative specimens to serum or plasma. - APPs response in different inflammatory diseases. Increased concentrations of different APPs were detected in a number of diseases involving inflammation such as polyarthritis, infectious diseases (parvovirus infection, leptospirosis, leishmaniasis, babesiosis), inflammatory bowel disease and in tumours. Haptoglobin in dogs is also raised in case of increased levels of endogenous (hyperadrenocorticism) or exogenous (anti-inflammatory treatments) corticosteroids. - Use of APPs indexes for treatment monitoring. Measurement of concentration of selected acute- phase proteins can be used to evaluate the response to treatment of dogs with different specific diseases such as leishmaniosis, inflammatory bowel disease, hyperadrenocorticism or selected neoplasias. However it seems that the use of APPs indexes based in the use of various positive (CRP, SAA or Hp) or negative (albumin) acute phase proteins could improve the sensitivity of APPs when monitoring treatment. It is expected that in the near future the development of cheaper automated assays for determination of main APPs and the increase in the number of experimental studies about APPs response and evolution after treatment in different diseases will contribute to a wider use of these proteins as biomarkers of infection and inflammatory lesions and their inclusion in the routine biochemical profiles in canine species. References Conner JG, Eckersall PD. Acute phase response in the dog following surgical trauma. Res Vet Sci 1988; 45: 107-110. Eckersall PD. The time is right for acute phase protein assays. Vet Journal 2004; 168 (1): 3-5. Jergens A, Scheiner C, Frank D, Niyo Y, Ahrens F, Eckersall PD, Benson T, Evans R. A scoring index for disease activity in canine inflammatory bowel disease. J Vet Int Med 2003; 17: 291-297. Martinez Subiela, Bernal L, Ceron JJ. Serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term treatment. Am J Vet Res, 2003; 64, 8: 1021-1026. Martinez Subiela S, Tecles F, Parra MD, Ceron JJ. Proteínas de fase aguda: conceptos básicos y principales aplicaciones clínicas en medicina veterinaria. An Vet, 2001; 17: 99-116. Martinez Subiela S, Ceron JJ. Evaluation of acute phase protein indexes in dogs with leishmaniasis at diagnosis, during and after short-term treatment. Vet Med –Czech, 2005; 50: 39-46. Parra MD, Väisänen V, Cerón JJ. Development of a time-resolved fluorometry based immunoassay for the determination of canine haptoglobin in various body fluids. Vet Res. 2005; 36:117-129. 12 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 APP IN FELINE DISEASES: QUANTITATIVE CHANGES AND ALTERATION IN GLYCOSYLATION Paltrinieri S Dept of Veterinary Pathology, University of Milan, Milan, Italy Email:firstname.lastname@example.com In this presentation the current knowledge about acute phase proteins (APPs) in feline diseases will be reviewed. Several studies in the past few years have been focused on quantitative changes or on altered glycosylation of acute phase protein in feline. These studies were mainly focused on alpha-1-acid glycoprotein (AGP) and serum amlyoid A (SAA) while Haptoglobin (Hp) or other APPs such as C Reactive Protein (CRP) have been rarely investigated. AGP and SAA can be considered major feline APPs, and these molecules might to e used as a marker of disease in this species. Specifically, increases in blood concentration of AGP and SAA have been reported in experimentally induced inflammation, in post-surgical follow up and in several spontaneously occurring pathologic conditions, including injury, renal failure and other urinary disorders, tumours, diabetes, and infectious diseases (Kajikawa et al., 1999; Sasaki et al., 2003). Within this latter category, the behaviour of APP has been extensively investigated during Feline Immunodeficiency Virus (FIV), Feline Leukemia Virus (FeLV) and especially Feline Coronavirus (FCoV) Infections. Increases of circulating levels of AGP were found in cats with FeLV but not in those with FIV (Duthie et al., 1997). The most evident changes of APP concentrations in blood, however, have been reported in feline coronaviroses, Increased levels of AGP and Hp in cats with Feline Infectious Peritonitis (FIP), a lethal disease induced by high pathogenic FCoVs, were firstly reported by Stoddart et al (1988) and confirmed by Giordano et al. (2004), which also reported increased levels of Hp and SAA in cats with FIP. AGP increases in these cats are so high that this APP might be considered a good diagnostic marker for FIP both in blood (Duthie et al., 1997) and in peritoneal fluid (Bence et al., 2004). Interestingly, during FIP outbreaks in FCoV endemic catteries, APPs transiently increases also in seropositive cats that do not develop the disease (Giordano et al., 2004) and recent unpublished data from our group demonstrated that these fluctuations are correlated with fluctuations of anti-FCoV antibody titres and, to a lesser extent, with faecal shedding of FCoVs, thus suggesting that AGP might play a role in protecting FCoV infected cats from the development of clinical forms of the disease. Also AGP distribution in tissues is different in cats with FIP compared with what observed in cats with other types of inflammation, since AGP extravasation from intralesional vessels is frequently detected in FIP but not in other pathologic conditions, but this is likely a consequence of the type of vascular lesions that characterizes FIP, rather than a pathogenic factor (Paltrinieri et al., 2003). Apart from these quantitative changes, also the glycosylation pattern of feline AGP has been extensively investigated, due to the well know role of AGP glycosylation changes reported in humans. Again, the more evident changes were reported during FIP: symptomatic cats have a decreased syalylation compared to controls and to non- symptomatic FCoV-positive cats (Ceciliani et al., 2004), and preliminary data about other monosaccharides expressed on AGP revealed some difference between cats with FIP and controls (Cunningham et al., 2004). All these changes confirm that the glycan moiety of AGP play some important pathogenic role also in cats. We are now studying the possible presence of glycosylation changes in FCoV-positive, non-symptomatic cats with increased AGP levels in order to clarify whether the glycan moiety might play some role in protecting infected cats from the development of lesions and symptoms. Syalylation pattern of AGP has been found to be altered also in other viral diseases. Specifically, AGP from FeLV-positive cats showed increased syalylation only in those cats which developed lymphoid tumours (Ceciliani et al., 2005). In conclusion, the analysis of APP concentration and/or of APP glycosylation pattern in feline disease has been used in the past to support clinical diagnoses and to draw useful information about the pathogenesis of many diseases, mainly induced by infectious agents. Nevertheless, further studies should be designed in order to elucidate some unclear aspects of feline APPs biology and pathology (e.g. the possible expression of APPs on circulating cells or the characterisation of APP receptors) that have already been approached as regards human APP. References: Kajikawa et al. (1999) Vet Immunol Immunopathol, 68:91; Sasaki et al. (2003) J Vet Med Sci 65:545; Duthie S et al (1997) Vet Rec 141 :299 ; Stoddart ME et al. (1988) Vet Rec 123:622; Giordano A et al. (2004) Vet J 167:38; Bence LM et al. (2004) Vet Clin Path 33:258; Paltrinieri S et al (2003) Comp Clin Path 12:140; Ceciliani F et al. (2004) Vet. Immunol Immunopathol 99:229; Cunningham K et al (2004), Vet Clin Path 33 :258; Ceciliani F et al. (2005) Vet Immunol Immunopathol, in press 13 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 MEASURING NEGATIVE ACUTE PHASE PROTEINS TO ASSESS ACUTE PHASE RESPONSE AND STARVATION Toussaint MJM1, Campbell FM2, Piñeiro M3, Gruys E1 1 Department of Pathobiology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands 2 Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland 3 Department of Biochemistry and Molecular and Cellular Biology, Faculty of Sciences, University of Zaragoza Zaragoza, Spain.Email: email@example.com A host of different processes induce an acute phase reaction in the body. Most widely studied are infections and inflammations that induce the typical change in blood profile. Although at first efforts have been made to fully understand when and how acute phase proteins (APPs) increase in concentration (the positive reacting proteins), lately more interest is put on the negative reacting proteins. These proteins are normally present in healthy animals, but will decrease in concentration due to the acute phase reaction. As for the positive reacting proteins also the negatives display a species difference. Not all proteins react in all species to the same extend. An overview will be presented on the negative reacting proteins with most emphasis on the pig. Most proteins that change in concentration during the acute phase reaction play a role in the attempt of the body to regain the homeostasis. The general role of proteins increasing in concentration can be scavengers of free hemoglobin or free oxygen radicals; scavengers of (parts of) bacteria; can activate the complement reaction; help in the transport of cholesterol and more. Albumin (Alb, MW 66,000 Da) is generally accepted as negative APP present in most species. Furthermore, decreased levels of albumin are also associated with malnutrition. A decline of appetite is a phenomenon often seen in mammals suffering from an acute phase reaction. The decrease in albumin during the acute phase reaction will be a combined effect from the reaction itself and the starvation of the animals. The negative reacting protein transferrin (Tf, MW 80,000 Da) is possibly involved in the innate immunity, perhaps by sequestering ferric ions to prevent pathogens and parasites from using nutrients. Retinol binding protein (RBP) is a small molecular weight protein (MW 21,000 Da), which is the exclusive protein for transport of vitamin A (retinol) in the body. The synthesis and secretion of RBP by parenchymal hepatocytes is mainly controlled by the concentration of retinol in the body. Circulation of retinol bound to RBP occurs mainly in combination with a larger, tetramer protein: transthyretin (TTR, MW approx. 55,000 Da). The complex formation appears to be necessary to prevent extensive loss of the low molecular weight RBP through glomerular filtration. Apart from its role of facilitating the transport of retinol, TTR is one of the three major thyroxin-binding proteins. Measurements of RBP and TTR will strongly correlate; in human medicine these proteins are routinely measured as indicator of health status. Apolipoprotein A-I (ApoA-I) is the major component of α-lipoprotein of high-density lipoprotein. Lipoproteins can inhibit complement binding to the target membrane. Serum Amyloid A (SAA) is positive reacting acute phase protein, also related to high-density lipoprotein (HDL). SAA and ApoA-1 are shown to be expressed as each other's counterparts in an acute phase reaction; possibly due to the competition on the HDL molecule. An increase in SAA is matched by the decrease in ApoA-I. In human medicine an index was defined were positive reacting proteins were combined with negative reacting proteins. The index obtained was called PINI (Prognostic Inflammatory and Nutritional Index) and reveals a sensitive tool to assess health. For animals a comparable index was defined: the acute phase index (API) were quick and slow reacting positive reacting proteins are combined with quick and slow reacting negative proteins. As soon as the most suitable proteins are defined for different species this tool can be applied as indicator for health, non-health and even be used to define recovery from periods of illness. Acknowledgements: Support from the European Commission for these studies is gratefully acknowledged (Shared Cost Project QLK5-2001-02219) 14 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 ACUTE PHASE RESPONSE PROFILES AS INDICATORS OF DISEASE SEVERITY, RECOVERY OR VACCINE EFFICACY IN BOVIDS INFECTED WITH PASTEURELLA MULTOCIDA Hodgson JC Moredun Research Institute, Bacteriology Division, Pentlands Science Park, Bush Loan, Penicuik, UK. Email:firstname.lastname@example.com The potential diagnostic value of acute phase proteins as markers of inflammation and infectious disease may be assessed by measuring changes in their serum concentrations during experimental infectious challenge studies. Plasma concentrations of acute phase proteins change by at least 25% during inflammation and are useful markers with which to determine the progression of disease with time and to characterise initial host responses. Hp is particularly useful as a sensitive marker of bacterial infection, increasing between 10- and 100-fold after challenge. Such studies provide information also on the function and possible therapeutic role of acute phase proteins. The role of the acute phase response in pneumonic and systemic infectious disease in ruminants caused by the Gram-negative bacterium Pasteurella multocida is the subject of the following review. P. multocida serotype A:3 is a major cause of pneumonic pasteurellosis in cattle, the incidence of which is rising steadily in the UK whereas serotype B:2 causes haemorrhagic septicaemia (HS) in South and Southeast Asia, resulting in severe economic losses amongst livestock through morbidity and mortality, particularly in buffaloes (Bubalus bubalis). Disease severity Pneumonic pasteurellosis Pneumonic pasteurellosis in young ruminants due to P. multocida serotype A is an infectious disease of high economic and welfare importance, but the mechanism of disease is poorly defined and the prevention and treatment of disease remains inadequate. The involvement of acute phase proteins in the pathogenesis and resolution of disease has been studied in calves challenged intratracheally with about 109 colony forming units (cfu) P. multocida A:3 given either in low (60ml) or high (300ml) volumes of suspension. All experimental protocols were approved by the Moredun Research Institute Animal Experiments Committee, authorised under the Animals (Scientific Procedures) Act 1986 and access to veterinary care was available at all times. Concentrations in jugular vein blood samples of acute phase proteins haptoglobin (Hp, using purified bovine Hp as a standard), α1-acid glycoprotein (AGP, by radial immunodiffusion kit, Saikin Kagaku Institute, Japan) and serum amyloid A (SAA, by ELISA, Tridelta Phase range, Greystones, Ireland) against time were summarised using the area under the curve (AUC) adjusted to individual time-zero baseline values and analysed using REML. Plasma Hp concentrations increased linearly to a mean value of about 1g/l, significantly greater (P<0.05) than the mean of the pre- infection samples, falling gradually thereafter. Increases in the concentration α1-AGP were more gradual than those observed for SAA or Hp and maintained for longer, reaching a peak mean concentration of 435 mg/l at 48 h post-infection (p.i.) declining thereafter with a slight recovery between 72–96 h p.i. Concentrations of SAA increased rapidly between 5 and 23 h p.i., remaining elevated (approximately 400 –600 mg/l) until falling dramatically to about 5 mg/l between 72 and 96 h p.i. All treatments induced clinical signs characteristic of bovine pneumonic pasteurellosis as observed in natural cases of disease, including moderate depression, pyrexia, laboured breathing, mild nasal discharge, and anorexia. In addition, the gross pathological and microscopic changes observed were similar to those reported both for experimental induction and field cases of bovine pneumonic pasteurellosis associated with P. multocida. The acute phase protein results indicated that, of the two treatment variables used (dose and volume), volume was the more influential factor inducing pneumonic disease. It was interesting that animals given the high volume challenge exhibited higher (P<0.05 ) peak responses in Hp. The high volume challenge probably affected a greater area of lung, especially if an initially slow response in Hp production allowed more time for the bacteria to proliferate. Examination of α1-AGP concentrations for all treatments indicated that the high volume challenge gave rise to a greater increase from resting level than the low volume challenge. Similar correlation of α1-AGP with severity of disease has been reported for other disorders aside from pneumonic pasteurellosis, and in previous work examining natural acute versus chronic inflammation in cattle. However, in the present work there was no correlation between acute phase protein concentrations and lung bacterial clearance. Systemic disease Haemorrhagic septicaemia in buffalo, a form of endotoxin shock, is characterised by a short clinical course (hours) and signs may include severe depression, pyrexia, submandibular oedema, dyspnoea followed by recumbency and death. There is no reliable long-acting vaccine and the pathogenic mechanisms are poorly defined. To investigate the role of the acute phase response during the disease, changes in acute phase protein concentrations were monitored during development of haemorrhagic 15 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 septicaemia in buffalo after intranasal infection with a representative strain of P. multocida B:2. Mean serum Hp concentrations in infected animals rose by 24h p.i. and the mean increase was significantly higher (P<0.05) than that in control (non-infected) animals, reaching a peak of 2.5 ± 0.6 mg/ml at 72h p.i. Serum AGP concentrations increased significantly (P<0.05) in surviving animals to about 300 µg/ml between 48h and 120h after infection but showed no change from a mean baseline concentration of 80 ± 6.6 µg/ml in control animals or in infected animals that succumbed to infection Recovery phase Studies of the pathogenesis of haemorrhagic septicaemia indicated that surviving animals went on to develop greater levels of Hp and α1-AGP. This was perhaps instrumental in restricting bacteraemia to levels below those that trigger endotoxaemia as acute phase proteins have been shown to enhance protection against bacterial infections possibly by modifying the inflammatory responses through effects on cell trafficking and mediator release. Responses after vaccination In efficacy studies in calves using an attenuated live AroA mutant of P. multocida B:2 as a potential vaccine against haemorrhagic septicaemia, SAA concentrations after intramuscular (i.m.) or intranasal (i.n.) vaccination were compared. Different patterns of response were observed between i.m. and i.n. vaccinated calves: concentrations increased significantly (P < 0.05) in i.m. vaccinated calves but showed no significant changes in i.n. vaccinated calves. The SAA concentrations declined in i.m. vaccinated calves after 24 h but increased again significantly (P < 0.001) in response to a second vaccination. No significant changes due to a second vaccination were noted for i.n. vaccinated calves. The concentrations of SAA in individual calves varied considerably, and the means before challenge were highest in the control calves, followed by i.m. vaccinated and then by i.n. vaccinated calves. The mean SAA concentration in i.n. vaccinated animals increased 13-fold by 10 h after challenge but by only 66 and 11% in i.m. vaccinated and control calves, respectively, and this difference between i.n. vaccinated calves and the others was statistically significant (P < 0.01). At 23 h p.i., SAA values exceeded 158 µg/ml in the two calves, one each from the i.n. vaccinated and control groups, that had survived, whereas the maximum increase observed for calves in the i.m. vaccinated group was only threefold (to 37 ± 15.6 µg/ml). High SAA concentrations took 6 to 8 days to return to prechallenge levels in the surviving calves. One calf from each of the otherwise vulnerable i.n. vaccinated and control groups did not develop disease. The reasons why not all animals succumb to infection are not clear, although it may that a gradual but sustained rise in acute phase proteins may play a part in helping to control disease. Thus, the large increase in SAA concentrations after challenge observed in these particular calves may have contributed to their survival. On the other hand, changes in the concentrations of SAA may also be used to indicate the progress and severity of infection. In the present work, the large increase in SAA concentrations observed in i.n. vaccinated animals upon challenge, compared with the moderate response of the i.m. group, may well have reflected a less well-developed immune protection in the former group. However, there was no clear indication that a higher SAA level prechallenge might represent a marker for subsequent survival, as an i.n. vaccinated calf that subsequently survived challenge had a low SAA level of 4.3 µg/ml while a control calf that survived had a high level of 128 µg/ml at the point of challenge. The acute phase response to the initial i.m. vaccination paralleled the clinical response (increased rectal temperature and overall clinical score), indicating that the attenuated bacteria persisted long enough to promote an inflammatory response. Other work showed strong evidence (P<0.001) that calves that had high levels of Hp or α1-AGP before challenge tended to have high levels of Hp or α1-AGP, respectively, throughout the first five days p.i.. A rapid rise in plasma SAA concentrations for calves immunised via intratracheal instillation of killed P. multocida was similar to that reported for cattle challenged intratracheally in other work with live Mannheimia haemolytica but less than the response observed following live P. multocida challenge. The lack of any statistically significant differences in Hp concentrations between immunised and control animals contrasted markedly to that following a live challenge of P. multocida when Hp concentrations increased linearly to a peak at 5 h p.c of 1 g l-1. Conclusions Concentrations of the acute phase proteins Hp, SAA and α1-AGP increase following experimental infectious challenge with P. multocida A:3, suggesting a role for these proteins as markers of the onset and progress of bovine pneumonic pasteurellosis or, in the case of α1-AGP, as contributors to controlling bacterial infection. Similar responses are noted in experimental challenge studies in cattle and buffalo infected with P. multocida B:2. Future detailed studies of host acute phase response together with host innate immune responses in vivo and in vitro will help extend our understanding of the pathogenic mechanisms involved and how to combat disease caused by P. multocida. 16 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Acknowledgements This presentation is a synthesis of work done over recent years with a number of colleagues, including Aileen Dowling (a former PhD student now working at the University of Thessalonika), David Eckersall (Glasgow University Veterinary School), Neil Horadagoda (University of Peradeniya, Sri Lanka), Anna Finucane, Saeed Ataei, Roger Parton, and John Coote (Glasgow University) Iain McKendrick and Jill Sales (Biomathematics and Statistics, Edinburgh), Alex Schock (Veterinary Laboratories Agency, Edinburgh) and Mark Dagleish (Moredun, Edinburgh). Funded by the Scottish Executive Environment and Rural Affairs Department, the Biotechnology and Biological Sciences Research Council, Intervet (UK) Ltd and the Wellcome Trust. 17 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 OVOTRANSFERRIN AS AN AVIAN ACUTE PHASE PROTEIN AND ITS IMMUNOMODULATORY FUNCTION Rath NC Agricultural Research Service, Poultry Science Center, University of Arkansas, Fayetteville, USA Acute phase proteins (APP) are humoral components of natural immunity elicited in response to physical, chemical, or infection-induced physiological injuries. The functions of these proteins are to recognize and protect the organism against invading pathogens, and restore physiological homeostasis. Although a substantial body of literature exists with respect to several mammalian APPs and their diagnostic potential, such information are limited in birds. To understand the avian acute phase response, we experimentally induced inflammation in broiler chickens by (1) endotoxin and bacterial cell wall administration and (2) croton oil. An analysis of serum protein patterns between 24-48 h after injection showed a consistently up regulated 65kD-band protein in birds with inflammation. Using a 2D gel and protein sequencing we identified the protein to be conalbumin also called, ovotransferrin (oTF). A commercially available anti-chicken transferrin antibody was used to develop a competition ELISA for ovotransferrin and measure its time course changes in blood during croton oil-induced inflammation and after viral and bacterial infections of SPF birds. Ovotransferrin levels were increased to different degrees under different infections. Transferrins are antibacterial proteins that relates to their iron sequestering properties. To find whether ovotransferrin has other functions beyond its anti-bacterial efficacy, we examined its effects on phagocyte functions using HD11 macrophage and avian peripheral blood heterophil cultures. Ovotransferrin upregulated interleukin-6, nitric oxide, and matrix metalloproteinase production and stimulated respiratory burst activities. Macrophages required priming by phorbol myristate acetate, a protein kinase C activator, to stimulate their respiratory burst activities. Heterophils did not produce nitrite in response to oTF treatment but showed degranulation as measured by the loss of granules and increase in myeloperoxidase release. Most stimulatory activities were limited to concentrations above 100 ug/ml. Despite small conditional differences in immunostimulatory effects of oTF on macrophages and heterophils, the results suggest that oTF may be a natural immunomodulator which protects the organism not only by its direct action against bacteria but by upregulating other defense systems. 18 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 ACUTE PHASE REACTANTS, CHALLENGE IN THE NEAR FUTURE Gruys E1, Toussaint MJM1, Upragarin N1,2, van Ederen AM1, Adewuyi S1, Candiani D1,3, Nguyen TKA1,4, Sabeckiene (Balciute) J1,5 1 Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht The Netherlands, 2Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Thailand, 3Department of Veterinary Morphophysiology, University of Torino, Torino, Italy, 4Department of Embryo Transfer and Reproduction, the National Institute of Animal Husbandry, Hanoi, Vietnam, 5Department of Infectious Diseases, Lithuanian Veterinary Academy, Kaunas, Lithuania Email:email@example.com Introduction By many different scientists from various disciplines the acute phase reaction known to occur on infection, inflammation, trauma, burns, malignancies and tissue damage in general, has been studied. Last decade emphasis has been given to application of tests for acute phase reactants to monitor animal health in general, as well as for human patients suffering from specified classes of diseases. However, basic mechanistic patterns associated with biological reaction patterns still remain to be discovered, such as local production of acute phase proteins by cells of organs involved in specific physiological mechanisms and disease processes. This may be associated with defence functions and with development of, even worse, tissue alteration. Precolostrum mammary tissue (McDonald et al, 2001) and mastitic mammary epithelium have been shown to form mammary serum amyloid A (mSAA) (Vivanco et al, 2002; Nguyen et al, 2002, Balciute et al, 2005). Moreover, haptoglobin (Hp) and other acute phase reactants are found in the milk. These factors are supposed to have functions in regulating the inflammatory process and to be beneficial for the enteric milieu of the young mammal including protection of the gut mucosa by mucus formation. In birds with chronic arthritis the synovial cells may reveal SAA upregulation, SAA protein formation (Ovelgönne et al., 2001; Upragarin et al, 2002, 2005a) and amyloid formation (Landman, 1998, Upragarin et al, 2005a). The present paper facing future developments expected, will be based on i. present scientific activities concerning basic mechanistic patterns not discovered sofar, ii. development of technology to measure quantities of proteins or cellular upregulation of their formation, and iii. assessment of disorders, health and welfare, and iv. it will regard some topics in these fields. Basic mechanistic patterns Specific organs Differentiated reactivity patterns of the parenchymal cells per organ involved, such as mammary gland, will depend on cytokines locally active and are to be unraveled. Moreover, cell specific factors may be liberated. Just like enzymes known from clinical enzymology, specific cell proteins such as the fatty acid- binding gut protein (Niewold et al, 2004), may be discovered and find their applications in diagnostics and therapy. Functional aspects of acute phase reactants in milk and blood need further attention. When activities which activate or just mitigate the inflammatory reaction, are known, this knowledge can lead to new ways for therapy and prevention of inflammatory processes in organs involved, such as lung or mammary gland. Local SAA formation and its precipitation as amyloid as has been found in avian amyloid arthropathy (Landman, 1998), need further investigations. From the pathogenetic mechanisms preventive measures may be developed. Once the beta-pleated sheets of amyloid have been formed, the substance has tremendous food safety implications. In murine studies orally administered AA-amyloid appeared to enhance inflammation- / acute phase reaction-induced amyloidosis, whereby the administered material acted as nidus for amyloidogenesis. This indicates a strong ban of this pathological material for risk groups of consumers comparable to that of prions. In mammary tissue, colostrum and fresh milk corpora amylacea may occur which contain amyloid (described to be derived from casein [Niewold et al, 1999]). Recently this amyloid was found to be positive for SAA (Toussaint et al, 2004; Balciute et al, 2005), and thus, as well as locally formed mSAA as unwanted beta pleated factors in colostrum and milk related to this SAA, need to be investigated. 19 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Various proteins In birds acute phase proteins such as (ovo)transferrin appear to have special characteristics differing from mammals. In mammals and possibly in avian species as well, some acute phase proteins may reveal differences in glycosylation patterns associated with different diseases and stages of those diseases, as has been shown for feline α1-acid glycoprotein (AGP). Further analysis may unravel basic biological mechanisms, indicate specificity of the glycosylation patterns for disease and reveal new concepts for therapy. Reaction of different analytes in various situations, separate and combined Viral, bacterial, and protozoal infections may be associated with different patterns in cytokine release and acute phase reactivity. Inflammatory processes in internal organs appeared to result in more severe reactivity patterns than diseases of the skin and the enteric system. Specific knowledge of pattern details may lead to implication of the parameters in diagnostics and stageing of the disease. Calculation of an index from values of rapid- and slow-reacting positive and negative APPs has been repeatedly mentioned (Gruys and Toussaint, 2001; Gruys, 2002; Tousaint et al., 1995, 2002, 2004; Niewold et al., 2003), because it appeared to increase statistical sensitivity and specificity for detecting non-healthy subjects. It covers a broad time span and includes changes in blood values resulting from the body's reactivity as well as starvation. In layer chickens on induction with Staphylococcus aureus or turpentine an acute phase protein reaction was induced. Measurement of values for SAA, transferrin, serum albumin and apolipoprotein A-1 in blood samples of these birds (Upragarin et al., 2005b) showed that calculation of an acute phase index, offers promising results in this species. Outcome was as has been calculated for cows with various diseases (Toussaint et al., 1995) and for pigs with a Streptococcus suis infection (Toussaint et al., 2002). Infections and vaccination To prevent spontaneous disease often vaccination is propagated. It has been shown, however, that upon vaccination an acute phase protein reaction may develop. This appears to limit the profitability of vaccines, because acute phase reactions are known to be contraproductive in view of muscle anabolism. Future interest will be more on amino acid pattern differences in muscle and APPs (Table 1) and on the negative acute phase reactants. On starvation and negative energy balance associated with most diseases, muscle proteins are catabolised for amino acid supply of the hepatic APP formation and as source of energy. Especially for those APPs which rapidly and quantitatively increase in blood, their formation may have amino acid impact. An increased hindquarter protein catabolism exceeding the hepatic protein synthesis, and efflux of glutamine and alanine from the hindquarter was measured during a porcine induced endotoxemia study (Bruins et al., 2003). For growth during and after recovery from a disease, food requirements for amino acids thus may differ from the formula in ordinary food. Some pig studies indicate positive influences of additional dietary tryptophan (Le Floc'h et al., 2004) or L-arginine (Bruins et al., 2002). Negative APPs may be associated with a change in concentration of bound compounds. A decrease of retinol-binding protein and of vitamin A values may be vice versa interrelated, vitamin A-deficiency being well known to decrease immune reactivity of children in developmental countries. It is striking to encounter a huge negative variation from normal blood vitamin A values of around 1-0.75 µMol/l to around <0.1 µMol/l in fattening pigs, as was revealed in a local investigation by a Dutch practitioner (Hogendoorn, 2004). In cattle an association of APPs with parturition, starvation and ketosis has been described. Rise in non- esterified fatty acids (NEFAs) occurs; their level increase might parallel those of some APPs. It is to be expected that due to negative APPs, blood vitamin A levels decrease as suggested for the pig. The NEFAs are toxic and have a negative influence on metabolism. It is hypothesized that the NEFAs may decrease on increased muscular acitivity (walking). An inversive association with walking activity has been shown (Adewuyi, 2004). 20 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Table 1. Amino acid composition differences between muscle protein and major acute phase proteins after a gross amino acid composition list (Reeds et al, 1994) Amino acid skeletal muscle protein CRP SAA Phe 40 105 103 Tyr 36 50 67 Trp 13 42 45 Arg 69 36 116 Ala 59 31 106 Development of technology to measure quantities of proteins or cellular upregulation of their formation After radio-immunoassays (RIA) and enzyme-linked methodology (ELISA) at present several groups develop methods for rapid measurements of APPs. Nephelometry, turbidimetry, and other methods on liquid phase analysis, and protein array methodology on slides, as well as 3D electrophoresis with MAS spectrometry have been shown to be applicable on samples with acute phase reactants. Especially these technological developments are of crucial importance for the future. When rapidly and with low costs many samples can be handled, the APPs have a diagnostic future. Assessment of disorders, health and welfare Specific groups of patients, such as castrated horses, cows with mastitis, periparturient sows, sheep with mastitis, or dogs and cats with infectious disorders have benefit form acute phase reactant measurement. When larger groups of animals are involved and this may concern a more wide variety of diseases, multi- analysis technology coupled with pattern recognition software has the power of selective diagnostics. At least, analysis of reaction pattern differences on the same agent may be used for selection and breeding purposes. Conclusion Future possibilities for acute phase reactants depend on basic new mechanistic findings of known proteins, new discoveries such as organ specific components, and on technological possibilities for rapid chemical multianalyses with computer analysis of the patterns found. The shared cost EU project (number QLK5-2001-02219) on porcine acute phase proteins has helped to spread the knowledge to specified scientists of member states involved, and to develop a base for practical applications. For other species such as cattle, horse, dog and cat, but also chicken and even human, a similar field is open for development of clinical and health management applications. Finally, new fields of research and application are in the negative metabolic influences of acute phase processes and their relationship with growth and nutrition. References 1. Adewuyi AA, 2004. Relationship between plasma NEFAs concentration and physical activity in postpartum ruminants. Thesis at Van Hall Institute, Leeuwarden, The Netherlands, project number 33410. 2. Balciute J et al., 2005. Serum amyloid A (SAA) in bovine tissues with inflammatory processes and in mammary corpora amylacea. To be published. 3. Bruins MJ et al., 2002. L-Arginine supplementation in pigs decreases liver protein turnover and increases hindquarter protein turnover both during and after endotoxemia. Am J Clin Nutr 75:1031-1044. 4. Bruins MJ et al., 2003. Aspects of organ protein, amino acid and glucose metabolism in a porcine model of hypermetabolic sepsis. Clin Sci 104:127-141. 5. Gruys E, 2002. Acute phase proteins in bovine medicine. In: Proceedings AVMA congress 2002, Nashville USA: AVMA 2002 Convention Notes, pp 317-321. 6. Gruys E and MJM Toussaint, 2001. Monitoring animal hygiene, welfare and health by analytes of the acute phase reaction. Non-specific assessment of infection, inflammation, bruising, stress and starvation. In: Proceedings 19th ESVP meeting, Thessaloniki, Greece 25-28 Sept. 2001, pp 113-131. 7. Hogendoorn MP, 2004. Vitamin A in fattening pigs (in Dutch). Internal report, 2004. 8. Landman WJM, 1998. Amyloid arthropathy in chickens. PhD thesis, Utrecht; ISBN 90-393-1667-8. 9. Le Floc'h N et al., 2004. The importance of dietary tryptophan for preserving growth and controlling inflammatory response of weaned pigs submitted to immune stress. In Proceedings ISAH congress, October 11-13, 2004, St Malo, France, Vol I, pp 239-240. 10. McDonald TL, 2001. Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A3 (M-SAA3) into colostrum. Vet Immunol Immunopathol 83:203-211. 11. Niewold TA et al., 2003. Monitoring health by acute phase proteins. In: Animal welfare and acute phase proteins. Proceedings of Fourth European Colloquium on acute phase proteins Segovia, Spain, 25- 26 September, 2003, pp 57-67. 12. Niewold TA, et al., 2004. Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs. Res Vet Sci 77:89-91. 21 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 13. Nguyen TKA et al., 2003. Serum amyloid A in acute phase response of cows with mastitis experimentally induced by Streptococcus uberis. In: Animal welfare and acute phase proteins. Proceedings book of Fourth European Colloquium on acute phase proteins, Segovia Spain 2003, pp 104- 105. 14. Ovelgönne JH et al., 2001. Identical amyloid precursor proteins in two breeds of chickens which differ in susceptibility to develop amyloid arthropathy. Amyloid: J Prot Fold Disord 8:41-51. 15. Toussaint MJM et al., 1995. Implication of clinical pathology in assessment of animal health and in animal production and meat inspection. Comp Haematol Int 5:149-157. 16. Toussaint MJM et al., 2002. Combination of values for acute phase proteins (APP) in an index in a pig model with induced Streptococcus infection. In: Abstract Book of First international congress on transthyretin in health and disease. April 22-25, Strasbourg, p 100. 17. Toussaint MJM et al., 2004. Serum amyloid A (SAA) in mammary tissues with inflammatory processes and in mammary corpora amylacea. In congress book Xth International symposium on amyloid and amyloidosis, 18-22 April 2004, Tours, p 53. Proceedings in press. 18. Upragarin N et al., 2002. Serum amyloid A (SAA) mRNA and protein expression in primary culture chicken synoviocytes. In Proceedings Third Colloquium on acute phase proteins, Kaap Doorn, Doorn the Netherlands, p 72. 19. Upragarin N et al., 2005a. AA-amyloid formation by primary chicken fibroblast-like synoviocytes. To be published. 20. Upragarin N et al., 2005b. Acute phase protein reaction in layer chickens. A calculated acute phase protein index as measure to assess health during the rearing period. To be published. 21. Vivanco V et al., 2003. Immunohistochemical investigation on serum amyloid A (SAA) in bovine tissues with inflammatory processes. In: Animal welfare and acute phase proteins. Proceedings book of Fourth European Colloquium on acute phase proteins, Segovia Spain 2003, pp 106-107.Invited 22 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Open Communications - Oral 23 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 MONITORING THE HEALTH OF FATTENING PIGS AT SLAUGHTER: INVESTIGATIONS ON A HERD CUT-OFF FOR HAPTOGLOBIN CONCENTRATION IN MEAT JUICE Van den Berg A, Danuser J, Regula G Swiss Federal Veterinary Office, Schwarzenburgstrasse 161, Switzerland Introduction The presence of a large number of animals with an acute phase response within a slaughter group can indicate acute or chronic health problems within a herd. Testing haptoglobin concentration in meat juice may therefore be a useful screening test to assess animal health and welfare status of a herd at slaughter. Our aim was to investigate the association between clinical findings at the abattoir and haptoglobin concentrations in meat juice on a herd level. In addition, we explored the ability of a herd test based on different cut-off levels for haptoglobin concentrations to identify herds with clinical health problems. Materials and Methods In a cross-sectional study at two larger abattoirs in Switzerland, 107 groups of slaughter pigs were examined. At the ante-mortem examination, the percentage of pigs with clinical signs (lameness, peritarsitis, tail biting, othaematomas and injuries or abscesses) per slaughter group was determined. From an average of 20 pigs per slaughter group, a post-mortem examination was done at which alterations of lungs, livers, hearts, and carcasses were recorded. Meat samples of the neck were taken from an average of 20 pigs per slaughter group, in total 2192 samples. Meat juice was obtained by freezing and thawing of the meat samples. Meat juice was single tested for haptoglobin concentration with RIDASCREEN® Haptoglobin from R-Biopharm. The different health indicators (clinical signs and lesions) were screened for statistical association with haptoglobin concentration with the Mann-Whitney test. Variables with a p-value <0.05 were entered in multinomial logistic regression models. Herds were classified as positive or negative based on different cut-off values for haptoglobin. Results Of all samples, the mean haptoglobin concentration was 0.15 mg/ml (s=0.25), the median 0.06 mg/ml (25%tile=0.03, 75%tile=0.16). Pigs from groups in which tail biting, lameness, othaematomas, peritarsitis, abscesses, pneumonia, pleurisies, pericarditis or condemnations of livers, lungs or carcasses occurred, showed higher levels of haptoglobin than pigs from slaughter groups without any of these clinical signs (p<0.05). A good agreement between presence of clinical signs and test for elevated haptoglobin concentration result was obtained when herds were classified as positive if at least 10% of the pigs had a haptoglobin concentration above 0.4 mg/ml meat juice. If a herd cut-off of 5% of pigs with a haptoglobin concentration above 0.6 mg/ml was chosen, similar results were obtained. The multinomial logistic regression model for a cut-off of 0.4 mg/ml and a maximum of 10% of the pigs tested above the cut-off included the variables lameness (OR=2.8), tail biting (OR=2.7) and pneumonia (OR=2.1). The model with a cut-off of 0.6 mg/ml and a maximum of 5% animals above the cut-off identified the variables lameness (OR=3.1), tail biting (OR=2.5) and othaematomas (OR=6.2) as risk factors for positive herds. Both models correctly classified 66% of all slaughter groups according to the presence of clinical signs. Discussion The associations which were observed between clinical signs observed at slaughter and haptoglobin concentration demonstrate a potential of measuring haptoglobin concentration in meat juice as a screening test to identify problem herds at slaughter. However, misclassification of herds is likely to occur for the following reasons. Many acute infections with an acute phase response do not result in clinical signs which can be observed at slaughter. On the other hand, clinical signs can remain visible after the acute phase response has terminated. As the current model classified 66% of the slaughter groups correctly, the test is not yet applicable in practice, and further investigations are needed 24 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 ACUTE PHASE PROTEIN RESPONSE IN PIGS EXPERIMENTALLY INFECTED WITH AFRICAN SWINE FEVER VIRUS Carpintero R1, Alonso C2, Iturralde M1, Alava MA1, Piñeiro A1, Lampreave F1 1 Departamento de Bioquímica y Biología Molecular y Celular,F.Ciencias,. Universidad de Zaragoza, Zaragoza, Spain; 2Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain. Email: email@example.com Introduction Viral infection is a recurrent disease in pigs causing important economic looses. At the present, little is known about acute phase protein (APP) response in infection induced by virus. In this work the response of some APPs, pig-MAP, haptoglobin, C-reactive protein (CRP) and apo A-I have been studied in pigs experimentally infected with African swine fever (ASF) virus. Materials and Methods Seven 3 months old pigs were inoculated intramuscularly with 5x102 tissue culture infective doses of the high virulent ASF virus. Clinical signs of ASF were monitored daily until day 7 in survivors. Blood samples were collected previously to virulent virus inoculation and at everyday post infection (p.i) since 3 days. Viremia was determined tritiating the virus in porcine alveolar macrophages by detection of infected cells using specific antibodies. The concentration of APPs was measurement by radial immunodiffusion using specific antiporcine APP antisera. Results Clinical signs of ASF virus (fever, anorexia, letharhy, shivering, cyanosis and recumbency) appeared in the experimental infection since 3-4 days p.i. and these signs progressed until death in all cases. The concentration of APPs showed significant variation with correlated with the severity of disease along days 3 to 6 p.i. Related to day 0 before infection, pig-MAP concentration increased 6-9 times, Hp 3-7 times and apo A-I decreased 4 to almost 10 times in ones of the pigs. Interestly, CRP did not showed significant response, perhaps because of the variability it showed at day 0. Conclusion The response to the African swine fever virus was faster and severe and unleashed the death of the pigs. Our results indicated that response of APPs (with the exception of CRP) followed up the clinical symptoms of the outcome of this viral disease. R. Carpintero holds a fellowship from Fundación Cuenca Villoro. We thank Nieves Gonzalez-Ramon for her contribution in the initial part of this work. 25 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 EFFECTS OF TWO TEETH RESECTION METHODS ON PLASMA C-REACTIVE PROTEIN AND CORTISOL IN PIGS Llamas Moya S1,2, Boyle L1, Lynch B1 ,Arkins S2 1 Pig Production Department, TEAGASC – Moorepark Research Centre, Fermoy, Co. Cork, Ireland. 2 Department of Life Sciences, University of Limerick, Limerick, Ireland. E-mail: firstname.lastname@example.com Introduction Resection of newborn piglets 'needle' teeth is widely practised in order to reduce facial injuries to piglets during the establishment of the 'teat order' and to minimise damage to the sows udder. Teeth are generally clipped to the gum line using side-cutting pliers. This can cause trauma in the oral mucosa and consequently result in infection and/or inflammation. The use of rotating electric grinders reduces this problem. However, grinding takes more time and involves more handling. This study aimed to determine the effect of two teeth resection methods on concentrations of C-reactive protein (CRP) and cortisol in pigs. Material and Methods Litters from 21 multiparous sows were used. All piglets from the same litter were subjected to the same treatment and had their teeth either clipped (CLIP), ground (GRIND) or left intact (INT). Clipping was done using clean, sharp side-cutting pliers. Grinding was performed using a high-speed diamond coated cylinder, grinding no more than one third of each tooth [Pigmatic 110, SFK Technology A/S, Herlev, Denmark]. The same trained technician applied all treatments within 12 h of birth. Twenty-four hours after treatments were imposed, blood samples from 1 male and 1 female piglet selected from each litter were collected by jugular puncture into lithium heparinised syringes [VacutainerTM, Unitech Ltd., Dublin 24, Ireland]. These animals were also blood sampled 24 h after weaning at 28 days of age. Blood samples were immediately centrifuged at 2000 g for 10 minutes at 5ºC, and plasma was stored at –20ºC until analysis. Plasma samples were analysed for their concentration of CRP [Tridelta Development Ltd., Maynooth, Co. Kildare, Ireland] and cortisol [DGR-Diagnostics, Marburg, Germany]. Data from 1-day- old piglets were subjected to analysis of variance (ANOVA) using the general linear model (GLM) procedures of SAS® to test for main effects of treatment and gender, and their interaction. Data from samples collected on day 29 were analysed similarly using data from day 1 as a covariate. Results Plasma CRP levels on day 1 did not differ between treatment or gender groups (pv>0.1, Table 1). No significant treatment by gender interaction was found (pv>0.1). A tendency towards a significant effect of treatment (pv=0.060) was found on day 29 (Table 1). CLIP pigs had higher plasma concentrations of CRP than GRIND pigs (pv<0.05). There was no effect of gender or treatment by gender interaction in pigs at this age (pv>0.1). A significant effect of treatment was found in plasma cortisol concentrations on day 1 (pv<0.05, Table 1). GRIND piglets had significantly higher levels of cortisol in plasma in comparison to CLIP piglets (pv<0.05). GRIND piglets also tended to have higher plasma cortisol concentrations in comparison to the INT group (pv=0.082). No gender effects were found and the treatment by gender interaction was also not significant (pv>0.1). On day 29 no significant effects of treatment, gender or their interaction were found (pv>0.1, Table 1). Table 1. Plasma concentrations of C-reactive protein (CRP) and cortisol of pigs from three treatments on days 1 and 29 of age. Data are presented in means ± SEM. Intact Clipping Grinding pv Day 1 13.91 ± 3.75 16.54 ± 2.48 15.23 ± 2.10 0.814 CRP, µg/ml Day 29 183.34 ± 33.94 229.47 ± 46.50 108.25 ± 14.10 0.060 Day 1 338.65 ± 52.65 271.07 ± 60.06 494.56 ± 70.01 0.047 Cortisol, ng/ml Day 29 17.38 ± 4.21 32.28 ± 10.72 23.46 ± 6.95 0.124 Discussion CLIP and INT piglets were more affected by infection and/or inflammation at weaning than GRIND piglets. This reflects the higher incidence of mouth and facial injuries respectively in these animals. The lack of a difference between the CLIP and INT groups indicates that both facial and mouth injuries activate the immune/inflammatory response to the same degree. Blood sampling of 1 day-old piglets is 26 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 an acute stressor that results in high plasma cortisol levels. This HPA axis activation was enhanced due to the longer handling times inherent to the GRIND treatment. Plasma CRP determination is a useful indicator to assess the welfare consequences of two different teeth resection methods in pigs at weaning. 27 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 MEASURING MILK QUALITY BY MILK AMYLOID Berry EA1, Hillerton JE1, Hett B2, Harte D2 1 Institute for Animal Health, Compton, Newbury, UK 2Tridelta, Maynooth, Ireland Introduction Amyloid A is an acute phase protein synthesised both in the liver - serum amyloid A (SAA), and the mammary gland - milk amyloid A (MAA). It has been shown to increase in concentration in various inflammatory conditions. SAA is synthesised by hepatic cells and MAA by mammary epithelial cells after the production of pro-inflammatory cytokines, both TNF and interleukin 6 (IL-6). The initial neutrophil response occurs earlier than the increase in SAA and MAA but is not usually measurable in a short time scale. As the presence of MAA occurs in milk early in the inflammatory process and its presence is apparently specific to local and not systemic inflammation then it may be a useful indicator of infection. Conventionally the infection status is measured by the concentration of somatic cells (SCC) in milk. SCC in milk are largely leucocytes derived from blood and generally increase with infection of the mammary gland. Here, the association between SCC and MAA is examined Materials and Methods A longitudinal study on 21 dairy cows was conducted over 33 days. All cows were pregnant and yielding between 18 and 26 litres of milk daily. At the beginning, after 14 days and at the end of the trial and on detection of clinical mastitis, foremilk samples were collected aseptically from each quarter and examined for bacteria. Representative samples (minimum 100 ml) of whole udder milk from each cow were collected daily from the parlour milk meters during morning milking. These samples were divided after thorough mixing into five aliquots with four of the samples sent to three laboratories (one laboratory received a duplicate sample on a three day rotation) and the fifth sample was sent for estimation of the concentration of milk amyloid A (Tridelta Ltd, Maynooth, Ireland). At least three data points were available for the somatic cell count allowing this data set to be cleaned and remove erroneous data points due to laboratory errors, however this was not possible for the MAA results. Results were categorised either as no inflammatory response or as a potential inflammatory response by selecting cut- levels of 200,000 cells/ml for somatic cell count (SCC) response and 800 ng/ml for MAA. Results Seven cows had persistent minor bacterial infections in at least one quarter affecting cell count and MMA throughout the sampling period. Five cows had an infection with either a streptococcus species or a coliform species either for the whole period or just detected at one sample point. Clinical mastitis developed in three of the cows due to these infections. Rises in cell count and MAA were observed in the uninfected cows lasting on average 1.8 days. The cell count of milk correlates with the concentration of MAA. On 24 cow days both SCC and MAA were increased whilst on five episodes of these occasions the MAA response was one day later than SCC, the preceding MAA result was increased but did not exceed 800 ng/ml. On five occasions the MAA response was sustained for at least one day longer than the SCC response. On four cow days the SCC exceeded the threshold but MAA was only 700 to 800 ng/ml MMA (14% false negative results). For 12 cow days SCC exceeded the 200,000 cells/ml threshold but no MAA rise was found (SCC 33% false positive results). Discussion The MAA test may provide promising information on the inflammatory response of the mammary gland complementary or as a substitute for cell count. 28 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 IDENTIFICATION OF THE BOVINE α1-ACID GLYCOPROTEIN IN COLOSTRUM AND MILK Ceciliani F, Pocacqua V, Provasi E, Bronzo V, Moroni P, Saltorelli P Dipartimento di Patologia Animale, University of Milan, Milano, Italy Email: email@example.com Introduction The acute phase protein alpha1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. AGP features at least two different biological activities, apparently very different each from the other: AGP may immunomodulate the inflammatory response, and, meanwhile, act as a plasma transport protein. As the other acute phase proteins, AGP is produced mainly by hepatic cells, but local expression has been reported in human breast epithelial cells, stimulated alveolar macrophages and human endothelial cells. The detection of two of the major bovine APP, haptoglobin and serum amyloid A (SAA) in milk during mastitis may suggest a role for APP also in the immunomodulation of local inflammatory reaction in the udder. This communication presents the detection of the bovine AGP (boAGP) in mammary secretions (colostrum and milk) and mammary gland tissue. Material and Methods Bovine AGP was purified from whey by means of ionic exchange and reverse-phase chromatographic procedure. The amount of boAGP was determined by means of RID test. The cDNA sequence was determined following conventional molecular biology procedure. Results Bovine AGP was detected by Western blotting in all the samples analyzed, and could be quantified in colostrum at 162 (± 63.7) mg/ml and 114.5 (±67.8) mg/ml during the first 12 hrs and 24 hrs respectively. In mature milk the boAGP concentration clearly decreased and was no longer detectable using the Radial Imunodiffusion (RID) technique. Mature milk boAGP's concentration was therefore semi-quantified using an anion-exchange chromatographic procedure that allowed the concentration of the protein to be determined. The presence of AGP in bovine milk was confirmed by the internal sequence analysis performed following the purification to homogeneity of the protein from milk. The concentration of AGP in bovine milk with low SCC (< 250000) was very similar to that from bovine with high SCC (> 250000). In order to investigate the origin of AGP in bovine milk, a search for mRNA was carried out in somatic cells and mammary gland tissue and cells: mRNA expression of boAGP gene was detected in mammary gland tissue, but not in somatic cells. Finally, cDNA sequence of boAGP was determined, and hereby presented: the cDNA sequence (658 bp) codes for 219 aminoacid residues that accounts for a MW of 20410 kDa. Prosite analyses revealed five potential N-glycosylation sites and, interestingly seven potential phosphorylation sites. Discussion What can be the biological significance of the presence of AGP in bovine milk? As an immunomodulatory molecule, boAGP could play several roles during inflammatory challenges in the mammary gland, including the modulation of the undesiderable inflammatory reactions during mastitis due to excessive phagocytic activities that can cause severe mammary secretory cell damages. Bovine AGP can be found also at high concentrations in colostrum in non-pathological conditions. Therefore, it is conceivable that boAGP may also contribute to the complex framework of the immunoregulatory molecules expressed in bovine colostrum. 29 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 EVALUATING THE STRESS OF PRODUCTION IN CATTLE USING HAPTOGLOBIN Slocombe LL, Colditz IG Cooperative Research Centre for Cattle and Beef Quality, University of New England, Armidale, Australia, CSIRO Livestock Industries, FD McMaster Laboratories, Armidale, Australia Email: email@example.com Introduction Cattle production, whether intensive or extensive, exposes animals to a variety of stressors. These stressors can reduce growth through, altered priorities for nutrient utilization, and reduce disease resistance through immunosuppression. Evaluating the stress placed on cattle by a production environment or during a management procedure is not easy, because commonly measured indicators such as cortisol peak and decline rapidly. The acute phase protein haptoglobin (Hp) is a good candidate for measuring stress in cattle due to its half life of 2-4 days (Jain 1993) and its latency to peak (approximately 24-48 hours). These characteristics provide a broad window in which animals can be blood sampled. The aims of this research were firstly to develop a method for measuring Hp which can deal with variable levels of haemolysis in samples and secondly to quantify Hp expression in response to different production stressors. Materials and Methods The method used for measuring Hp in cattle plasma was based on the method of Jones and Mould (1984), which uses the preserved peroxidase activity of the haemoglobin-haptoglobin complex to oxidise a chromogen. A method was developed to correct for the variable levels of haemolysis that can occur at the time of sampling. This is achieved by measuring the endogenous peroxidase in each sample and free haemoglobin (Hb) resulting from haemolysis. Endogenous peroxidase is measured in each sample by substituting methemoglobin with saline. The Hb level within the plasma sample is then measured using a modification of the method described by Levinson and Goldman (1982), before being converted into its contributed Hp interference from the predetermined relationship: (Hp=0.118Hb +0.015; r2=0.97). The Hp values attributed to endogenous peroxidase activity and free Hb are then subtracted from the apparent Hp value determined in the Hp assay to yield a corrected Hp value. Thus Corrected Hp = Apparent Hp – (Hp due to endogenous peroxidase activity + Hp due to free haemoglobin). Blood samples from cattle undergoing different production stressors (weaning, transportation, social re- grouping and intensive management in a feedlot) were assessed for their Hp content. Comparisons were: unweaned versus weaned for 7 days post weaning; before and after transport for 150 km; prior to and during feedlot finishing, and control groups and groups mixed with unfamiliar cattle during feedlot finishing. Hp values were transformed using a natural log scale to obtain a normalised distribution. Results The effect of the different production stressors on Hp levels (range) are shown in Table 1. Table 1: Production Stressors and their haptoglobin ranges Stressor Number of Animals Hp Control Range (mg/ml) Hp Treatment Range (mg/ml) T-value* P-value* Weaning 38 0.000 to 0.259 0.000 to 1.476 t = -2.276 P = 0.025 Road Transportation 110 0.016 to 0.829 0.000 to 0.565 t = -1.986 P = 0.050 Intensive Management 96 0.011 to 0.148 0.000 to 0.853 t = -3.553 P = 0.001 Re-grouping 210 0.000 to 2.853 0.001 to 3.930 t = -3.293 P = 0.001 * The values presented here are from the transformed data Discussion Preliminary analyses have examined the main treatment effect on Hp but not on the other parameters measured (haematology and production traits such as weight gain). It appears as though production stressors increase plasma Hp concentration to varying degrees. Further analyses of these data will examine associations between Hp levels and production variables. Jain NC (1993). Essentials of Veterinary Hematology; p354 Jones GE and Mould DL (1984). Research in Veterinary Science. 37:p87-92. Levinson SS and Goldman J (1982). Clinical Chemistry. 28(3):pp471-474. 30 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 INTERPRETATION OF SAA CONCENTRATIONS IN MILK FROM DAIRY EWES AND GOATS Winter P, Miny M, Baumgartner W Department for Farm Animals and Herd Management, Veterinary University, Vienna, Austria Email: email@example.com Introduction The prevalence of clinical mastitis in dairy sheep and goat flocks is below 5 %, whereas the prevalence of subclinical intramammary infections is estimated about 20-30 %. The detection of subclinical infections within a herd is very poor, the available tools for diagnosis are of limited interest. California mastitis test (CMT) and bacteriological examination cannot be regularly and systemically applied to large herds. Recently, the potential of Serum Amyloid A (SAA) as an early diagnostic marker for clinical and subclinical mastitis in cattle was described. Additionally it could be demonstrated that concentrations in milk are highly elevated during experimentally induced mastitis in ewes. The current study examined SAA concentrations in indivdual milk from dairy ewes and goats to evaluate the potential for SAA as indicators of subclinical mastitis. Furthermore the bulk milk SAA concentrations were measured to check the use for monitoring the udder health status within a herd. Materials and Methods Milk samples from 267 ovine udder halves and from 234 caprine udder halves were collected. The fat- and cell-free fraction of milk was prepared by centrifugation and stored at -20 °C until assayed. Samples were examined for presence of bacteria, and somatic cell counts (California mastitis test) were estimated. SAA was determined by a commercial ELISA kit (Phase SAA kit, Tridelta) according to the manufacturer's instructions. Bulk milk was collected from 7 sheep flocks and from 3 goat herds 5 to 6 times in a monthly interval. For checking the udder health within a herd a randomly assigned group of animals was sampled at each farm visit. SCC (Fossomatic) and SAA were detected in bulk milk samples. For the statistical analysis the Kruskal Wallis test, the Mann-Whitney test and the Pearson's correlation coefficient were calculated. Results The results of the SAA concentrations in individual milk samples are listed in table 1. There was a significant correlation between the SAA concentrations of ovine samples and the CMT results and the bacteriological results, respectively. In goat milk no correlation between the SAA concentrations and the udder health could be found. S. aureus positive goat samples showed significantly higher SAA concentrations. Table 1: SAA concentrations (ng/ml) in individual milk samples ewes (n = 267) goats (n = 234) CMT n mean SD n mean SD negative 156 8,578.9 6,562.1 108 9,088,6 13,710.8 + 34 18,052.6 4,624,0 61 15,627.5 32,189.4 ++ 32 * 99,925.5 212,806.0 42 23,887.7 70,643.5 +++ 45 * 177,242.4 308,125.7 23 20,936.2 34,531.9 BE negative 188 1,096.7 1,017.1 170 10,218.4 19,104.4 CNS 75 * 43,469.2 77,417.3 45 15,896.0 34,512.7 S. aureus 0 17 * 56,170.3 104,281.3 CMT = California mastitis test; SD = standard deviation; BE = bacteriological examination; CNS = coagulase negative staphylococci; * = p < 0.05 No correlations between the SAA concentrations of bulk milk and the SCC and the udder health status in the herd were observed. Conclusions: SAA concentrations in milk are positive correlated with the CMT scores ++ and +++ in ewes. Intramammary CNS infections cause an increase of SAA in ovine milk. SAA is a valuable marker for screening udder health status from individual ewes. SAA cannot be used as a diagnostic tool in goats. Bulk milk SAA concentrations do not have any potential for monitoring the udder health of herds. 31 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 HAPTOGLOBIN IN SERUM AND MILK OF COWS AND ITS RELATION TO UDDER HEALTH AND METABOLIC DISORDERS Weinkauf C, Hachenberg S, Hiss S, Müller U, Sauerwein H Institute of Physiologie, Biochemistry and Animal Hygiene, University Bonn, Germany Introduction The relationship between experimentally induced bovine mastitis and increased Haptoglobin (Hp) levels in milk is already known (1). To clearify if Hp in milk is a reflector for metabolic disorders as well, Hp was determined in milk and ß-hydroxybutyrate (ß -OH-B), non esterified free fatty acid (NEFA) and Hp were analysed in serum in early lactation. Methods Quarter milk samples (n=1940) and blood samples (n=490) were collected weekly during week 3 to 12 post partum from 49 Holstein Friesian cows, being in their first to eighth lactation., ß -OH-B concentrations were determined by a veterinary diagnostic laboratory (VLK, Koeln). Serum and milk Hp concentrations were determined using an ELISA (1). NEFA concentrations were analysed by a commercial test kit (Roche Diagnostics, Mannheim). Quarter milk samples were diagnosed healthy or diseased on the basis of the Hp value (3,4 µg/mL) for udder health (2). To evaluate the relationship between Hp concentrations in milk and metabolic blood parameters, five Hp groups were composed (see table 1). Results Table.1: Results of Serum Hp and metabolic parameters (Mean±SD) Serum number of quarters per cow above cut-off value [>3,4 µg/mL] four n=13 three n=26 two n=71 one n=102 none n=273 a b b b Hp [mg/mL] 2.4±1.87 0.42±0.60 0.39±0.68 0.24±0.27 0.17±0.27b a b b b NEFA 0.4±0.37 0.17±0.15 0.19±0.17 0.19±0.18 0.20±0.14b [mmol/L] ß-H-B [mg/dL] 17±12.8a 8.5±3.6 b 7.8±3.6b 9.2±5,4b 9.1±4,8b a,b Means in the same row with different superscripts differ (p<0.001) Discussion Relationships between Hp in milk and metabolic parameters could be established. Hp serum concentrations as well as metabolic parameters were not significantly different in cows having elevated milk Hp in no quarter or even single quarters. Increased Hp levels in all quarters per cow and increased metabolic parameters may indicate that milk Hp also reflects extramammary disorders. These findings underline the importance of quarter milk samples in the assessment of Hp in mastitis. References 1) Hiss, S.; Mielenz, M.; Bruckmaier, RM; Sauerwein, H., 2004: J Dairy Science 87 (11), 3778-3784 2) Neu-Zahren, A.; Müller, U.; Hiss, S.; Sauerwein, H., 2004: 7th EAAP/ASAS/COST, Bled, Slovenia 32 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 THE PORCINE ACUTE PHASE PROTEIN RESPONSE TO EXPERIMENTAL STREPTOCOCCUS SUIS INFECTION Sørensen NS1, Tegtmeier C1, Andresen LO1, Piñeiro M2, Toussaint MJM3, Campbell FM4, Heegaard PMH1 1 Dept. of Veterinary Diagnostics and Research, Danish Institute for Food and Veterinary Research, Copenhagen, Denmark; 2Dept. of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Zaragoza, Spain; 3Dept. of Pathobiology, University of Utrecht,, Utrecht, The Netherlands 4 Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland Introduction Streptococcus suis (S. suis) is an important pathogen of pigs causing septicaemia with various localizations. The early detection of acute S. suis-infection in pig herds could help reduce morbidity and mortality and prevent spread of the infection. Here we studied the acute phase protein (APP) response to experimental S. suis-infection by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pigMAP) and the negative APPs albumin and apolipoprotein (Apo) A-I, in order to identify the most appropriate proteins for the sensitive detection of S. suis infection in the pig. Materials and Methods Five pigs, 5 weeks of age, with no history of S. suis serotype 2 infection were injected with 1010 CFU live S. suis serotype 2. Blood was sampled before and on days 1, 2, 5, 8, 12 and 14 after inoculation (p.i.), when the pigs were killed and autopsied. Rectal temperatures and clinical signs were recorded daily, and infection was confirmed by the re-isolation of S. suis serotype 2 from blood on day 1 and 8 p.i. The serum concentrations of pigMAP, ApoA1 and albumin (radial immunodiffusion) and CRP, Hp and SAA (ELISA) were determined. Results CRP and SAA showed fast increases in serum concentrations. CRP was increased from day 1-12 p.i. and peaked between day 1 and 8 p.i. with ten times the day 0-levels. SAA rose sharply on day 1 p.i. to peak levels of 30-40 times the day 0-levels on day 1-2, after which concentrations returned to pre-inoculation levels from day 5. Hp and pigMAP showed slightly slower responses. Hp was increased on days 1-14 p.i. and peaked at levels around ten times the day 0-levels on day 5 p.i., and pigMAP was elevated on days 1- 12 p.i. and peaked at levels of 5-10 times the day 0-levels on day 5 p.i. Apo A-I showed a fast decrease to minimum levels 60% lower than day 0 on day 1-2 p.i. and was decreased from day 1-8 p.i. No clear pattern of changes in albumin levels could be identified. Clinical signs, fever and lameness, were observed in four of the five pigs from day 2 p.i., and these pigs also had arthritic lesions at autopsy. One pig showed no clinical signs at any time and no arthritic lesions, but showed an APP-response comparable to the other pigs. Conclusion Both acute clinical and subclinical S. suis-infections can be revealed before the occurrence of clinical signs by the measurement of one or more of the APPs CRP, SAA, Hp, pigMAP and Apo A-I. Different patterns of APP-response kinetics were identified, and a combination of 2-3 APPs with different responses may be used to achieve the highest sensitivity for the detection of S. suis-infection. Acknowledgements: Support from the European Commission for these studies is gratefully acknowledged (Shared Cost Project QLK5-2001-02219) 33 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 COMPARISON OF PRODUCTIVE PERFORMANCE, HEALTH STATUS AND Pig- MAP SERUM CONCENTRATION OF PROGENIES FROM PRIMIPAROUS AND MULTIPAROUS SOWS DURING THE NURSERY PERIOD Piñeiro C1, Lorenzo E1, Morales J1, Piñeiro M1, Lázaro R2, Manteca X3 , Mateos GG2 1 PigCHAMP Pro Europa S.A., Spain, 2UP Madrid, Spain, 3UA Barcelona, Spain Introduction Piglets from primiparous sows (PS) have lower body weight at birth and higher mortality rate during lactation than piglets from multiparous sows (MS). The difference observed may affect pig performance late in their fattening period. One cause of the poorer viability observed in PS piglets might be related to the lower immune transmission via colostrum which results in higher susceptibility to pathogens and lower health status. Acute phase proteins (APPs) have been demonstrated to be valid biomarkers of health status but no data is available of its value as biomarker for immune status. Aim: To compare the productive performance and mortality rate of piglets from PS or MS, during nursery. Pig- MAP concentration during post-weaning was assessed as a health and productive performance biomarker. Material and Methods A total of 400 LW x LD weaned piglets (8.2 ± 0.97 kg BW; 28 d of age) was divided into two groups based on litter parity: PS or MS. The average daily gain, feed intake, feed:gain ratio and mortality of piglets were controlled, and blood samples were taken at 28 (weaning), 40 and 60 d of age to determine the concentration of Pig-MAP in serum by ELISA (commercial kit from PigCHAMP Pro Europa S.A. Segovia, Spain). Results Productive performance: During nursery piglets from PS had lower ADG and FI and worse feed to gain ratio than piglets from MS. PS showed a poorer performance than MS sows for average daily gain (391 vs 456 g/d; P<0.001), feed intake (453 vs 500 g/d; P<0.05) and feed:gain ratio (1.20 vs 1.10 g/g; P<0.05) during the nursery period (28-60 d of age). Mortality: A higher mortality was registered for PS than for MS piglets (2.5 vs 0.50 %; P<0.10), suggesting that the incidence of pathologies was higher for PS group. APP concentration: At weaning Pig-MAP concentration was higher in piglets from PS sows than in piglets from MS sows (1.35 vs 1.01 mg/mL; P<0.01) but no differences were observed afterwards. The evolution of Pig-MAP concentration throughout the experimental period is presented in figure 1. Figure 1. Pig-MAP (mg/mL) evolution in piglets from PS and MS. 1,5 1,3 1,1 mg/mL 0,9 0,7 0,5 28 40 60 days of age PRIM MULT Conclusions: Litters from primiparous sows showed poorer productive performance during the nursery period than litters from multiparous sows. Also Pig-MAP concentration at weaning, was higher in piglets from primiparous sows. Concentration of Pig-MAP in serum might be an indicator of health status and susceptibility to pathologies of piglets during lactation. Our data indicate that health status was lower and a susceptibility to pathologies higher for PS piglets than for MS piglets 34 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 CHANGES IN C-REACTIVE PROTEIN AND HAPTOGLOBIN IN DOGS WITH LYMPHATIC NEOPLASIA Mischke R1,2, Eckersall PD2 1 Small Animal Clinic, Hannover School of Veterinary Medicine, Hannover, Germany, 2Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland Introduction In humans, acute phase proteins (APP) are regarded as a useful diagnostic tool in patients with lymphoma, leukaemias and multiple myeloma, and can be used as a prognostic factor and as an early indicator of sepsis. The aim of this pilot study was to examine the acute phase proteins (APP) C-reactive protein (CRP) and haptoglobin in dogs with different malignant, lymphatic blood disorders. Materials and Methods Dogs with malignant multicentric (high grade) lymphoma (n=16), acute lymphoblastic leukaemia (ALL) (n=11), chronic lymphocytic leukaemia (CLL) (n=7) und multiple myeloma (n=9) were included in the study. 25 healthy dogs served as a control. Lithium-heparinate plasma was used as a sample material. Measurements of the CRP concentration was performed with an ELISA (Tridelta Development Ltd, Ireland), haptoglobin was measured with an assay based on its haemoglobin binding capacity (Eckersall et al, Comp Haem Int, 1999, 5:117-121). Results Global group comparisons using Kruskal-Wallis test revealed significant group differences for both APPs (p<0.0001). Median CRP concentrations were increased in all groups with neoplastic lymphatic disorders (lymphoma: 37.2 mg/L, ALL: 47.8 mg/L, CLL: 35.5 mg/L, myeloma: 21.5 mg/L) compared to the control (1.67 mg/L; p<0,001). Compared to the 95 % quantile of the healthy control dogs (9.6 g/L), CRP concentration in 13 of 16 dogs with malignant lymphoma were increased, in 10 of 11 dogs with ALL, 6 of 7 dogs with CLL and 6 of 9 dogs with multiple myeloma. Compared to the healthy control (median=0.59 g/L), haptoglobin level was especially increased in dogs with ALL (6.8 g/L, p<0,001) followed by dogs with malignant lymphoma (3.8 g/L, p < 0.001) and CLL (2.0 g/L, p=0.0287) (Fig. 2), whereas the results in dogs with multiple myeloma did not differ significantly from the healthy control (median=2.5 g/L, p=0.0610). The median values in the dogs with ALL were significantly higher than in dogs with other neoplastic lymphatic disorders (p<0.05), whereas no significant differences were observed between the other patient groups. Similar to CRP, a wide range of values was found in all patient groups. Compared to the 95 % quantile of the control (2.45 mg/L), 13 of 16 dogs with malignant lymphoma had increased values, 10 of 11 dogs with ALL, 3 of 7 dogs with CLL and 5 of 9 dogs with multiple myeloma. Conclusion Particularly severe and acute lymphatic neoplasia as high grade lymphoma and ALL are causing significant acute phase reactions in dogs. This fact has to be considered when using APP levels in dogs with lymphatic neoplasia as an indicator of infection or sepsis. Whether levels of acute phase proteins in dogs with neoplastic lymphatic disorders can be used as prognostic factors as is the case in humans has to be investigated in further studies. 35 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 EQUINE SERUM AMYLOID A RESPONSE FOLLOWING VACCINATION AGAINST INFLUENZA AND TETANUS Andersen SA1, Petersen HH1, Ersbøll AK1, Falk-Rønne J2, Jensen AL1, Jacobsen S1 1 Department of Large Animal Science, Royal Veterinary and Agricultural University, Copenhagen, Denmark, 2The Equine Clinic, Charlottenlund, Denmark. E-mail: email@example.com Introduction Serum amyloid A (SAA) is a major acute phase protein in horses reflecting trauma, infection and inflammation by increased serum concentration. The aim of this study was to assess the SAA response after vaccination against equine influenza and tetanus using 2 commercially available equine vaccines. Materials and Methods Nine horses from a heterogeneous population (horses and ponies, age 4-22 years) were assigned to one og two vaccination groups after stratification for age and stable. After 5 daily clinical examinations and blood samplings, 4 horses were vaccinated against influenza and tetanus using inactivated (Equip® FT Vet., Schering-Plough, injection volume 2 ml) and 5 horses using live recombinant (ProteqFluTM-Te, Merial, injection volume 1 ml) vaccines. The horses were examined and blood samples obtained 9, 24, 48, 72 and 96 hours after vaccination. Serum was tested for SAA using a human Turbidometric ImmunoAssay (1). Results 160 150 140 vaccine 1 130 vaccine 2 120 110 100 SAA (mg/L) 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Day Figure 1. The equine serum SAA response following vaccination against influenza and tetanus using 2 different vaccines, a live recombinant virus vector vaccine (1) and an inactivated ISCOM vaccine (2). The horses were vaccinated on day 8 (black arrow). Before vaccination, the median serum SAA concentration was 0.2 mg/L (5% quartile 0.0 mg/mL, 95% quartile 0.4 mg/L). All serum SAA concentrations from 24 hours after vaccination were significantly higher than before vaccination (p < 0.001) and the serum SAA concentration between the 2 vaccination groups were significantly different (p = 0.030). There was no interaction between vaccine type and time after vaccination (p = 0.10). Discussion and Conclusion The serum SAA concentration in horses increased following vaccination against influenza and tetanus. The increase persisted for 96 hours and the magnitude depended on type of vaccine. The SAA response was higher for the 2 ml inactivated vaccine than for the 1 ml live recombinant vaccine. Differences in adjuvant, injection volume or influenza virus types are some possible reasons for the differences in the response. Reference 1. Jakobsen S, Kjelgaard-Hansen M, Petersen HH, Jensen AL. Equine serum amyloid A measurements using a commercially available turbidometric immunoassay. 5th Colloquium on Acute Phase Proteins, Dublin, 2005. 36 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 IMMUNOHISTOCHEMICAL DETERMINATION OF BOVINE HAPTOGLOBIN IN EXPERIMENTAL MASTITIS Hiss S1, Petzl W2, Zerbe H3, Schuberth HJ2, Seyfert HM4, Sauerwein H1 1 Institute of Physiologie, Biochemistry and Animal Hygiene, University Bonn, 2Immunology Unit, School of Veterinary Medicine, Hannover,3Clinic for Ruminants, LMU München, 4FBN, Dummerstorf, Germany Introduction The origin of milk Hp can be attributed to serum Hp passing the blood milk barrier during the acute phase reaction but also to local sources within the mammary gland (1). The objective of the present study was to evaluate the relationship between systemic Hp and immunohistochemically detectable Hp in mammary glands infected with two different pathogens. Methods Eight lactating Holstein Friesian cows were used for the experiment. Each cow was inoculated three times with either 500 colony-forming units (cfu) E. coli or 10,000 cfu Staph. aureus (4 cows each) through the teat canal. The right front quarter was inoculated first (24 h until slaughter), the right rear quarter was inoculated 12 h later, the left rear quarter was inoculated after 18 h (6 h until slaughter) and the left front quarter served as the control quarter. Blood samples were taken before, 12, 18 and 24 h after the first inoculation. All cows were slaughtered 24 h after the first inoculation. Milk and tissue samples were collected from each quarter. Serum and milk Hp concentrations were determined using an ELISA (1). Tissue samples were fixed in Bouin`s solution and embedded in paraffin. Tissues were cut into serial sections of 5 µm. Immunhistochemistry (IHC) was performed using polyclonal rabbit antibodies against bovine haptoglobin (1). For negative controls non-immune rabbit serum was used. Slides were counterstained in Mayer´s Haemalaun. IHC was quantified by counting cells positive for Hp with a reference grid in the microscope ocular. Due to the restricted number of animals, the presentation of results is limited to descriptive statistics. Results Tab.1: Hp concentrations in milk and serum (medians) Hp in quarter milk samples (µg/ml) serum Hp (µg/ml) hours after inoculation of hours after first inoculation individual quarter 0 6 12 24 0 12 18 24 E. coli 2.0 3.8 80.7 537.5 15.1 33.4 70.4 1195.0 Staph. aureus 2.9 5.1 2.8 2.0 22.9 30.0 30.1 15.0 An intense immunostaining was observed in the lumina of alveoli, blood vessels and in the cytoplasm of leukocytes. Few epithelial cells were positive for Hp. Hp staining showed no obvious differences neither between control quarters and infected quarters nor between the two pathogens used. Discussion: The inoculation of E. coli into the mammary gland lead to increased somatic cell counts (SSC, not shown) and increased levels of milk and serum Hp whereas the response towards Staph. aureus (SSC and Hp) was negligible. However the immunohistochemical results do not reflect these differences. The response towards Staph. aureus inoculation has to be confirmed in further studies. Reference 1) Hiss, S.; Mielenz, M.; Bruckmaier, RM; Sauerwein, H., 2004: J Dairy Science 87 (11), 3778- 3784 37 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 EVALUATION OF THE HEALTH STATUS OF SUBCLINICALLY DISEASED POULTRY BY THE USE OF BLOOD PARAMETERS Vandendriessche J, Arnouts S Inve Technologies nv, Dendermonde, Belgium Email: email@example.com Introduction The intensive animal production of the last decades was mainly focused on the achievement of maximal production results (eggs, milk and meat). In recent years the European consumer has expressed his concern towards animal welfare and food safety. The intensive animal production is forced to produce a high quality product with special attention towards animal health and food safety. Growth, product quality and animal welfare are influenced by subclinical multifactorial diseases and stress. The common veterinary diagnostic techniques (autopsy, histology,…) are therefore not always useful to screen the health status of the animal. Other diagnostic bloodparameters, for example biochemical enzymes and acute phase proteins, can be an alternative option. Especially in poultry, little is known about the value of blood parameters in the screening of the animal's health. Aim Analyse the value of blood parameters to evaluate the health status of poultry in case of subclinical diseases and stress. Materials and Methods During a two year period several poultry trials were set up with special attention towards the induction of stress and subclinical diseases by oral application of lectins, cyclophosphamide, lipopolysaccharides and vaccination. A total of 377 animals were slaughtered at the age of 28 days for autopsy scoring and blood sample collection. The following bloodparameters were determined: total protein, albumin, phosphatase alkaline (AP), alanine aminotransferase (ALAT), creatine kinase (CK) and alpha-1-acid glycoprotein (AGP). All parameters were analysed in serum using Vettest, Idexx Laboratories, except for alpha-1-acid glycoprotein which was analysed using the alpha-1-acid glycoprotein assay from Tridelta Development Ltd., Ireland. Based on the autopsy score (evaluation of different organ systems and Eimeria lesions), the live weight and the bursa/live weight ratio the animals were divided into two groups: healthy or subclinically diseased. The animals were classified as healthy if the autopsy score was 0 (no abnormalities) and both the live weight and bursa/live weight ratio were situated in the range of the average of the control animals (no stress induction) +/- (2 x the standard deviation). Animals that showed a high score for the autopsy and/or had a live weight and/or bursa/live weight ratio that was out of the range of the average of the control animals (no stress induction) +/- (2 x standard deviation) were classified as subclinically diseased. The subclinically diseased animals were in turn subdivided into subgroups. Subclinically diseased animals with a score ≥ 3 for coccidiosis (Eimeria ascervulina, maxima and tenella) were classified in the subgroup lesion. Subclinically diseased animals were classified in the subgroup systemic disease if the autopsy score was ≥ 5. Results There were no significant differences in the concentration of total protein, alpha-1-acid glycoprotein and the 3 enzymes (AP, ALAT and CK) between both groups. The concentration of albumin in the serum of the subclinically diseased animals was significantly lower compared to the healthy animals. Power analysis revealed that a significant difference in the alpha-1-acid glycoprotein concentration can be detected in subclinically diseased animals if a total amount of 500 animals is used. The serum ALAT- concentration of the subgroup lesion decreased significantly compared to the healthy animals. The concentration of CK, ALAT and AGP in the serum of systemic diseased animals showed major non significant differences compared to the healthy animals. According to statistical analysis the p-value of these three parameters was low, with values of respectively 0.37, 0.39 and 0.15. 38 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Table 1. Average (standard deviation) of the determined blood parameters in the groups healthy and subclinically diseased and the subgroups lesion and systemic disease (Means with different letters are significantly different at p < 0.05). Total Autopsy Albumin # animals protein CK (IU/l) AP (IU/l) ALAT (IU/l) AGP (µg/ml) score (g/l) (g/l) 30,3 a 7,95 a 4460 a 12900 a 33,5 a 284 a Healthy 145 0 (1,27) (2,38) (2140) (10985) (13,6) (182) 29,8 a 7,34 b 4445 a 12310 a 31,3 a 306 a Subclinical 232 3 (5,10) (2,55) (2146) (8437) (12,3) (160) 30,3 a 7,61 a 4378 a 10998 a 29,3 b 293 a Lesion 92 3 (5,40) (2,82) (2231) (7361) (10,8) (143) 30,8 a 7,62 a 5203 a 10678 a 29,0 a 385 a Systemic 13 5 (5,30) (2,02) (1902) (8411) (7,9) (167) Discussion and Conclusion Determination of the health status of poultry in case of a subclinical disease or stress is not possible by using total protein and the 3 enzymes AF, ALAT and CK. In case of a large amount of serum samples, alpha-1-acid glycoprotein is a useful blood parameter, together with albumin, to make a distinction between healthy and subclinically diseased animals. The interference of several mild pathologies is a possible explanation for this conclusion. In the subgroup lesion, animals with a high score for coccidiosis were selected. To make a distinction between healthy animals and animals with coccidiosis the serum ALAT-concentration is the only useful parameter. Due to the fact that subclinically diseased animals have no major pathologies in either one or several organ systems, deviations in the level of the parameters from normal values may be too small to be significant. The animals, classified as systemic diseased, had a high autopsy score (≥ 5), which indicates severe pathologies on either one or several organ systems. These animals had a major non significant difference in the serum concentration of CK, ALAT and AGP compared to the healthy animals, even if only a small amount of animals was used. Furthermore the p- value of these three parameters was low. The results of this trial indicate that it should be possible to make a distinction between healthy and systemic diseased animals by using blood parameters, even if a low number (50 for ALAT and CK, 20 for AGP) of animals is used. This work was funded by IWT-Flanders. 39 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 ACUTE PHASE PROTEIN REACTION IN LAYER CHICKENS. A CALCULATED ACUTE PHASE PROTEIN INDEX AS MEASURE TO ASSESS HEALTH DURING THE REARING PERIOD Upragarin N1,2, Toussaint MJM1, Tooten PCJ1, van Asten AJAM1, Wajjwalku W3, Gruys E1 1 Department of Veterinary Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Tthe Netherland. 2Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand. 3Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand. E-mail: firstname.lastname@example.com Introduction Acute phase proteins (APPs) have been defined into two groups, positive and negative APPs. With their levels an acute phase index (API) may be calculated to increase the sensitivity of non-healthy condition assessment. APPs and API have been used as a biomarker to assess animal health so far; for poultry, however, limited data are available. The aim of the present study was to investigate kinetics of a small set of APPs (serum amyloid A [SAA], transferrin [TFR], serum albumin [Alb] and apolipoprotein A-I [apoA-I]) in commercial layer after injection with turpentine (a sterile reaction model, TM) and with Staphylococcus aureus (a septic reaction model, SM) and to evaluate the sensitivity of the API as a health monitoring method. Materials and Methods Forty seven-week-old birds were randomly assigned into 4 groups of 10 chickens each: a turpentine- injected brown chicken group, turpentine-injected white chicken group, S. aureus-injected brown chicken group, and a S. aureus-injected white chicken group. Serum samples were collected before injection and 6 h, 12 h, 24 h, 48 h, and 72 h after injection. SAA was examined by gel electrophoresis and immunoblotting and ELISA. TRF, Alb, and apoA-I were examined by gel electrophoresis and immunoblotting. Results In untreated birds the SAA levels was less than 20 ng/ml. At 12 h after injection levels increased up to 500 to 10000 times (mean 28.93 µg/ml), and they were even much higher after 72 h (mean 84.56 µg/ml). There was no difference of the SAA levels between chicken groups. The mean serum TFR levels in all chicken groups were less than 4 mg/ml before treatment, increased slightly at 24 h after injection, and increased 5 times of their levels to pretreatment at 72 h after injection. The TRF levels in TM chickens were higher than those in the SM chickens. The Alb levels in all chicken groups before treatment were 50% to 60% of TSP and were slightly decreased 12 h after injection. After 72 h injection the Alb levels were about 10% to 20% lower than those before injection. The apoA-I levels in all birds ranged 2 to 3.29 mg/ml before injection, decreased slightly at 24 h after injection, and decreased 50% from normal levels 72 h after injection. The API values in all chickens were less than 0.5 before injection, increased 8 to 25 times at 24 h after injection, and increased 100-times 72 h after injection. Conclusions The results showed that SAA is the fastest positive APP, while TFR reacts less vigorously. ApoA-I and Alb are negative APPs. Similar to SAA, API is a suitable biomarker to assess the chicken health conditions. 40 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 SERUM AMYLOID A AND OTHER ACUTE PHASE REACTANTS IN HORSES WITH NORMAL OR PROTRACTED RECOVERY AFTER CASTRATION Jacobsen S, Frei S, Jensen JC, Thoefner MB Department of Large Animal Sciences, The Royal Veterinary and Agricultural University,Copenhagen, Denmark Email: email@example.com This Abstract has been removed from this presentation at the request of the author for reasons of copyright. Please contact the author if you would like further information. Thank you. 41 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 OPTIMIZATION OF INTERNAL QUALITY CONTROL OF VETERINARY ACUTE PHASE PROTEIN ASSAYS - EXEMPLIFIED FOR CANINE SERUM C-REACTIVE PROTEIN Kjelgaard-Hansen M, Jensen AL, Kristensen AT Department of Small Animal Clinical Sciences, The Royal Veterinary and Agricultural University, Copenhagen, Denmark. E-mail: firstname.lastname@example.com. This Abstract has been removed from this presentation at the request of the author for reasons of copyright. Please contact the author if you would like further information. Thank you. 42 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 ACCURACY OF MILK AMYLOID A (MAA) CONCENTRATION AND SOMATIC CELL COUNT FOR DIAGNOSING BOVINE MASTITIS Petersen HH1,2,Gardner IA2, Rossitto P3, Larsen HD4 , Heegaard PMH4 1 Department of Large Animal Science, Royal Veterinary and Agricultural University, Copenhagen, Denmark,2Department of Medicine and Epidemiology, University of California, Davis, USA, 3Veterinary Medicine Teaching and Research Centre, Tulare, California, USA, 4Department of Veterinary Diagnostics and Research, Danish Institute for Food and Veterinary Research, Copenhagen, Denmark. E-mail: firstname.lastname@example.com Introduction Acute phase proteins are inflammatory serum glycoproteins that undergo substantial changes in concentration following inflammation, infection or trauma (1). Recently, acute phase proteins have also been reported to be present in bovine milk (2,3) indicating a potential for detecting mastitis in dairy cattle. Our aim was to estimate the accuracy of the acute phase protein milk amyloid A (MAA) for diagnosing bovine mastitis. Materials and Methods One-hundred-twenty-nine dairy cows from 5 herds in the Central Valley of California were selected based on somatic cell count and mastitis records, days in milk in the current lactation and lactation group to ensure an adequate spectrum of mastitis cases and non-affected controls. All cows were clinically examined, and milk was sampled from 2 contra-lateral quarters after pre-stripping. From cows with clinical signs of mastitis, the affected and the contra-lateral quarter were sampled. Milk was tested for MAA concentration using a commercial kit (Tridelta ltd, Ireland) and submitted for somatic cell count and microbiological examination at a diagnostic laboratory. Quarters were classified as healthy if no clinical signs (pain, swelling, oedema, abnormal milk) were observed and if the microbiology was negative. A quarter was defined as mastitic if one or more of the clinical signs were present or if the sample was positive when cultured (pure culture of known mastitis pathogen). Results Of 43 mastitic quarters, 19 had clinical signs only, 12 were only microbiologically positive (sub-clinical cases) and 12 were positive on both. Clinical signs were present without positive microbiology in 54% of the clinical cases of mastitis. When all paired samples from 2 contra-lateral quarters were compared, a weak correlation (r2 = 0.08, n = 129) was found. The MAA content in samples from contra-lateral, uninfected quarters was better correlated (r2 = 0.24, n = 90) with no significant differences in concentration. The MAA concentration was higher in quarters with mastitis compared to corresponding contra-lateral healthy quarters (p = 0.0001, n = 28). The test performance estimated using the area under the ROC-curve showed no significant differences between the MAA and SCC. However, when optimizing the cut-off value based on the ROC-curves, MAA were generally a more sensitive and less specific parameter than the SCC (Table I). Table I. Comparison of milk amyloid A (MAA) and somatic cell count (SCC) used for diagnosing bovine mastitis in single quarter milk samples (n=230). Sensitivity (Se), specificity (Sp), area under the curve (AUC) and 95% confidence interval of AUC (95% CI) for different definitions of a positive mastitis diagnosis. Parameter Mastitis Clinical mastitis Sub-clinical mastitis Se Sp AUC 95% Se Sp AUC 95% Se Sp AUC 95% CI CI CI MAA 86.0 74.5 0.81 0.75- 90.3 73.2 0.83 0.78- 83.3 63.8 0.67 0.61- 0.85 0.88 0.73 SCC 67.4 87.2 0.77 0.74- 71.0 87.0 0.80 0.75- 66.7 77.7 0.71 0.65- 0.84 0.85 0.76 Conclusion The MAA concentration in milk is quarter specific and reflects quarter infection status. Despite no overall difference in the ROC-curves for SCC and MAA, MAA was found to be a more sensitive and less specific mastitis parameter than SCC indicating a possible future use of MAA in diagnosing bovine mastitis. 43 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 References 1. Gruys E., Obwolo M.J., Toussaint M.J.M. Diagnosic significance of the major acute phase proteins in veterinary clinical chemistry: a review. Vet. Bull. 64, 1009-1018, 1994. 2. Eckersall P.D., Young F.J., McComb C., Hogarth C.J., Safi S., Weber A., McDonald T., Nolan A.M. and Fitzpatrick J.L. Acute phase proteins in serum and milk from dairy cows with clinical mastitis. Vet. Rec. 148, 35-41, 2001. McDonald T.L., Larson M.A., Mack D.R. and Weber A. Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A 3 (M-SAA3) into colostrums. Vet. Immunol. Immunopathol. 83, 203-211, 2001. 44 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 EQUINE SERUM AMYLOID A MEASUREMENTS USING A COMMERCIALLY AVAILABLE TURBIDOMETRIC IMMUNOASSAY Jacobsen S1, Kjelgaard-Hansen M2, Petersen HH1, Jensen AL2 1 Department of Large Animal Sciences, 2Department of Small Animal Clinical Sciences, The Royal Veterinary and Agricultural University, Copenhagen, Denmark Email: email@example.com This Abstract has been removed from this presentation at the request of the author for reasons of copyright. Please contact the author if you would like further information. Thank you. 45 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 QUANTITATIVE CYTOKINE AND ACUTE PHASE PROTEIN mRNA EXPRESSION IN LIVER AND MAMMARY GLAND FROM DAIRY COWS EXPERIMENTALLY INFECTED WITH Streptococcus uberis Nielsen L1, Røntved CM1, Pedersen LH2, Ingvartsen KL1 1 Danish Institute of Agricultural Sciences, Animal Health, Welfare and Nutrition, Foulum, Denmark 2 Danish Dairy Board, Århus C, Denmark Introduction Streptococcus uberis cause a significant proportion of clinical and subclinical intramammary infections in lactating dairy cows. In particular, pro-inflammatory cytokines and acute phase proteins are involved in the mammary glands immune response to invading microorganisms. Cytokines stimulate the production of acute phase proteins, primarily produced by the liver. But recently, focus has been on a suggested local production of acute phase proteins in the mammary gland. The objective of this study was therefore to elucidate the gene expression of cytokines and acute phase proteins in the liver and compare that to the gene expression in the mammary gland. Furthermore, we wanted to study in which region of the mammary gland the inflammatory factors were expressed. Materials and Methods Mastitis was induced in the right front quarter of six Holstein-Friesian cows in mid-lactation with no prehistory of S. uberis mastitis. The cows were inoculated with approximately 107 colony-forming units of S. uberis into the teat cistern of the right front quarter. The left front quarter was simultaneously inoculated with sterile milk (control). The six cows were euthanized consecutively at 2-h intervals at 2, 4, 6, 8, 10 and 12 h after inoculation. After euthanasia, liver and mammary tissue was collected and snap frozen in liquid nitrogen. Mammary tissue was collected from the right front mammary gland at four different sections (A: glandular cistern, B: distal, C: central and D: proximal mammary gland). RNA was extracted from liver and mammary tissue using an RNeasy Protect Mini Kit (Qiagen), and concentrations were measured on a GeneQuant pro spectrophotometer (Amersham Pharmacia Biotech) and adjusted to 0.2 µg/µl. Specific primers and probes were designed for tumour necrosis factor α (TNF-α), interleukin (IL) 1β (IL-1β), IL-6, IL-10, haptoglobin (Hp), serum amyloid A (SAA) and α1-acid glycoprotein (AGP) mRNA, and the mRNA expression was quantitatively evaluated by real-time RT-PCR (Taqman). Results TNF-α, IL-1β, IL-6, IL-10, Hp, SAA and AGP mRNAs were found both in the liver and in the mammary gland. Expression levels of liver IL-6, SAA and AGP remained constant, except for cow no 5 (euthanized at 10 h), which had a much higher expression level than other cows. We observed increased expression levels of liver TNF-α, IL-1β, IL-10 and Hp depending on time of euthanasia. TNF-α expression was increased as early as 4h after inoculation (cow 2), IL-10 were highest in cow 4 and 5 (8 and 10h after inoculation) and Hp was highest in cow 6 (12 h after inoculation). Compared to expression profiles in the mammary gland, there were no indications of a relation between liver and mammary gland gene expression for any of the measured genes. Expression of some cytokines and acute phase proteins appear to change considerably due to time of inoculation, but comparison of average expression levels between liver and mammary tissue showed a higher expression of AGP, Hp, IL-1β and IL-10 in liver tissue (Table 1). The opposite was the case for SAA, IL-6 and TNF-α, which were higher expressed in mammary tissue than in liver tissue (Table 1). Table 1. Differences in gene expression between liver and mammary tissue for the seven tested genes. AGP Hp IL-1β IL-10 SAA IL-6 TNF-α Fold difference in gene expression 131072 446 1.6 9.2 (Liver/mammary tissue ratio) Fold difference in gene expression 4 274 2.3 (Mammary tissue/liver ratio) The acute phase proteins Hp, SAA and AGP were primarily expressed in the glandular cistern and the distal region of the mammary gland. IL-1β and IL-10 had a similar expression pattern, showing the highest expression in the distal part of the gland, while TNF-α primarily was expressed in the central and proximal region of the mammary gland. IL-6 was equally expressed in the mammary gland. 46 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Discussion TNF-α is one of the fastest responding cytokines. Thus, it was expected to find the early increase in liver expression. Surprisingly, mammary gland TNF-α increased much later, but Pfaffl (2003) have suggested, that "milk cells" are the main source of TNF-α rather than the mammary gland. Although, the contribution of TNF-α mRNA in milk may primarily be from the leukocytes. Comparison of gene expression between liver and mammary tissue has not been done before. We were surprised to find a higher expression of SAA in the mammary gland than in the liver, since liver is thought to be the main source of SAA. If the production of acute phase proteins in the mammary gland is regulated in a similar way as in the liver, it is surprising to find the acute phase proteins and the regulatory cytokines expressed in different regions of the mammary gland. Though, IL-6 is thought to be the main stimulator of the acute phase proteins and this cytokine was expressed equally throughout the mammary gland. Since the presented results are only from one animal at each sample time, data should be verified by further experiments. Furthermore, expression data from the mammary gland should be compared with expression analysis from the control quarter. 47 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 THE PLASMA PROTEOME OF EQUUS CABALLUS: PATTERNS UNDER BASELINE CONDITIONS AND DURING ANTHELMINTIC TREATMENT Miller I1, Fountoulakis M2, Duvigneau C1, M Hartl R1, Dobretsberger M3, Sieber K4, Prosl H4, Gemeiner M1 1 Department of Natural Sciences, University of Veterinary Medicine, Vienna, Austria 2Foundation for Biomedical Research of the Academy of Athens, Greece; formerly: F. Hoffmann-La Roche Ltd., Center for Medical Genomics, Basle, Switzerland 3Research Estate Kremesberg, University of Veterinary Medicine, Vienna, Austria 4Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria Email: firstname.lastname@example.com Introduction The present study focuses on the plasma proteome of Equus caballus, and investigates its changes during infestation and anthelmintic treatment, paying special attention to acute phase proteins and immunologic reactions. Intestinal parasitic infection causes a variety of clinical symptoms which may occur with different severity, and regular anthelmintic treatment is known to contribute to the animals' wellbeing. Materials and Methods Sera from several healthy horses of Norican breed were used to establish the serum protein map. Classical two-dimensional electrophoresis (2-DE) was applied; proteins were identified either by mass spectrometry methods or by immunoblotting and (cross-) reactive antibodies. For the infestation study, a group of six horses was subjected to oral treatment with ivermectin, blood samples were collected in regular intervals from day 0 to 14. Effectivity of the treatment was checked by parasitological investigations (nematode egg counts from feces). Besides determining hematological values as well as cytokine and IgE expression levels, serum protein patterns were studied as a function of time. A combination of different methods was applied, ranging from Elisa (for serum amyloid A) to SDS- PAGE and 2-DE (including differential image gel electrophoresis). Results Equus caballus is one of the species with yet rather incompletely characterized genomes, and thus protein databases lack informations on horse proteins. Part of these problems can be solved by cross-species comparison and check for similarity with homologues from other species. Thus, we were able to identify almost all moderate to high abundance proteins stained in the plasma 2-DE pattern. Compared to maps of other species (human, bovine, pig), the horse map shows several differences concerning positions/patterns and levels of single proteins. Known polymorphisms, e.g. for-antitrypsin (pi), could be detected in specimens from individuals. Horses in the anthelmintic study showed temporarily increased IgE levels and eosinophile counts as a reaction to treatment. Longer-term investigations of haptoglobin revealed a significant increase a few days after treatment, in all investigated animals. Similarly, a temporary increase of fibrinogen was noticed. In contrast to that, time-courses of serum amyloid A and overall IgG(T) were individual, with considerable to minor fluctuations. Selected samples, from the most interesting period between day 0 and 7, were investigated in more detail by 2-DE. This method allowed resolution and quantification of proteins which cannot be sufficiently separated in one-dimensional methods, and thus gave more insight into variations of single proteins. Conclusion The present study shows that - although used as a single dose administration - the drug causes an inflammatory response which is biphasic. This might be taken as a sign that the treatment was effective against different developmental stages of equine intestinal parasites. Part of the study was supported by a GEN-AU grant no. GZ 200.069/2-VI/1/2003 of the bm:bwk, the Austrian Ministry of Education, Science and Culture. 48 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Open Communications - Posters 49 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 1 THE EFFECT OF α-1-ACID GLYCOPROTEIN ON ROS PRODUCTION IN VITRO IS LIMITED BY STIMULUS NATURE AND SIMULTANEOUS PRESENCE. Stakauskas R1, Leibold W2, Pieskus J1, Schuberth H2 1 Laboratory of Immunology, Lithuanian Veterinary Academy, Kaunas, Lithuania 2Immunology Unit, School of Veterinary Medicine Hannover, Hannover, Germany Introduction Alpha-1-acid glycoprotein (AGP) is an acute phase protein with described anti-inflammatory and immunomodulating properties. AGP is described as a potent inhibitor of neutrophil functions like chemotaxis and production of reactive oxygen species (ROS) in human neutrophils. However published reports about the mechanism of inhibition are conflicting. The influence of bovine AGP on the different stimuli-induced ROS production by bovine peripheral blood polymorphonuclear leukocytes (PMN) was studied using a highly sensitive method approaching its inhibitory mechanism. Materials and Methods Bovine peripheral blood PMN were purified by ficoll separation of mononuclear leukocytes and hypotonic lysis of erythrocytes. Three different stimuli - phorbol 12-myristate 13-acetate (PMA), non- opsonized and opsonized Staphylococcus aureus bateria were used in different concentrations to induce ROS production in PMN. ROS production was quantified by intracellular oxidation of dihydrorhodamin 123 (DHR) and evaluated by flow cytometry. AGP was tested over a wide concentration range in various experimental set-ups. Results AGP efficiently suppressed PMA, but not bacteria or opsonized bacteria-induced ROS generation in vitro. The suppressive effect was concentration-dependent and adversely proportional to PMA concentration. The selective inhibitory potential of AGP in comparison with ovalbumin and bovine serum albumin showed that ROS inhibition was not mere protein effect. ROS production was suppressed only if AGP and PMA were simultaneously present with PMN. Pre-incubation of PMN with AGP did not alter the PMN response to PMA. Moreover, AGP could not suppress ROS production after pre-stimulation of PMN with PMA. Conclusion The AGP does not modulate neutrophil responsiveness to stimulus and ROS production directly, but likely blocks PMA, thus reducing the stimulus concentration available for triggering the PMN. 50 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 2 DETERMINATION OF FELINE SERUM AMYLOID A (SAA) BY MEANS OF A COMMERCIALLY AVAILABLE HUMAN SAA TURBIDIMETRIC IMMUNOASSAY Kjelgaard-Hansen M, Hansen AE, Schaap MK Department of Small Animal Clinical Sciences, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.E-mail: email@example.com. Introduction Serum Amyloid A (SAA) has been recognized as an acute phase protein in cats with a marked increase within 8 hours after the occurrence of an inflammatory stimulus. Maximum concentration is reported to be reached after 24-48 hours and a fast normalization was observed when no further stimulus was present. These features suggest feline SAA to be useful as a marker for inflammation as also sustained by previous studies. However, at the present no automated commercially available assay is validated with a practicability expected by modern laboratories for routine diagnostic purposes. The objective of this study was to evaluate whether feline SAA could be measured reliably using a turbidimetric immunoassay (TIA) designed for the determination of human SAA. Materials and Methods A commercially available TIA for human SAA (Eiken Chemical Co, Tokyo, Japan [Lot no. 47007]) was used for determination of feline SAA performed on an automated chemical analyzer (ADVIA 1650, Bayer, Newbury, UK) according to recommendations of the manufacturer for human SAA determination. Intra- and interassay imprecision were investigated by multiple measurements of feline serum samples and serum pools, respectively, and assay inaccuracy was assessed by investigation of linearity under dilution. Overlap performance was investigated by comparison of SAA levels of (A) clinically healthy cats (n=56) and cats with a clinical diagnosis of an inflammatory disease (n=13) and (B) cats with (n=31) and without (n=72) an acute phase response (as evidenced by an elevated level of alpha1-acid glycoprotein determined by a feline specific immunodiffusion assay [Ecos Institute, Miyagi, Japan]), respectively. Results The observed intra- and interassay imprecision were between 2.1–9.9% and 7.0–12.5%, respectively. Dilutions were measured in a linear and proportional manner indicating no significant inaccuracy. A significant difference (P<.05, non-parametric) in SAA concentration (median [range]) was observed between both (A) healthy cats (0.4 mg/L [0.0-3.8 mg/L]) and cats with a diagnosed inflammatory disease (46.6 mg/L [3.3-150.6 mg/L]) and (B) between cats with (21.3 mg/L [0.4-150.6 mg/L]) and without (0.4 mg/L [0.0-60.4 mg/L]) a present acute phase response. Discussion and Conclusion The observed analytical performance seemed acceptable for clinical purposes. In the overlap performance, the expected difference in SAA levels according to the presence or absence of systemic inflammation was detected. Thus, feline SAA could be determined reliably by use of the human SAA TIA. The high practicability of the assay, being commercially available and automated should facilitate the implementation of feline SAA measurements for routine diagnostic purposes. 51 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 3 THE LIPOPOLYSACCHARIDE-INDUCED BOVINE ACUTE PHASE RESPONSE IS DOSE-DEPENDENT AND VARIES SIGNIFICANTLY BETWEEN INDIVIDUALS Jacobsen S1, Toelboell T1, Andersen PH1, Heegaard PMH2 1 The Royal Veterinary and Agricultural University,Copenhagen, Denmark, , 2 Danish Institute for Food and Veterinary Research, Section of Immunology and Parasitology, Copenhagen, Denmark Email:firstname.lastname@example.com This Abstract has been removed from this presentation at the request of the author for reasons of copyright. Please contact the author if you would like further information. Thank you. 52 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 4 SERUM AMYLOID A IN SERUM AND MILK IN EXPERIMENTALLY INDUCED BOVINE CNS MASTITIS Pyörälä S, Orro T, Simojoki H, Hyvönen P, Suojala L Department of Clinical Veterinary Sciences, University of Helsinki, Helsinki, Finland E-mail: firstname.lastname@example.com Introduction A new experimental mastitis model was developed, in which a strain of coagulase-negative staphylococci (CNS), S. chromogenes was used to induce udder infection. The aim of this study was to investigate development and inflammatory reaction of mastitis caused by a minor pathogen, CNS. The results are compared with those from a serious experimental infection model by a major pathogen, E. coli. Materials and Methods Six first-lactating, clinically healthy dairy cows of Holstein-Friesian breed with a low somatic cell count in their milk were used in the study. The experimental challenge was done in early lactation. One udder quarter of each cow was inoculated using a CNS strain (S. chromogenes SL37-2/01) isolated from subclinical mastitis. The infection dose was approximately 2x106 CFU per quarter in 5 ml of saline. Infection dose was selected based on pilot challenge studies with different doses of CNS. One cow was excluded after challenge, due to mastitis in another quarter. Cows did not receive any treatment. Before the challenge and at regular intervals thereafter the cows were examined clinically using a scoring system. Clinical findings consisted of general attitude, appetite, temperature, rumen function, udder palpation and milk appearance. Milk samples were taken for bacteriological culturing, SCC, NAGase, and acute phase protein determination, and blood samples were taken for acute phase protein determination. Serum amyloid A (SAA) was determined from serum and milk using a commercial kit (Phase SAA kit, Tridelta Ltd, Ireland). Results All cows became infected with S. chromogenes. Clinical signs were mild and none of the cows showed systemic signs such as fever. Only mild local signs in the inoculated quarter such as slight swelling and a few clots in the milk could be seen, which disappeared within a few days. Infection was eliminated within 38 h post-challenge in all cows except one which developed chronic mastitis. Somatic cell count increased in the affected quarters, peaking at 24-32 h (mean peak value 3.68 million cells/ml). The concentration of SAA increased in serum, and the peak levels which were seen by 48 h post-challenge varied between the cows (19-131 mg/l). SAA concentration in the milk before challenge was under the detection limit but slightly increased at 24 h after challenge (mean 2.5 mg/l) and fluctuated during the following 3 days. The maximal SAA concentrations in serum of the cows with CNS mastitis were about one fifth and SAA concentrations in the milk about 1/100 of that seen in E. coli mastitis (data not shown). Conclusions Bovine experimental CNS mastitis has not been described before, and there certainly are differences between different species in this respect. A large inoculum dose was necessary to induce mastitis. Results from this study confirmed the mild character of CNS mastitis, however detectable changes in SAA could be seen in serum and milk, which could be used to diagnose mastitis. 53 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 5 CHANGES IN LIPOPOLYSACCHARIDE BINDING PROTEIN, SERUM AMYLOID-A AND HAPTOGLOBIN CONCENTRATIONS DURING AN OUTBREAK OF RESPIRATORY DISEASE IN YOUNG DAIRY CALVES. Orro T1, Pohjanvirta T2, Rikula U3, Huovilainen A3, Sihvonen L3, Pelkonen S2, Soveri T1 1 Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland 2National Veterinary and Food Research Institute, Kuopio, Finland 3National Veterinary and Food Research Institute, Helsinki, Finland. E-mail: firstname.lastname@example.com Introduction Experimental infection studies have shown that the bovine acute phase proteins (APP) lipopolysaccharide binding protein (LBP), serum amyloid-A (SAA) and haptoglobin (Hp) are potential markers of the acute phase response during respiratory infections of calves, and the use of APP to measure the inflammatory response in naturally acquired respiratory disease has been suggested. The objective was to evaluate LBP, SAA and Hp as potential inflammatory markers during an outbreak of bronchopneumonia caused by bovine respiratory syncytial virus (BRSV) in young dairy calves under field conditions. Materials and Methods Blood samples from a group of 10 calves (7 males, 3 females) from a research dairy farm were obtained weekly during a 6-week period starting one week (week 0) before the occurrence of first clinical symptoms of respiratory infection. Mean age of calves at the beginning of the observation period was 21 ± 7 days. Calves were housed in two pens with common air space. Tracheobronchial lavage (TBL) samples were taken at week 3. Serum samples were analyzed for SAA and LBP by commercial ELISA kits and Hp by a hemoglobin-binding assay. Results All calves presented mild to moderate symptoms of respiratory disease (cough, nasal discharge, fever, high respiratory rate and abnormal respiratory sounds) from week 1. More pronounced clinical symptoms were recorded at weeks 2 and 3. The mean OD–value of BRSV antibodies increased from less than the cut-off-point of ELISA (0.2) at week 0 to 1.47±0.63 at week 6. RT-PCR analysis of TBL samples was positive to BRSV in 5 calves and 9 calves had growth of Pasteurella multocida. Mean serum concentration of LBP at week 0 was 12.6±9.2 mg/l. There was an increase in mean concentration at weeks 1, 2, and 3 (26.9±11.3 mg/l, 25.32±17.2 mg/l and 27.7±20.2 mg/l respectively, p<0.01, p<0.05 and p<0.05). Nine calves had at least a 2-fold increase in concentration (range 2-10) at some point of observation period. Mean SAA concentration increased from week 0 to week 1 from 20.0±24.2 mg/l to 59.2±31.1 mg/l (p<0.01). High mean values were also seen at weeks 2 and 3 (60.0±51.0 mg/l and 69.8±73.7 mg/l, respectively), but these were not statistically different from the initial values. Seven calves had at least a 2-fold increase in concentration (range 5-32). Mean Hp concentration increased at week 3 from 0.14±0.03 g/l to 0.28±0.2 g/l (p<0.05), and 5 calves had at least a 2-fold increase (range 2- 4). Correlation coefficients between SAA and LBP, SAA and HP, LBP and Hp were 0.774, 0.498 and 0.602, respectively. Conclusions The results indicate that SAA and LBP are sensitive and early markers of respiratory infection. Hp seems to react in later stages probably in response to the secondary bacterial infection. Although SAA shows a high relative rise and early response to the infection, LBP may be more suitable for evaluation of inflammatory response in field conditions when the stage of infection and pathogens involved in the respiratory disease are unknown. 54 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 6 EVALUATION OF ACUTE PHASE PROTEINS AS POTENTIAL INDICATORS OF SOCIAL STRESS IN DAIRY CATTLE Bach A1,2, Iglesias C 2 , Devant M2, Manteca X3, Calsamiglia S4, Ferret A4, Gimenez M5 , Saco Y5, Bassols A5 1 ICREA (Institució Catalana de Recerca i Estudis Avançats), Barcelona, Spain 2Unitat de Remugants- IRTA (Institut de Recerca i Tecnologia Agrolamientàries), Barcelona, Spain 3Department of Physiology, Universitat Autònoma de Barcelona, Barcelona, Spain 4Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, Barcelona, Spain 5Departament de Bioquímica i Biologia Molecular i Servei de Bioquímica Clínica Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain Introduction It is generally thought that multiparous cows are dominant over primiparous cows and this may result in social stress due to competition for limited resources such as water, feed, or resting areas . Acute phase proteins (APP) have been proposed as potential indicators of stress, and thus, they might potentially be used to assess social stress in dairy cattle. The aim of this study was to assess the value of APP to quantify social stress in primiparous cows. Materials and Methods A total of 60 primiparous cows were randomly distributed into two groups: a group of 45 primiparous cows with no multiparous cows (PM) and a group composed by 15 primiparous cows and 30 multiparous cows (MIX). The ratio animal/feeding place was 1.6 in both groups, thus not all animals could eat at the same time. The two groups were kept on the same dairy farm, received the same ration, and were subjected to the same management conditions. Cows were blood-sampled 30 min after the morning feeding to determine serum amyloid A (SAA) and haptoglobin concentrations. On the same day, a milk sample was collected from each primiparous cow during milking to determine milk amyloid A concentrations. Amyloid A in serum and milk was analyzed using ELISA, and serum haptoglobin was determined by the hemoglobin-binding method using a random analyzer. Data were analyzed using an analysis of variance with days in milk (DIM), treatment (MIX or PM), and the interaction between these two fixed effects. Results Primiparous cows in the MIX treatment gave priority to multiparous cows to access the feed bunk, as the median time of day at which they accessed the feed bunk was delayed (P < 0.001) about 30 min with respect multiparous cows. This suggests that primiparous cows in the MIX group experienced a social pressure from their multiparous mates. Preliminary results show that serum haptoglobin decreased (P < 0.01) with DIM and tended (P = 0.09) to be lower in PM than in MIX cows during the first quartile of lactation (1.23 vs 0.24 µg/ml). Serum amyloid A concentrations were not affected by treatment nor by DIM. Milk amyloid A concentrations followed the same trend as SAA. In fact, these two measurements were positively correlated although the correlation was not strong (r2 = 0.34, P < 0.001). Conclusions These preliminary results indicate that haptoglobin might be a useful indicator of social stress. However, if serum haptoglobin concentration is to be used to assess stress in dairy cattle, it would be necessary to establish different thresholds according to DIM, as serum haptoglobin concentrations are also affected by the physiological state of the animal. 55 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 7 ACUTE PHASE PROTEINS IN MILK: LOCAL PRODUCTION OF MAMMARY ASSOCIATED SERUM AMYLOID A3 AND HAPTOGLOBIN Eckersall PD1, Young FJ1, Nolan AM1, Knight CH2, Scott EM3 *Fitzpatrick JL1 1 Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland *now Moredun Research Institute, Penicuik, Scotland 2Hannah Research Institute, Ayr, Scotland 3Department of Statistics, University of Glasgow, Glasgow, Scotland Introduction The acute phase response, triggered by infection, inflammation and trauma, is an integral part of the innate immune response and is stimulated by pro-inflammatory cytokines with production of acute phase proteins (APP) from the liver. Extra-hepatic synthesis of these proteins has been reported and bacterial infection of the mammary gland causes their secretion in milk from cows with mastitis. Objective To relate the pathophysiological changes of the APP haptoglobin (Hp) and serum amyloid A (SAA) and mammary associated SAA3 (M-SAA3) in milk and serum to alterations in the expression of their mRNA in liver and mammary tissue. Materials and Methods The concentrations of Hp and SAA or M-SAA3 were determined by ELISA in milk and serum from cows in an experimental model of Staphylococcus aureus induced mastitis. The expression of the mRNA coding for these proteins was assessed in both mammary and hepatic tissue from infected animals and the site of synthesis of M-SAA3 protein in mammary tissues was investigated by immunocytochemistry. Results The concentration of Hp and M-SAA3 in milk increased within 18 h of S aureus infection, with peak concentrations occurring 3 days after infection at concentrations (mean + SEM) of 20.9 + 11.9 µg/ml and 27.9 + 10.1 µg/ml for M-SAA3 and Hp respectively. The increase in the milk concentration preceded the increase found in serum concentrations of both proteins as the first detectable increase of Hp and SAA in serum occurred 24 h post infection. At 48 h after infection of mammary gland with S aureus the relative increase of M-SAA3 mRNA (1500 x β-actin) in the mammary tissue was greater than the increase in Hp mRNA expression (20 x β-actin), whereas in hepatic tissue the increase in SAA mRNA (2.5x β-actin) was less than that for Hp mRNA (1000 x β-actin). Immunocytochemistry identified M-SAA3 protein within secretory epithelial cells at significantly higher levels in infected mammary glands than in control tissues. Conclusion Inoculation of mammary glands with S aureus was associated with local production of APP within a few hours. In mammary tissue up-regulation of mRNA for M-SAA3 was greater than that of mRNA for Hp whereas in the liver the relative up-regulation was reversed. 56 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 8 HAPTOGLOBIN AND PIG-MAP AS INDICATORS OF STRESS IN PREGNANT SOWS Chapinal N, Ruiz de la Torre JL, Baucells MD, Gasa J, Manteca X School of Veterinary Science, Universitat Autònoma de Barcelona, Barcelona, Spain Email: email@example.com Introduction Council Directive 2001/88/EC forces to house pregnant sows in groups between day 29 of pregnancy and 1 week before parturition. This will affect production (management and feeding) as well as welfare. Acute phase proteins like Haptoglobin (Hp) and pig-MAP (pM) are proposed as possible indicators of chronic stress in pigs. In a previous work, 60 pregnant sows were housed in individual stalls (IS), with slow feeding system (SF) and with electronic feeding system (EF). Hp and pM were analysed on weeks 9th and 14th of gestation. Hp results suggested a lower level of chronic stress in EF animals, while PM did not showed differences among groups (Chapinal et al., 2003). The aim of this study was to analyse different physiological markers of stress in two different group housing systems, compared to sows kept in individual stalls. Materials and Methods One hundred and twenty pregnant sows (Lw x Ld) from first to eighth parity, were housed between day 29-30 of gestation and 1 week before parturition in three different systems: individual stalls (IS), slow feeding system (SF), and electronic feeding system (EF). Each group consisted on 20 animals; this design was repeated introducing the replica (season) effect: winter vs spring replicas. Blood samples were obtained the 7-9th and the 12-14th week of pregnancy from 6 to 10 sows per group. For statistical test, repeated measures ANOVA and Spearman correlation test were applied. Pig-MAP and haptoglobin plasma levels were analysed by immunodiffusion. Results In the second replica, pM showed neither time nor housing system effects, while Hp decreased in SF and EF (p<0.007) but maintained levels in IS. When using all data, both pM and Hp showed a replica effect (p<0.001) with higher values in winter than in spring. Discussion Our data suggest that individual stalls are more stressing than group housing systems, whereas no differences were found between the two group housing systems studied. When using all data, the correlation coefficients between the first and the second samples were significant and higher than 0.632 (p<0.002) for Hp and pM plasma levels in winter and spring replicas. The replica effect should be confirmed since other factors, such as stockmanship and timing of blood sampling, could be present. Nevertheless, our data suggest that animals were less stressed in spring than in winter. 57 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 9 ACUTE PHASE PROTEINS IN PIGS EXPERIMENTALLY INFECTED WITH AUJESZKY´S DISEASE VIRUS Carpintero R1, Madec F2, Iturralde M1, Alava MA1, Piñeiro A1, Lampreave F1 1 Departamento de Bioquímica y Biología Molecular y Celular, F.Ciencias, Universidad de Zaragoza. Zaragoza, Spain 2French Agency for Food Safety, Ploufragan, France. Email: firstname.lastname@example.com Introduction Viral infection is a recurrent disease in pigs causing important economic loses. In this work the response of some acute phase proteins (APPs), pig-MAP, haptoglobin, C-reactive protein (CRP) and apo A-I have been studied in pigs experimentally infected with Aujeszky´s disease virus. The effect of an experimental Aujeszky´s disease vaccine was also studied. Materials and Methods Fifteen 18 weeks old pigs were inoculated with three infectious doses of a reference strain of Aujeszky´s disease virus. Seven of this pigs had been previously vaccinates at ages of 10 weeks, with an experimental Aujeszky´s disease vaccine. The other eight pigs had not been immunized. Blood samples were obtained before the injection (day 0) and 4, 8, 15 and 22 days post injection (p.i.). The concentration of positive APPs, pig-MAP, Hp and CRP and negative APP, apo A-I, were measurement by radial immunodiffusion using specific antiporcine APP antisera. Results The effect of the vaccine has been shown in this experimental model. A minor APP response and only moderate clinical sings of disease has been shown in pigs previously immunized. In contrast, pigs no vaccinates developed a severe Aujeszky´s disease and a high variation in APP concentrations. In this case, related to the day 0 before the infection, pig-MAP increased 5 times, Hp in some animals more than 100 times, CRP 6 times and apo A-I decreased 3 times. Conclusion/Discussion A high acute phase response was observed in pigs experimentally infected with Aujeszky´s disease virus. Moreover, the experimental vaccine used here mean moderated clinical symptoms of the disease and this correlated with a minor variation of APP. Our results indicated that response of APPs followed up the clinical symptoms of the outcome of this viral disease. R.Carpintero holds a fellowship from Fundación Cuenca Villoro. We thank Nieves Gonzalez-Ramon for her contribution in the initial part of this work. 58 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 10 CAN ACUTE PHASE REACTANTS BE USED AT PARTURITION TO IDENTIFY SOWS WITH SUBCLINICAL MASTITIS? Zhu Y, Österlundh I, Hultén F, Magnusson U Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden E-mail: email@example.com Aims The objective of this study was to find out whether the blood concentrations of serum amyloid A (SAA), haptoglobin and tumor necrosis factor-α (TNF-α) could be used as markers of experimental coliform mastitis in sows at time of parturition Materials and Methods Blood samples were obtained from sixteen crossbred primiparous sows categorized into three groups: non-inoculated (n = 4), inoculated and affected (n = 4), and inoculated and non-affected (n = 8). The later group was regarded as subclinically diseased. Blood samples from the non-inoculated sows were collected for three consecutive days starting 10 to 5 days before farrowing, the day just before farrowing (day -1) and during four days after farrowing. Sows in the inoculated groups were inoculated with E. coli (serotype O 127) within 24 h before farrowing. Blood samples from these groups were collected for three consecutive days starting 9 to 4 days before inoculation and at 0, 24, 48, 72 and 96 h after inoculation. Sows were categorized as affected or non-affected based on rectal temperature, habitus, appetite and clinical udder changes. The concentrations of SAA in plasma and TNF-α in serum were measured by ELISA, serum haptoglobin concentration was assayed by use of a haemoglobin-binding method. Results In the non-inoculated sows there was a significant elevation (p < 0.001) of plasma SAA concentration on day 2 post farrowing as compared to the pre-farrowing concentrations. Also haptoglobin concentration was elevated on day 1 (p < 0.001) and day 2 (p < 0.001) post farrowing in this group. The serum concentration of TNF-α on the other hand did not vary around parturition in the non-inoculated group. At 24 h post inoculation, the blood concentrations of SAA (p < 0.001) and TNF-α (p < 0.01) were elevated in both inoculated groups as compared to the pre-inoculation values. In addition, the concentration of TNF-α in serum (p < 0.001) was lower in the subclinical group than in the affected group at 24 h post inoculation. Conclusions The data indicate that the blood concentrations of SAA and haptoglobin are altered by normal parturition itself, and are therefore not suitable for detection of mastitis at this time-point. In contrast, it seems that the concentration of serum TNF-α is not affected by normal parturition and that it increases more in sows clinically affected by coliform mastitis than in sows that are subclinically diseased. Therefore we suggest that TNF-α in blood should be further evaluated as a marker of subclinical coliform mastitis in the periparturient sow. Identifying these sows will likely be a major contribution to the improvement of piglets health and survival. 59 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 11 DEVELOPMENT OF A RAPID BIOSENSOR METHOD FOR DETERMINATION OF HAPTOGLOBIN IN BOVINE MILK Åkerstedt M1, Björck L1, Waller KP 2,3 , Sternesjö Å 1 1 Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden 2 Department of Ruminant and Porcine Diseases, National Veterinary Institute 3 Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden Introduction The somatic cell count (SCC) in bulk tank milk is widely used in milk quality programmes, serving as one of the parameters for quality payment of the raw milk. The dairy cooperatives often give farmers premium quality payment to encourage a low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory processes in the lactating cow includes the acute phase proteins haptoglobin (Hp) and serum amyloid A (SAA). So far these proteins have been studied from the diagnostic perspective, with little attension regarding their role in relation to milk composition and technological properties. The aim of the present study was to develop a rapid biosensor method to determine Hp in milk and to compare the results of the assay with results obtained by a commercially available immunoassay. Materials and Methods An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology (Biacore AB, Uppsala, Sweden). The resulting assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis (n=43) collected at the dairy research herd at Jälla, Uppsala. In addition, quarter milk samples from cows with clinical mastitis (n=23) were obtained from the National Veterinary Institute, Uppsala. All milk samples were also analysed with a commercial ELISA for determination of Hp in milk (Tridelta Development, Ltd., Co. Kildare, Ireland) for comparison with the biosensor assay. According to the manufacturer, the limit of detection of the ELISA was 0.3 µg/ml. Results The detection limit (LOD) of the developed biosensor assay was determined to 1 µg/ml. With-in and between-day variations (CV) determined at 4 µg/ml were 3.6 % and 5.9 %, respectively. In the analysis of Hp in composite milk samples from cows without clinical signs of mastitis, 36 of the 43 samples did not contain detectable levels of Hp. The ELISA detected Hp in the remaining 7 samples (1-12 µg/ml) whereas the developed biosensor assay only detected Hp in the 3 samples with highest concentrations. In the analysis of quarter milk samples from cows with mastitis, 19 of the 23 samples had Hp concentrations below 50 µg/ml (1-48 µg/ml) and 4 samples contained higher Hp levels (51-143 µg/ml). For these samples the results obtained by the biosensor assay agreed satisfactory with the results in the ELISA. Conclusions Detection of sub-clinical cases of mastitis in the herd is of great importance, not only to the producer but also to the dairy industry. Milk from cows with sub-clinical mastitis has altered composition and processing properties, which will affect the quality of dairy products. New indicators of inflammation are needed to allow early identification of infected quarters. The overall aim of our work is to study if the acute phase proteins can be used as indicators not only for udder health but also for raw milk quality. For such studies we need rapid and simple methods. To our knowledge this study is the first application of an optical biosensor for determination of acute phase proteins e.g. Hp in milk. The main advantage of the biosensor in comparison with ELISA is the automated and rapid detection; each analysis requiring approximately 8 minutes. The results of this study show that the developed assay has potential to be useful in screening of Hp in milk, however, further optimization is required to achieve a lower detection limit. 60 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 12 HAPTOGLOBIN IN SERUM OF COWS: RELATIONS TO METABOLIC PARAMETERS DURING EARLY LACTATION Hachenberg S, Weinkauf C, Hiss S, Müller U, Sauerwein H Institute of Physiology, Biochemistry and Animal Hygiene, University Bonn, Germany Introduction: Haptoglobin (Hp), one of the most sensitive acute phase proteins (APP) in cattle, is synthesized in response to infection, inflammation or fatty liver (1). The elevation of non-esterified fatty acids (NEFA) observed in the conditions of negative energy balance is characteristic for high yielding dairy cows in early lactation. To clarify whether a negative energy status increases serum Hp, Hp was determined in cows without abnormalities in differential haemogram during that time. The data for ß- hydroxybutyrate (ß-OH-B), non-esterified fatty acids (NEFA) and of NEFA/ß-OH-B ratio presented here were tested for potential relationships with serum Hp concentrations. Methods: Blood samples were collected weekly by jugular venipuncture, during the first 12 weeks post partum, from 28 multiparous Holstein Friesian cows, average age 4.64 ± 1.23 years of two different feeding intensities. Group I received 154 ± 13 MJ NEL/d, group II was fed with 145 ± 19 MJ NEL/d. Serum ß-OH-B concentrations were determined by a veterinary diagnostic laboratory (VLK, Cologne). NEFA concentrations were analysed with a commercial testkit (Roche Diagnostics, Mannheim). Serum Hp concentrations were determined using an ELISA (2). The NEFA/ß-OH-B ratios were calculated (3). The metabolic parameters in blood samples (n=14 animals of each group) were compared using analysis of variance (SPSS 12.0). Results: Serum concentrations of Hp as well as the metabolic parameters were affected by time (p<0.05). For Hp and the NEFA/ß-OH-B ratio maximal concentrations were observed during the first two weeks after delivery, while NEFA and ß-OH-B were elevated for the first half of the experimental time. In serum, Hp and NEFA were related (r=0.4; p<0.001). Differences between the two feeding-groups could be found between the metabolic parameters NEFA and the NEFA/ß-OH-B ratio (p<0.01). Conclusions: Higher NEFA concentrations as well as higher NEFA/ß-OH-B ratios according to (3), demonstrate the negative energy status of the animals in group II. Although no differences of Hp concentrations could be established between the two feeding-groups, there seems to be an interrelation between conditions of negative energy balance and increasing Hp levels. References (1) Nakagawa, H.; Higuchi, H.; Watanabe, A.; Katoh, N., 1997: Res. Vet. Sci. 62, 137-141 (2) Hiss, S.; Mielenz, M.; Bruckmaier, RM.; Sauerwein, H., 2004: J Dairy Sci. 87 (11), 3778-3784 (3) Sato, H.; Matsumoto, M.; Hanasaka, S., 1999: J Vet Med Sci. 61 (5), 447-51 61 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 13 RELATIONSHIP BETWEEN HAPTOGLOBIN, FIBRINOGEN AND CLINICAL STATUS IN HORSES Oukacha F1, Bertrand P2, Bustin J1, Humblet M1, Amory H2, Godeau J1 1 Department of Functional Sciences ,2Department of Clinical Sciences, University of Liege, Liege, Belgium Email: email@example.com Introduction Haptoglobin (Hp) and fibrinogen (Fb) are positive acute phase proteins (APPs) that increase in cases of infections, inflammatory or trauma conditions (Conner and al.,1986 ; Skinner and al., 1991). These proteins measurements have been used to identify clinical and subclinical diseases in horses (Hulten et al., 2002). Elevated concentrations are not specific to a disease, but are rather the outcome of tissue dammage resulting from infection or inflammation (Eckersall and Conner, 1988). The aim of this study was to compare Hp with Fb in horses. Hp and Fb reference values were also determined in healthy horses. Material and Methods Blood samples were collected from clinically healthy and diseased horses, on the first day of clinical examination, for the determination of Hp and Fb concentrations. They were measured in order to assess their ability to identify horses suffering from acute inflammation. None of these horses was pregnant. The animals were classified according to clinical examination : they were healthy or diseased horses. Diseased horses were gathered on the basis of the category of pathologies: digestive, circulatory, musculoskeletal, genital, nervous, respiratory, cutaneous and cardio-pulmonary. Results Hp reference intervals were from 0 to 600 mg/L and Fb reference values from 0.00 to 2.50 g/L and Fb mean concentrations in healthy horses, 375±194 mg/L and 1.78±O.36 g/L respectively, were significantly lower than diseased horses, respectively, 946±748 mg/L and 2.74±1.49 g/L, ( P≤0,001). When all diseased animals were considered together, Hp sensitivity reached a 60.0% value against 48.0% for Fb. Meanwhile, respectively 84.0%, 77,3% and 73.0% of musculoskeletal, respiratory and digestive diseases presented a positive response in Hp. On the other hand, Fb mean concentrations were significantly higher in 80.0% of cutaneous and 72,7% of musculoskeletal pathologies. Hp and Fb were not significantly correlated. Discussion Hp was well in agreement with the clinical status of disease in case of musculoskeletal, respiratory and digestive pathologies. Meanwhile, the presence of an inflammation process was better confirmed by Fb in case of cutaneous and musculoskeletal pathologies. Considering all diseased horses, it appeared that Hp presented a better sensitivity than Fb. In conclusion, serum haptoglobin concentration seemed to be more sensitive than Fb in the detection of various inflammatory diseases in horses. 62 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 14 USING HAPTOGLOBIN AS PRELIMINARY INFORMATION TO ASSESS CARCASS´S EXAMINATION RESULTS AND SALMONELLA STATUS OF SLAUGHTER PIGS Gödderz A1, Witten I1, Knura S1, Wendt M2, Petersen B1 1 Department of Preventive Health Management, University of Bonn, Bonn, Germany 2Forensic Medicine and Ambulatory Service, School of Veterinary Medicine, Hannover, Germany Email:email@example.com Introduction Haptoglobin (hp) is one of the major acute phase proteins in swine. Several authors have reported an increase of blood hp concentration in pigs due to acute inflammatory infections but also due to subclinical diseases. Therefore hp is discussed as a screening parameter to assess swine´s health status along the pork production chain. In order to get additional information of slaughtered pig´s health status this study examined whether there is a relation between measured hp concentrations in slaughter blood and meat juice (mj) and findings of the carcass`s examination and the Salmonella status of the slaughtered pigs. Material and Methods At a German abattoir 330 fattening pigs were randomly selected and carcass and the appendant organs were tested for pathological findings by means of checklists. Slaughter blood was collected in EDTA covered vacutainers from each pig. The samples were centrifuged for 10 minutes at 2500 U/min and the supernatant was aliquoted. Two mj samples of each pig were obtained by deep freezing (-21°C, 24 hours) walnut sized pieces of musculus brachiocephalius (mj 1) and of the diaphragmatic pile (mj 2) and following thawing at room temperature. Hp concentrations of blood and mj samples were determined by an ELISA (HISS et al. in Vet. Immunol. Immunopath. 2003). Additionally the Salmonella antibody titer of mj 2 was determined (Salmotype® Fleischsaft ELISA, Labor Diagnostic Leipzig). A pig was assumed sero positive if optical density was above 40 %. Faecal samples were extracted by incision of the illeum and qualitative checked for the presence of Salmonella spp. Statistics were done by using SPSS 12.0 for windows and the calculation of the Odds Ratios (OR) by using Win Episcope 2.0. For the calculation of OR two categories of the measured hp concentration were composed: One below the according mean concentration and one above. The level of confidence was determined at 95 %. Results and Discussion According to previous investigation correlations (p ≤ 0.01) between hp concentration in blood and mj samples were found (blood and mj 1: r = 0.75 / blood and mj 2: r = 0.70). The statistical significant OR and the according Confidence Interval (CI) for a carcass or organ finding depending on an increased hp concentration (> overall mean) are shown in table 1. Table 1: OR and CI for a carcass/organ finding, if hp exceeds the mean concentration of all 330 pigs hp (> overall mean scratches pericarditis pleurisy pneumonia concentration) OR CI OR CI OR CI OR CI blood (>1.08 mg/ml) 0.553 0.322-0.920 3.962 1.568-10.010 - - - - mj 1 (> 0.14 g/ml) 0.573 0.337-0.974 3.876 1.580-9.507 2.320 1.236-4.355 2.199 1.347-3.589 mj 2 (> 0.12 mg/ml) 0.522 0.312-0.871 4.061 1.514-10.890 - - - - These results indicate increased risks for specific organ findings of animals having higher hp concentrations. The long time interval between the origin (acute disease) of detected pathological findings and the time of blood resp. mj sampling must be taken into consideration when assessing the results. The lower risk for scratches of pigs with increased hp concentrations may be attributed to reduced activity due to disturbances of health. For the hp and the Salmonella status of the pig as well as all other findings no statistical significant OR could be calculated. This might be the consequence of the fact that most of these findings were chronical. 63 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Confirming actual literature an OR of 1.837 (CI: 1.029-5.279) was determined by regarding Salmonella sero-positive animals in relation to detected milk-spots in the liver. Furthermore Salmonella sero-positive pigs showed higher (p = 0,054) mean blood hp concentration (1,03 mg/ml) than sero-negative pigs (1,26 mg/ml). This tendency could also be shown in pigs with Salmonella detection in faeces (shedders). The elevation of the hp concentration after induction of the acute phase reaction caused by several pathogens had already been shown in former investigations. In this studie it could be suggested that the presence of Salmonella might cause increased hp levels. In conclusion Hp quantification in mj or slaughter blood might be an useful parameter to assess meat quality at slaughter and further along the processing chain in terms of animal health. Acknowledgment: We would like to thank the Bakum Field Station for Epidemiology of the School of Veterinary Medicine Hannover for the measurement of the salmonella antibody titer as well as the salmonella status of the faeces. 64 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 15 POST-TRANSLATIONAL MODIFICATION OF FELINE ALPHA1-ACID GLYCOPROTEIN IN RETROVIRUS (FIV, FELV) AFFECTED CATS Pocacqua V, Provasi E, Paltrinieri S, Ceciliani F Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, University of Milan, Milano, Italy Introduction AGP (Alpha1-acid glycoprotein) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this communication we present the feline AGP's glycan moiety modifications in the course of two very frequent feline diseases, the feline acquired viral immunodeficiency (FIV-associated) and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I (SNAI) and Maackia amurensis agglutinin (MAA) lectins were used to detect sialic acid residues, Aleuria aurantia lectin (AAL) was used to detect L-fucose residues and Concanavalin A (ConA) was used to evaluate branching degree. In order to determine the possible number and position of oligosaccharide chains, the cdna sequence of fagp was also determined. Materials and Methods Serum samples (0.5 to 1 ml) were obtained from 12 clinically healthy cats, as positive controls, 12 FIV affected cats and 8 FeLV-positive cats. The purification of fAGP was carried out following the protocol described in Ceciliani et al. Purified fAGP was subjected to SDS-PAGE in a discontinuous pH system using a Miniprotean II apparatus (Bio-Rad) and then stained with Coomassie blue, or electroblotted onto nitrocellulose. Structural characterization of glycoprotein carbohydrate chains was determined by using digoxigenin- labeled lectins on blots and four lectins were used in this study; the digoxigenin-conjugated lectins SNAI and MAA (Roche) that are specific for sialic acid alpha(2-6)-linked to galactose and alpha(2-3)-linked to galactose, respectively. The biotin-conjugated lectin AAL (Vector Laboratories) shows affinity for alpha(1–6), alpha(1–2) and alpha(1–3)-linked fucose while Con A (Vector Laboratories) binds to the diantennary glycans. In order to determine fAGP coding sequence, total RNA was extracted from samples of liver collected at necrospy from cats that did not show inflammatory or neoplastic changes and then transcribed into cDNA by reverse transcription (RT). PCR was performed using primers that were designed on the basis of the most conserved portions of the coding regions of known AGP sequences available in Genbank. The segments obtained with predicted molecular weights were gel-purified and sequenced directly with ABI technology using an automated DNA sequencer (ABI PRISM 310 Genetic Analyzer). The predicted amino acid sequence was obtained using the ExPASy proteomic server. Results The molecular weight of fAGP, determined by electrophoretical mobility, ranged from 42 kDa to 46 kDa; since the molecular weight of the aminoacidic sequence accounts for 21 kDa, we may deduce that the glycan component of fAGP accounts for approximately 50%. The present study found that fAGP expressed during FIV did not present any fucose residues on its surface and its branching was not increased during the course of the disease. On the contrary, several differences in the degree of sialylation were detected among FIV affected cats. The expression of both alpha(2-6)- and alpha(2-3)-linked sialic acid were increased in FELV-cats which developed lymphoma, while clinically healthy FeLV-positive cats did not present any increase in the expression of sialic acid on the protein surface: on the contrary, a reduction of alpha(2-3)-linked sialylation was detected. There were also several differences in the sialylation degree of fAGP among FeLV affected cats. The three segments of fAGP obtained were overlapped together to form a 702-bp cDNA sequence and the translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues with five potential N-Glycosylation sites, but also with seven potential phosphorylation sites. Comparison of the deduced primary structure of feline AGP with that of other proteins belonging to the same family showed that 89 out of 183 aminoacids, and all the cysteine residues, are conserved in all the species so far sequenced. 65 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Conclusion The lectin affinity staining of electroblotted fAGP is a technique that allows a rapid screening of a large number of samples. By means of this simple technique, we have shown here that the glycan pattern of fAGP is modified during disease. We report that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in lymphoma affected cats, while FeLV -positive cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of purified fAGP, which however varied widely among individuals. The determination of the primary structure also revealed a very interesting feature of fAGP; like other AGP's also fAGP exhibits several (seven) potential phosphorylation sites. We can also conclude that the three dimensional structure of these proteins is well conserved. References Ceciliani F., Grossi C., Giordano A., Pocacqua V. and Paltrinieri S., Decreased sialylation of the Acute Phase Protein a1-Acid Glycoprotein in feline infectious peritonitis (FIP), Vet Immunol Immunopathol. 99 (2004) 229-36. 66 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 16 TIME TREND IN HAPTOGLOBIN, C-REACTIVE PROTEIN AND PIG-MAP CONCENTRATION AFTER LONG DISTANCE TRANSPORT OF PIGS Mellia E1, Salamano G1, Candiani D2, Ru G1, Ingravalle F1, Bruno R2, Doglione L1 1 IZS-State Veterinary Institute of Piedmont, Turin, Italy 2Dept. of Veterinary Morphophysiology, Turin University, Turin, Italy. Email: firstname.lastname@example.com Introduction The synthesis of acute phase proteins (APPs) is a physiological event that occurs following a threat to organism's homeostasis. Body's cytokine system activates these proteins and initiates the Acute Phase Response (APR), which leads to non-specific host defence and to repair mechanisms. Positive APPs rise to concentrations several times the normal level in response to challenge; in swine the major positive APP include haptoglobin (Hp), C-reactive protein (CRP) and pig major acute phase protein (pig-MAP). The aim of the present study was to investigate the effect of long distance transport on Hp, CRP and pig- MAP serum concentration and the changes of these APPs over a period of 28 days. Materials and Methods A total of 60 Landrace x Large White 4-month old gilts were transported from Denmark to Italy (a 48 h journey) and then followed up for 28 days. These animals were randomly allotted to three different groups (A, B, C) of 20 pigs. Each group was located in a 6 x 3.5 meter pen. Food and water were given ad libitum throughout the study. Blood samples were collected from all animals from the jugular vein immediately upon arrival and 28 days later (T1, T28). Group B was also bled on fourteenth day (T14) while group C was also bled on third, fifth and fourteenth day (T3, T5, T14). In order to rule out concurrent inflammatory processes complete hematology profile was determined in fresh whole blood using a HemaVet 3500 analyser (CDC, Technologies, Oxford, CT) calibrated for swine and furthermore the serum from each blood sample was separated by centrifugation (3000 rpm, 10 min) and stored at –80°C. Serum haptoglobin and CRP concentrations were measured with commercial assay kits (Tridelta Development, Greystones, Ireland). Serum pig-MAP concentration was assayed with a sandwich ELISA test (PigCHAMP Pro Europa S.A., Segovia, Spain). The distributions of APPs were described using boxplots. After checking normality and homogeneity of variances for all data, non parametric methods (Friedman, Kruskal-Wallis and Wilcoxon tests) were applied. Results Hematology profiles supported the absence of inflammatory processes throughout this field study. APP concentrations on T1 in the three groups were similar (P>0.05) whereas group C showed higher levels on T28. The time trend about pig-MAP shown in the figure 1 below can paralleled to the other proteins. Hp, CRP and pig-MAP showed similar behaviour: in groups A and B their concentrations on T28 compared to those on T1 were decreased; in groups B and C increased median levels of APPs were detected on T14, this was particularly evident in group C. With regard to pig-MAP and Hp the increase was statistically significant. Furthermore in group C data showed a trend toward lower serum concentrations of APPs on T5 compared to those on T1 and T14. 67 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 g rou p A g rou p B 2 .5 2 .5 2 2 pi gM AP in m g/ ml p i gM AP in m g/ ml 1 .5 1 .5 1 1 .5 .5 0 0 T1 T2 8 T1 T1 4 T2 8 g rou p C 4 3 p i gM AP in m g/ ml 2 1 0 T1 T3 T5 T1 4 T2 8 Fig.1 - Pig-MAP boxplots in groups A, B and C Discussion The quantification of Hp, CRP and pig-MAP concentrations in swine seems to be useful for evaluating stressing conditions unrelated to inflammatory processes even if statistical significance is not always reached. Unexpectedly it has not been observed a constant trend toward lower concentrations of APPs from T1 through T28. The increase observed on T14 could be likely due to the fact that gilts require a period of adaptation to a new environment. This kind of stress in group B seems worked out within an additional 14-day period and probably the same happens in group A that was not bled on T14. Serial blood samplings carried out on group C represent further stressors for gilts. That might explain why this group showed levels of the APPs particularly elevated on T14 and why on T28 higher concentrations persist. Further studies are needed in order to get better understanding about the behaviour of Hp, CRP and pig- MAP after the move of swine to a new environment. Moreover it would be interesting to obtain data about APPs concentrations immediately before a long distance transport. This study was supported in part by a grant from the Italian Health Ministry (Ricerca corrente 2002). 68 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 17 THE GLYCOSYLATION PATTERN OF FELINE ALPHA 1 ACID GLYCOPROTEIN (AGP) Cunningham K1, Smith KD2, Addie DD3, Eckersall PD1 1 Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland 2Department of Bioscience, University of Strathclyde, Glasgow, Scotland. 3Division of Infection and Immunity, University of Glasgow, Glasgow, Scotland. E mail: email@example.com Introduction Alpha 1 acid glycoprotein is a positive acute phase protein in humans and cats as its plasma concentration increases 2-5 fold during the acute phase response. Alpha 1 acid glycoprotein is extensively glycosylated with 5 complex type N-linked glycan chains that account for 45% of its 41-44 kDa molecular weight. In normal plasma AGP does not exist in a single form but as heterogeneous population due to subtle changes in the N-linked oligosaccharide chains. Since the oligosaccharide chains attached influence the functional role of AGP, the existence of structural distinct glycoforms in plasma implies functional diversity. During several physiological and pathological conditions in man, not only the total concentration of AGP is altered but the relative proportions of the normal AGP glycoforms have also been found to change and abnormal glycoforms are expressed with the oligosaccharide "fingerprint" of AGP being altered. The plasma concentration of AGP is known to be increased in feline infectious peritonitis (FIP) and is now used as part of a panel of tests to diagnose FIP. At present there is relatively little information on the diagnostic significance of AGP glycosylation in this diseases. Materials and Methods The concentration of AGP was measured in peritoneal fluid and plasma submitted to the feline virus diagnostic laboratory of Glasgow Veterinary School using radial immunodiffusion. Thereafter AGP was purified from the remnant of each sample after all diagnostic tests were complete using a method which ensured that no desialylation of the oligosaccharide chains or denaturation of the protein structure occurred. The samples were initially precipitated with 30 % w/v Polyethylene glycol (PEG) Mwt 8000, followed by three chromatography steps, which utilized affinity, anion and cation exchange chromatography resins and desalting. After purification samples were either hydrolysed with acid or digested with PNGase F which releases the oligosacchrides. After acid hydrolysis the monosaccharide composition was determined using High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Samples digested with PNGase F were also analysed using HPAEC-PAD. Results Initial analysis of feline AGP revealed differences in glycosylation between healthy and diseased cats. Fucose, Mannose, Galactose and Glucosamine were found on feline AGP from FIP infected cats however little or no fucose was found on normal feline AGP. Oligosaccharide analysis revealed that feline AGP purified from peritoneal fluid of FIP infected cats had mainly bisialylated biantennary glycan chains attached to the polypeptide backbone. Conclusion The glycosylation pattern of feline AGP has novel features and could be diagnostic for the appearance of FIP, opening up possibilities for treatment. 69 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 18 A NEW ASSAY FOR FELINE α1-ACID GLYCOPROTEIN Bence LM1, Eckersall PD1, Addie, DD2 1 Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland 2 Division of Infection and Immunity, University of Glasgow, Glasgow, Scotland. Email:firstname.lastname@example.com Introduction Feline infectious peritonitis (FIP) is the leading infectious cause of cat death, currently diagnosed using a panel of tests, including measurement of acute phase proteins. Alpha-1-acid glycoprotein (AGP) is an acute phase protein in cats and is elevated in FIP. AGP levels in peritoneal fluid or serum are routinely measured using a single radial immunodiffusion test (RID). Immunoturbidimetric assays are more precise, rapid and efficient and would allow AGP to be included in the panel of tests currently measured on biochemical analysers. The objective of this study was to develop an immunoturbidimetric assay to measure AGP and to compare the results with those obtained from RID. Materials and Methods AGP was purified from a pool of peritoneal fluid, with an elevated concentration of AGP, obtained from cats diagnosed with FIP. Antisera to AGP was raised and a method was then developed on the COBAS MIRA (ABX Diagnostics) analyser. The AGP concentration in a pool of peritoneal fluid was established and this was used as a standard curve to develop the immunoturbidimetric assay. Samples of serum or peritoneal fluid were obtained from approximately 170 cats, the AGP concentration measured using the developed immunoturbidimetric assay and compared to the concentration measured by RID. Results The concentration of AGP in the pool of peritoneal fluid was measured as 3.65g/L, which was diluted to give a standard curve ranging from 0-1g/L AGP. Samples were applied at a range of dilutions. Purified AGP, at known concentrations, were also added as QC controls. This study shows that there is good correlation, in peritoneal fluid (r2 = 0.91; p<0.001)) and in serum (r2 = 0.92; p<0.001), between results obtained using the RID and results obtained using the immunoturbidimetric assay. Conclusion A rapid immunoturbidimetric assay for measurement of feline AGP in peritoneal fluid or serum has been developed, which will be of value in diagnosis of FIP. 70 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 19 AUTOMATION OF A METHOD TO DETERMINE ACID SOLUBLE GLYCOPROTEIN IN PORCINE SERUM AFTER ACID PRECIPITATION Tecles F, Martínez Subiela S, Parra MD, Cerón JJ Animal Medicine and Surgery Department, University of Murcia, Murcia, Spain E-mail: email@example.com Introduction After perchloric acid precipitation, the acid soluble glycoprotein (ASG) fraction left in supernatant can be easily measured using Coomassie brilliant blue (Nagahata et al 1989) or bicinchoninic acid (Eckersall et al., 1996). In most species, the acute phase protein alpha-1 acid glycoprotein (AGP) is the predominant protein in ASG, so determination of ASG can reflect the acute phase status of pigs (Lampreave et al 1994, Eckersall et al 1996). The purpose of this work was to validate an automated protocol for the measurement of ASG after acid precipitation in porcine serum and prove its usefulness to detect acute phase response in pigs. Material and Methods All analyses were performed as follows: samples were precipitated using perchloric acid. After centrifugation, total protein concentration in supernatant was determined using bicinchoninic acid in Cobas Mira Plus analyzer. Calibration was performed using a secondary pooled porcine serum standard with a concentration of ASG of 19.15 g/L, diluted to 9.58, 4.79, 2.39, 1.2, 0.6 and 0.0 g/L. These concentrations were established using human ASG (Sigma Chemical) as primary standard. For analytical validation of the method, 3 porcine serum samples with high ASG value, and 3 with low ASG concentration were used. Within and between run coefficients of variation were determined by repeated measurements performed the same day and in different days, respectively. Accuracy was determined by linearity under dilution method, diluting samples at 50, 25 and 12.5%. Limit of detection was determined by repeated analysis of sample diluent. To assess clinical validation, 10 porcine samples from healthy animals and 12 from animals with different diseases were measured using the same protocol described above. A non parametrical statistic analysis was performed using SPSS for Windows. Results Within and between run coefficients of variation were lower than 5% and 10%, respectively. Linearity under dilution showed a coefficient of linear regression higher of 0.99 in all the samples tested. Limit of detection estimated for the test was 0.23 g/L. Values for healthy samples ranged between 2.71 and 5.32 g/L, with median of 3.57 g/L; for pathologic samples values ranged between 4.46 and 11.45 g/L, with median of 8.35 g/L. Statistical differences were seen between healthy and pathologic samples (p<0.001). Conclusion The automated method described can be used for an accurate, cheap, rapid and easy quantification of ASG in porcine serum samples, and it could contribute to a wider use of ASG as acute phase protein in porcine medicine. References. Eckersall PD, Saini PK, McComb C. 1996. The acute phase response of acid soluble glycoprotein, alpha1-acid glycoprotein, ceruloplasmin, haptoglobin and C-reactive protein, in the pig. Veterinary Immunology and Immunopathology 51: 377-385. Lampreave F, Gonzalez-Ramon N, Martinez-Ayensa S, Hernandez M, Lorenzo H, Garcia-Gil A, Pineiro A. 1994. Characterization of the acute phase serum protein response in pigs. Electrophoresis 15: 672-676. Nagahata H, Taguchi K and Noda H. 1989. Preliminary studies on the acid soluble glycoproteins in serum and their diagnostic value for acute inflammatory disease in cattle. Vet. Res. Comm., 13:257-263. 71 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 20 MEASURING THE NEGATIVE ACUTE PHASE REACTANT TRANSTHYRETIN IN PORCINE SERUM Campbell FM, Waterston MM, Eckersall PD Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland Email firstname.lastname@example.com Introduction Transthyretin (TTR) is a thyroxine binding protein found in blood which forms a complex with retinol binding protein for the transport of vitamin A. TTR is a negative acute phase reactant in humans and the concentration of TTR in serum falls due to decreased synthesis in inflammation, infection or trauma. Serum levels of TTR are measured as a health status indicator using commercially available assays. We have developed an assay for TTR in pig serum the method and use of which is described below. Materials and Methods Microtiterplates (96 well, Corning Costar, Cambridge UK) were coated with pig serum samples or purified human prealbumin (TTR) (Sigma-Aldrich, Poole, UK) diluted in 50 mM NaHCO3 (pH9.6), 100 µl/well for 20 hours at 4oC. The samples were then decanted and unbound sites blocked by adding 250µl of 5% (w/v) non fat dried milk in assay buffer, (0.12 M NaCl, 0.02 M Na2HPO4, 0.1% (v/v) Tween 20, pH 7.4) at room temperature for 30 minutes. The plates were washed with assay buffer and then 100 µl of sheep anti-human TTR (ICN Biomedicals, Basingstoke UK) diluted 1/1000 in assay buffer was added to each well and the plates incubated for 1 hour at 37oC. The wells were then decanted and washed three times in assay buffer. Then 100µl of HRP conjugated anti-sheep IgG (Sigma, Poole, UK) 1:2000 in assay buffer was added to each well and incubated for 30 minutes at 37oC. After washing three times with assay buffer the wells were filled with 100 µl freshly prepared TMB substrate solution (KPL, Guildford, UK) the reaction was stopped after 30 min by the addition of 50µl of 1 M HCl and then the absorbance was read at 450nm. The TTR concentration in the serum samples after a 1:400 dilution was compared to a standard curve of human TTR over a range of 0.03-2.00 µg/ml. This assay was used to determine TTR concentrations in porcine serum samples from individual pigs in order to give an idea of the range of concentrations of TTR present in porcine serum. Results The lower detection limit for porcine serum TTR was determined as 0.031 µg/ml. Detection limits were determined by measuring 15 replicates of saline and replicates of the lowest concentration standard and calculating the mean concentration of saline +2 SD of the lowest concentration using human TTR as standard. The interassay precision determined by calculation of coefficient of variance (CV) of 8.4 % at 129.4 µg/ml and 13.8% at 83.6µg/ml was obtained by measuring the same two porcine serum samples in 18 separate assays. The intraassay CV was ascertained to be 2.2 % being the mean of the CVs of 28 duplicate samples in the same assay at a concentration range of 117 – 292 µg/ml. Serum samples taken from individual pigs (n=272) were assayed for TTR and values for the individual pig serum samples measured ranged from 32 to 690 µg /ml with a median of 129.5 µg /ml. Conclusions We have developed a means to measure TTR in pig serum. Measuring the levels TTR in serum in addition to other acute phase proteins to monitor pig health status may provide extra information particularly as its may be a useful monitor of growth related phenomena as it is in man. Acknowledgements: Support from the European Commission for these studies is gratefully acknowledged (Shared Cost Project QLK5-2001-02219) 72 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 21 BIOMARKERS FOR BOVINE MASTITIS USING SURFACE ENHANCED LASER DESORPTION-IONISATION TIME OF FLIGHT MASS SPECTROMETRY Eckersall PD1, Hogarth, CJ2 1 Division of Animal Production & Public Health, University of Glasgow, Glasgow, Scotland 2Ciphergen Biosystems Ltd, Guildford, Surrey, UK Introduction Recent studies have shown that during bovine mastitis, acute phase proteins are produced by the mammary gland and are secreted in milk. However, there are likely to be other changes to the proteins in milk during the development of the disease which could be exploited as biomarkers for mastitis. Proteomic studies seek to examine the full complement of protein expression in a cell, tissue or fluid under a given set of physiological conditions. Previously, proteomic approaches combining two- dimensional gel electrophoresis (2-DE) and mass spectrometry have been used for the analysis of bovine milk. Low molecular weight proteins and peptides are notoriously difficult to analyse in conventional 2- DE based proteomic approaches. Surface enhanced laser desorption-ionisation time of flight (SELDI- TOF) mass spectrometry provides a methodology to extend the examination of the milk proteome to include these low molecular weight proteins. Material and Methods In the current study SELDI-TOF mass spectrometry was used to examine differentially expressed proteins in bovine milk from quarters of cows in an experimental model of Staphylococcus aureus induced mastitis. Milk samples were collected from infected quarters of 6 cows 42-48 hours after inoculation with S aureus and were compared to the milk from opposite, healthy, quarters in the same cows that had been inoculated with sterile saline. Following centrifugation to remove lipid and cellular material, 2 µl of the milk samples were applied to each individual spot of an 8-spot CM10 (weak cation exchange) array and incubated for 1h at room temperature. The arrays were then washed (3 x 5min) in a 100mM Na Acetate buffer, pH4 and left to dry at room temperature. After drying, 0.5 ml of saturated sinapinic acid (in 50% acetonitrile and 0.5% TFA) was applied to each spot twice, prior to final air-drying for 10 minutes. Arrays were analysed by SELDI-TOF mass spectrometry on a Series 4000 instrument (Ciphergen Biosystems Ltd). Results Comparison of the the SELDI-TOF mass spectrograms between milk samples from healthy and mastitic quarters in the same cow showed a clear increase in the number and peak heights for low molecular weight peptides (mass/charge (M/Z) ratio <20k). In total 63 statistically significant (p-value < 0.05) protein biomarkers were identified as being able to discriminate between mastitic and healthy milk. Of these, 44 were of low molecular weight). Discussion This preliminary study has shown that these is a considerable increase in the number and concentration of low molecular weight proteins and peptides in milk during the first 48 hours of bovine mastitis. Identification of these potential biomarkers should be undertaken to establish whether these are related to the acute phase proteins that are known to be secreted in milk during this condition or whether they are the result of protease action on milk proteins. Acknowledgements: Prof Julie Fitzpatrick, Prof Chris Knight and Dr F Young are thanked for provision of the milk samples used in this study. 73 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 22 HAPTOGLOBIN LEVELS IN FATTENING PIGS RECEIVING CONVENTIONAL ANTIBIOTIC PROPHYLAXIS IN COMPARISON TO A VITAMIN SUPPLEMENTATION Gymnich S1, Hiss S2, Petersen B1 1 Department of Preventive Health Management, 2 Department of Physiology and Hygiene, University of Bonn, Bonn, Germany Email:email@example.com Introduction In pig production antibiotics are used in prevention as well as therapy since decades. The emphasis of medical treatment is mainly at weaning as well as stabling into the fattening. Thereby the use of antibiotics is increasingly criticized because • with inappropriate application food might be loaded with residue • antibiotic resistances in humans might be induced • the spectrum of the effective medicaments for application in livestock production increasingly limits itself. The aim of the work was to look at the effect of a vitamin supplementation in contrast to an antibiotic prophylaxis using the acute phase protein haptoglobin (Hp) as an indicator for pig's health. Materials and Methods 120 fattening pigs from one rearing farm were divided into two groups (A/B) and 10 clinical healthy pigs of each group were marked individually. In the fattening farm the pigs were housed in the same partition. Group A got an antibiotic prophylaxis (Tetracyclin, Amoxicilin, Neomycinsulfat) for on week whereas group B was fed with a vitamin supplementation (Miravit, AGRAVIS Raiffeisen AG) for three weeks. Blood sampling was performed three times: • 3 days before moving the pigs into the fattening (Hp 1) • 21 days after housing (Hp 2) • 42 days after housing (Hp 3) Hp was determined using the method of HISS and co-authors (Vet. Immunol. Immunopath., 2003). Furthermore performance data were recorded. Results Table 1 shows the Hp levels as well as the performance data per pig group. Pigs of group A had significantly lower Hp levels at the first sampling time. Three weeks after housing pigs of group A showed significant higher Hp concentrations (2.34 mg/ml) than pigs in group B (1.29 mg/ml). At the last sampling time Hp levels did not differ significantly. Furthermore pigs of group B showed a better performance in the fattening period. Table 1: Overview over determined Hp concentration (mean + s) as well as performance data in the two pig groups Group A Group B Significance Parameter Antibiotic prophylaxis Vitamin supplementation (p) Hp 1 (mg/ml) 1.02 + 0.48 1.46 + 0.38 0.026 Hp 2 (mg/ml) 2.34 + 0.73 1.29 + 0.65 0.004 Hp 3 (mg/ml) 1.61 + 0.63 1.48 + 0.58 0.633 Daily weight gain 748 800 (g) Losses (%) 8,9 0 Feed efficiency 1 : 2,94 1 : 2,75 Fattening days 120 116 Discussion Francisco and coauthors (Swine Health and Production, 1996) found higher Hp levels in piglets after treatment with enrofloxacin and tiamulin in comparison to the control group. In addition no benefit in performance in the nursery phase was observed. Our study confirms this phenomenon. Nevertheless the reason for significantly higher Hp levels after antibiotic prophylaxis versus vitamin supplementation can not be determined from this study.5 74 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 23 EFFECTS OF HAEMOLYSIS, LIPAEMIA AND BILIRUBINAEMIA IN A TR-IFMA FOR CANINE CRP Parra MD, Martínez-Subiela S, Tecles F, Cerón JJ Animal Medicine and Surgery Department, University of Murcia, Murcia, Spain Email: email@example.com Introduction C-reactive protein (CRP) is one of the major acute phase proteins in the dog. Currently a commercial ELISA is the method of choice for measuring the protein in canine specimens, however this assay showed interferences when using haemolysed, lipaemic or hiperbilirubinaemic samples. Recently, a new method for measuring canine CRP has been developed (Parra et al. 2005), but not assessed for haemolysis, lipaemia or bilirubinaemia. The aim of the present study was to evaluate the effect of increasing concentrations of haemoglobin, lipids and bilirubin in CRP measurements by this new technique. Materials and Methods The effects of these interfering substances in canine CRP determination were assessed following previously reported protocols (Jacobs et al. 1992; Lucena et al. 1998) except for the haemolysis trial. In this case, haemolysis was produced by freezing red cells at –20°C in order to not incorporate distilled water to the assay. The haemolysate was added to pooled serum at final concentrations of 0.0, 0.25, 0.5, 1.0, 2.0 and 4.0 g/dL. A commercial emulsion of triglycerides (Lipofundina MCT/LCT 20%, Braun Medical, S.A.; Barcelona) was added to homologous pooled sera at 0.0, 31.2, 62.5, 125, 250 500 and 1000 mg/dL. Bilirubin (Sigma-Aldrich, Spain) was initially dissolved in dimethyl sulfoxide (DMSO) and then added to pooled sera at 0, 3.75, 7.5, 15, 30 and 60 mg/dL. DELFIA Diluent I was used to prepare haemoglobin and lipid series and DMSO to the bilirubin series. Samples were analyzed by a Wilcoxon Signed-Rank test (SPSS software, SPSS Inc, Chicago, Ill). Results The effect of the interfering substances can be appreciated in the corresponding interferographs (Figures 1-3) where data points represent the mean of duplicate determinations, X axes increasing concentrations of haemoglobin, lipid or bilirubin and Y axes percentage change of C-reactive protein for a given concentration of the added substances. Figure 1. Effect of added haemoglobin. Figure 2. Effect of added triglycerides. CRP % (final /original result)x100 CRP % (final /original result)x100 120 120 100 100 80 80 60 60 40 40 20 20 0 0 0 0.5 1 1.5 2 2.5 3 3.5 4 0 200 400 600 800 1000 Hemoglobin (g/dL) Tryglicerides (mg/dL) Figure 3. Effect of added bilirubin CRP % (final /original result)x100 120 100 80 60 40 20 0 0 10 20 30 40 50 60 Bilirrubin (mg/dL) Addition of fresh haemolysate, tryglicerides or bilirubin to serum samples did not affect CRP concentrations (P ≥ 0.18). 75 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Discussion The stable europium-chelate used as label in the assay enables the measurement of the time-resolved fluorescence that is virtually free from the background signal derived from the sample components and plastics. Furthermore, the dilution factor used in our study is higher than that used for the commercial assays and this is likely to have a positive contribution in reducing any assay interference from blood components. The TR-IFMA could be an alternative to the traditional tests for canine CRP, with the advantage of not being affected by haemolysis, lipaemia and bilirubinaemia. References Jacobs RM, Lumsden JH, Grift E. Effects of bilirubinemia, hemolysis, and lipemia on clinical chemistry analytes in bovine, canine, equine, and feline sera. Can Vet J 1992, 33:605-608. Lucena R, Moreno P, Pérez-Rico A, Ginel PJ. Effects of hemolysis, lipaemia and bilirubinemia on an enzyme-linked immunosorbent assay for cortisol and free thyroxine in serum samples from dogs. Vet J 1998, 156:127-131. Parra MD, Tuomola M, Cabezas-Herrera J, Cerón JJ.Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood. AJVR 2005, 66: 62-66 76 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 24 ALBUMIN A MAMMARY GLAND SECRETING CELL KEEPER Shamay A1, Feuermann Y1, Homans R1, Mabgeesh SJ2 1 Agricultural Research Organization (ARO), Institute of Animal Science, Bet Dagan, Israel 2Department of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences, the Hebrew University of Jerusalem, Jerusalem, Israel E-mail:email@example.com Introduction Albumin, a well-characterized product of the liver is the most abundant extracellular protein. It is a single polypeptide with 585 amino acids and a molecular weight of 66,200D. Albumin is manufactured in the liver at a rate 9-12g/day and there is no storage and no reserve. Albumin is catabolized at a rate of 9 - 12 g/day (the same rate as it was produced) by pinoctosis in cells adjacent to the vascular endothelium. Albumin is a major plasma protein, responsible for binding and transport of many biologically active molecules (Peters, 1985). Albumin is synthesized largely in the liver, though non-hepatic expression has been documented in several other tissues, such as the mouse retina (Dodson et al., 2001), the mouse skeletal muscle (Wagatsuma and Stromberg, 2001; Wagatsuma et al., 2002) human ovarian epithelial cells (Varricchio et al., 1994) and bovine tracheal gland serous cells (Jacquot et al., 1988). Milk whey albumin has the same amino acid sequence as the blood serum molecule. Therefore, increase in milk albumin was taken as evidence for tight junction disruption (Nguyen and Nevill, 1998). Albumin concentration in milk increases during functional transitions from lactation to involution and from involution to lactogenesis (Sordillo et al., 1987) and during inflammation (Riollet et al., 2000; Watanabe et al., 2000). During these periods albumin may augment the immune defenses of the gland (Bounous, 2000). Materials and Methods Preparation of mammary gland culture Explants: Mammary gland tissue from cows was obtained from the slaughterhouse. The tissue was transferred into medium M-199 containing 100 U penicillin, 100 ug streptomycin, 0.25 µg fungizone and insulin at 1 µg/mL and was brought to the laboratory. Explants were prepared as previously described (Shamay et al., 1987). The mammary gland tissue was cut into small pieces (3 to 6 mg) and placed on an impregnated lens paper in a 50-mm plastic dish. Five mL medium M-199 with 1 µg insulin, 10,000 U penicillin, 10 mg streptomycin, 0.025 mg fungizone and 0.5 µg cortisol/ml and the experimental additives. Cells: Bovine mammary gland tissue was transferred to the laboratory, cut into small pieces and placed in a 500-mL Erlenmeyer flask containing medium M-199 supplemented with collagenase (1 mg/mL), hyaluronidase (1mg/mL), and bovine insulin (1µg/mL) in a ratio of 10 mL medium to 1 g tissue. Cells were washed 3 to 5 times and grown in a 10-mm plastic dish in DMEM F-12 (HAM) 1:1. The cells were used for RNA isolation. Albumin secretion to the medium was determined by ELISA assay. Real Time RT-PCR Assay Real time Reverse Transcription-PCR for albumin mRNA was carried out with forward (5' AGG GAG GTC TGG GCT ATC ATC 3') and reverse (5' TTC GTG AAA CCT ATG GTG ACA TG 3') primers employing a SYBR green measurement of the accumulation of double-stranded DNA. ELISA Assay Analysis of BSA was done as described by Stelwagen et, al 1994. Enzyme linked immunosorbent assay (ELISA) was performed on medium collected from tissue cultures of healthy, mastitic and dry mammary gland tissue explants and on medium collected from primary culture cells, with or without LPS (lipopolysaccharide; Sigma, St. Louis MO USA). The ELISA was carried out with two 96-well polystyrene microtiter plates coated with BSA incubated with mouse monoclonal anti-bovine serum (Sigma, St. Louis MO USA B201) at a final dilution of 1:8000. After washing the plate was incubated goat anti-mouse IgG peroxidase conjugate (Sigma, St. Louis MO USA) at a final dilution 1:2500 in ELISA diluent for 1.5 h at 37°C. A substrate solution (0.1% 2' 2'-azino-diethylbenzo-thiazoline in citrate- phosphate buffer, pH 4.0, containing 0.003% (wt/vol) H2O2) was added to each well. After a final incubation at 20°C for 30 min, absorbance was measured at 405 nm. 77 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Results Albumin synthesis: Incubation of mammary gland explants with the labeled amino acid L- [35S] methionine resulted in formation of labeled immunoprecipitable albumin, newly synthesized in the explant. Immunoprecipitable albumin in the medium verified that newly synthesized albumin was also secreted into the medium. This shows that the gland itself is a source of milk albumin. Albumin expression and secretion: Mammary gland tissue from healthy and mastitic dairy cows were examined for albumin mRNA expression by means of Real-Time RT-PCR. Albumin mRNA expression was approximately 4 times higher in mastitis mammary gland tissue compared to expression in healthy tissue. Mammary gland explants from healthy, mastitis, and dry dairy cows were incubated for 3 d in medium. The effect of mastitis on the secretion of albumin from the mammary gland tissue was determined by ELISA. The secretion of albumin was increased 3.5-fold (P < 0.05) in the mastitic mammary gland tissue explants, relative to the healthy mammary gland tissue explants. Hormonal effect on BSA: we demonstrate here for the first time that leptin in the presence of prolactin (simulation of lactation) enhance the expression and accumulation of albumin in the bovine mammary gland (fig 1). Conclusion The results shown here suggest that the synthesis and secretion of albumin by the mammary gland is part of the innate nonspecific defense system. Albumin synthesis and secretion in the mammary gland is influence by the tissue health status and by hormones. Arbitrary units of optical density Insulin 1µg/ml + + + + + + + + Prolactin 1µg/ml - - - - + + + + Leptin ng/ml - 1 10 100 - 1 10 100 IF IFP 78 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 25 PSYCHOLOGICAL STRESS CAUSED BY CHANGES IN FEEDING MANAGEMENT DOES NOT MODIFY THE CONCENTRATION OF ACUTE PHASE PROTEINS IN PIGS Piñeiro C1, Piñeiro M1, Morales J1, Ruiz de la Torre JL2, Mateos GG3 , Manteca X2 1 PigCHAMP Pro Europa S.A., Spain, 2UA Barcelona, Spain, 3UP Madrid, Spain Introduction In a previous study, Pineiro et al. (2003) demonstrated that acute phase proteins (APP) can be used to detect stressful situations in pigs. In that study, pigs (26.3 ± 0.4 kg BW) were allotted to two treatments based on two feeding systems: ad libitum (AL) or disorderly (DIS) which consisted in alternating periods of AL food administration with periods of no feeding at all. DIS fed pigs showed higher APP concentrations (Pig-MAP, haptoglobin, SAA and CRP), than AL fed group. Also, the concentration of negative APP increased slightly with time in the AL pigs, but not in the DIS group. Differences in APP values between the two groups were evident in males, whereas in females the concentrations were similar for both groups. These results were associated with lower growth rates in DIS fed males than in AL fed males. However, in this trial pigs were allotted to pens in groups of 12 and, it was not possible to distinguish if the observed increases in APP's levels in the DIS group were caused directly by psychological stress or were the result of an increase of fights and lesions in this group of pigs. Therefore, two studies with individual penned pigs have been performed with the aim to ascertain if the effect was due to psychological or social stress. Aim To assess the use of APPs as biomarkers of psychological stress promoted by changes in feeding management during the growing (study 1) or the finishing (study 2) period of pigs housed individually. Material and Methods Twenty-four and twenty pigs were used in the growing (about 25 kg BW) and finishing (about 80 kg BW) studies, respectively. Pigs were housed individually and distributed in two groups that differ in the feeding pattern used (AL and DIS). The trial lasted 20 days. At days -5, -2, 1, 3, 6, 8, 10, 13, 15 and 21 in the growing study and at days -5, 1, 4, 15 and 21 in the finishing study, pigs were bled and Pig-MAP serum concentration analysed by ELISA using a commercial kit (PigCHAMP Pro Europa, S.A., Segovia, Spain). Data were analysed as repeated measures including feeding pattern, time and their interaction as main effects. Results and Discussion Feeding pattern did not affect APP concentrations in any of the two periods studied. Evolution of Pig- MAP with time for the growing and finishing periods are shown in figure 1 and 2, respectively. Psychological stress did not affect APP concentrations, suggesting that the stress previously reported was a response to social stress in the DIS fed group. Figure 1a. Changes of Pig-MAP concentration Figure 1b. Changes of Pig-MAP concentration with time. Growing period with time. Finishing period. 3,5 1,6 3,0 1,4 AL DIS 2,5 1,2 AL 2,0 1,0 mg/ml mg/ml DIS 1,5 0,8 1,0 0,6 0,5 0,4 0,0 0,2 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Time Time 79 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Conclusion APP were not valid biomarkers to assess the psychological stress caused by changes in feeding management. Reference: Pineiro C, Lorenzo E, Morales J, Gomez E and Mateos GG (2003) Effect of stressors on serum concentration of acute phase proteins and performance in pigs. J. Anim. Sci (81) Suppl. 1. Abstract nº 621, p 157. 80 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 26 ACUTE PHASE PROTEIN CONCENTRATION IN SERUM IS NOT AFFECTED BY TIAMULIN MEDICATION IN HEALTHY GROWING PIGS Piñeiro C1, Lorenzo E1, Morales J1, Piñeiro M1, Alloza L2, Carpintero R2 , Mateos GG3 1 PigCHAMP Pro Europa S.A., Spain, 2Universidad de Zaragoza, Spain,3UP Madrid, Spain Introduction The concentration of acute phase proteins (APP) in serum changes during the acute phase response to inflammation. An increase in basal APP concentration has been reported in response to experimental or natural infection in the early stages of the immune response. In fact, differences in APP serum levels have been found between high and low health status herds. Several studies have demonstrated that APP serum concentrations can be used as biomarkers to assess the health status of pigs and the efficacy of therapeutical treatments against several pathologies. Medicated early weaning is a common strategy for disease control in swine production and Francisco et al. (1996) have described increases in APP serum levels as a consequence of preventive medication with enrofloxacin and tiamulin in apparently healthy piglets. Aim To determine the effects of tiamulin administration on serum concentration of two APP (Pig Major Acute phase Protein; Pig-MAP and Haptoglobin; HPT) in healthy growing pigs from 74 to 116 d of age. Material and Methods A total of 132 pigs LW x LD (31.3 ± 0.22 kg BW; 74 d of age) were randomly divided into six groups with two sexes (males and females) and three therapeutical treatments (non-treated control group; tiamulin administration group, 50 ppm in diet; and injected tiamulin administration group; 162 mg for 5 consecutive days). Average daily gain (ADG), feed intake and feed:gain ratio were measured. Blood samples were obtained at day 0, 6, 12 and 28 of trial and the serum concentration of Pig-MAP and HPT was determined by radial immunodiffussion. Results: Performance: No signs of disease were observed throughout the experimental period. No differences (P>0.10) in productive performance traits were observed between treatments throughout the experiment. A treatment x sex interaction was observed for ADG (P<0.10) for the global period (74- 116 d of age); males performed better than females in the non treated and orally treated pigs but not for injected pigs. APP concentration: APP concentration was not affected by tiamulin medication through the experiment (table 1). Table 1. APP concentrations (mg/mL) through the experiment (0, 6, 12 and 28 days of trial) Pig-MAP, days HAPTOGLOBIN, days 0 6 12 28 0 6 12 28 Control 0.52 0.60 0.63 0.65 1.05 1.95 1.63 1.81 Oral treatment 0.70 0.73 0.74 0.71 1.52 1.44 1.55 1.46 Injected treatment 0.60 0.58 0.61 0.93 1.25 1.10 1.20 2.42 SEM 0.075 0.086 0.059 0.128 0.364 0.323 0.251 0.388 P NS NS NS NS NS NS NS NS Conclusions Tiamulin treatment, either oral or injected, of healthy growing pigs did not affect APP 's serum concentrations. The results suggest that both APP are good biomarkers of health or productive performance in pigs, independently of the use of this antibiotic. Reference: Francisco CJ, Bane DP and Unverzagt L (1996).The effects of enrofloxacin and tiamulin on serum haptoglobin and a-1-acid glycoprotein concentrations in modified medicated-early-weaned pigs. Swine Health and Production 4: 113-117. 81 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 27 COMPARISON OF LABORATORY ACUTE PHASE MARKERS IN CANINE BABESIOSIS Matijatko V, Mrljak V, Kiš I, Kučer N, Foršek J, Barić Rafaj R, Potočnjak D, Brkljačić M, Grden D Faculty of Veterinary Medicine, Clinic for Internal Diseases, Zagreb, Croatia Introduction Babesiosis is the disease that, like human falciparum malaria, can be classified as "protozoal sepsis". Although haemolytic anaemia is the hallmark of babesiosis, the clinical presentation is highly variable. The many and varied clinical manifestations of canine babesiosis are difficult to relate to an organism that is solely restricted to the erythrocyte. It is likely that the inflammatory mechanisms in this disease are similar to those of other septic conditions that lead to SIRS and MODS. Aim This study was designed to test whether sequential measurement of acute phase proteins would prove valuable in assessing response to antibabesial treatment and to compare the acute phase proteins with commonly used inflammatory markers such as erythrocyte sedimentation rate, WBC count, neutrophil to lymphocyte ratio and platelet count. Materials and Methods In this study, serum concentrations of C- reactive protein, haptoglobin and serum amyloyd A and the presence of SIRS were investigated in fifty dogs naturally infected with B. canis before and 1st, 2nd, 3rd, 4th and 7th day after the antibabesial treatment in order to document acute phase reaction and its potential value in the diagnosis and monitoring of the disease. Results Table 1 - Laboratory acute phase markers in control group Control CRP 5,7±0,82 HPT 8,15±6,66 WBC 8,75±1,39 ESR 1,00±0,78 SAA 2,61±4,44 Ratio 2,82±0,44 PLT 397,0±45,41 Table 2 - Laboratory acute phase markers in canine babesiosis before and 1st, 2nd, 3rd, 4th and 7th day after the antibabesial treatment Before 1st day 2nd day 3rd day 4th day 7th day CRP 173,14±57,59 153,5±57,64 87,19±40,93 53,32±32,28 33,82±30,36 14,23±12,7 HPT 29,31±22,29 60,17±47,01 79,75±55,28 83,05±50,03 81,76±59,34 50,46±46,2 WBC 6,68±2,95 3304±3629 1678±2036 877±1433 447±597 79±69 ESR 28,36±28,36 11,56±4,58 13,12±4,03 11,9±2,87 11,81±2,61 11,41±3,37 SAA 2819±4596 20,09±20,86 13,10±19,29 9,09±14,66 7,36±11,92 3,86±5,95 Ratio 4,02±4,13 1,56±1,14 0,97±0,67 1,3±0,57 1,5±0,63 2,54±2,09 PLT 16,5±12,34 18,09±11,46 30,73±15,03 75,95±43,99 124,5±69,18 223,4±78,03 Conclusions These results indicate that the parasite induces marked acute phase response in the host. The results of this study support the idea that sequential measurement of C-reactive protein and serum amyloid A concentrations could be used to monitor the clinical course of the disease and its response to treatment. Such measurements are likely to be more objective than clinical observations and standard hematology and biochemistry. 82 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 28 AA AMYLOID FORMATION BY PRIMARY CHICKEN FIBROBLAST-LIKE SYNOVIOCYTES Upragarin N1,2, van Asten AJAM1, Wajjwalku W3, Gruys E1 1 Department of Veterinary Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht,Tthe Netherlands.2Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand. 3Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom,Thailand.E-mail: firstname.lastname@example.com Introduction Some strains of commercial layer chickens are prone to develop AA-amyloid arthropathy in response to chronic arthritis, whereas others are resistant. In previous studies on experimental brown layers during amyloidogenesis in the joint the possibility of extrahepatic production of serum amyloid A (SAA) has been suggested. Isolated fibroblast-like synoviocytes (FLS) appeared to form SAA protein after stimulation with LPS. Recent findings of in vitro studies by various authors reveal that small β- pleated sheet structure proteins accelerate amyloid fibrillogenesis from precursor proteins. The so called 'amyloid enhancing factor" which can be obtained from β-pleated proteins such as amyloid fibrils or silk, may act as nidus for the accelerated fibrillogenesis. The aim of the present study was to study extrahepatic AA amyloid formation by synoviocytes, and to investigate the mechanism of AA- amyloidogenesis. Materials and Methods Primary chicken FLS originating from brown layer were isolated and cultured in vitro. Recombinant chicken SAA (rchSAA) was generated using the pGEX bacterial expression system and fibril-derived amyloid enhancing factor (FAEF) solution was prepared. Fibril formation of rchSAA was assessed by Congo red staining and by electron microscopy. The capability of FLS to degrade rchSAA was examined by gel electrophoresis and immunoblotting. Finally, AA-amyloid fibril formation in cultured FLS was assessed by Congo red staining and immunohistochemistry. Results After incubating rchSAA in acidic buffer without cells, small fibril fragments formed which on Congo red staining revealed green birefringence. When FLS were incubated with rchSAA under neutral conditions, degradation products of rchSAA were detected in culture medium within 48 hours. However, prolonged culture time (15 days) did not result formation of amyloid fibrils. Addition of FAEF to this culture system under a neutral environment, evident amyloid fibrils were formed after 48 h and more abundantly after 5 days. Conclusions The present findings suggest that chicken FLS which earlier were found to act as a source of SAA in the joint during infection and inflammation, can favour amyloid formation from the formed SAA. This process may be mediated by fragments of degraded amyloid or other β-pleated units which may be derived from inflammatory cells. The amyloidogenesis process itself appeared to be independent of macrophages. 83 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 29 SERUM AMYLOID A IN GOOSE Kovacs BM1, Szilagyi L2, Janan J3, Toussaint MJM4, Gruys E4, Rudas P1 1 Department of Physiology and Biochemistry, Szt. Istvan University, Budapest, Hungary. 2Department of Biochemistry, Lorand Eotvos University, Budapest, Hungary.3Department of Applied Ethology, Szt. Istvan University, Godollo, Hungary.4Department of Pathobiology, Utrecht University, Utrecht, The Netherlands Email: firstname.lastname@example.com Introduction Amyloidosis - which is a heterogeneous group of disorders described by the pathologic extracellular deposition in various tissues of some fibrillar protein, amyloid - is a common disease among domestic mammals and birds. Acute phase protein serum amyloid A is the precursor protein of AA amyloidosis, the type of amyloidosis in geese. The amyloid fibril protein deposited in secondary amyloidosis is amyloid protein A, which is derived from serum amyloid A (SAA) and is generally found to be modified by proteolytic removal of C-terminal amino acids. Chronic SAA level increase is an essential but not sufficient precondition in this secondary disease resulting from a variety of infectious and non- infectious inflammatory conditions. Therefore goose SAA might be considered as a significant signal protein of the disease. Materials and Methods Cloning and expression of recombinant goose serum amyloid A: Total RNA was isolated from goose liver and used to synthesise first strand cDNA. The coding region of the goose SAA cDNA was amplified by PCR using primers corresponding to the appropriate conservative regions of duck SAA mRNA. The product was subcloned into pET-15b expression vector to result in a HisTag fusion protein expression. The protein was purified by affinity chromatography. The nucleotide sequence of the construct was verified by automated dideoxy sequencing. Immunisation and detection of SAA-reactive antibodies: New Zealand White rabbits were immunised intracutaneously. Subsequent bi-weekly immunisations were followed for 6 weeks. The appearance of the antibody was detected by using standard immunodiffusion techniques. The quality was tested by Western blotting. After testing the sera were purified by ion-exchange chromatography. ELISA assay: The antibody was used for developing an ELISA assay measuring SAA concentration in goose sera samples. The method was direct ELISA, coating the sera samples onto a high bonding 96 wells' plate. For the calibration curve wells were coated with the recombinant protein. The purified antisera were used as primary antibody, and then anti-rabbit peroxidase antibody was used for the secondary immuno-reaction. For detection 2,2'azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) was used as substrate. Results We defined the nucleotide-sequence of the full-length goose serum amyloid A and compared it to SAA sequences of the duck. The aim of this work was to clone and express recombinant goose SAA and to produce useful antibody against this protein for specific ELISA. The anti-SAA serum was proved to be a highly specific antibody. Using this antibody and the recombinant SAA protein, a sensitive direct ELISA assay was developed. Conclusion Our work is the first study on the cloning of goose SAA and on the production of specific antiserum against that protein, thus our ELISA method is the first assay for measuring serum SAA level in geese. This assay will be used to monitor health conditions of goose flocks. 84 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 30 AGGRESSIVE BEHAVIOUR RELATED ACUTE PHASE RESPONSE IN PIGS Candiani D1,3, Salamano G2, Mellia E2, Doglione L2, Bruno R3, Toussaint MJM1 ,Gruys E1 1 Dept. of Veterinary Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.2 IZS-State Veterinary Institute of Piedmont, Turin, Italy 3 Dept. of Veterinary Morphophysiology, Faculty of Veterinary Medicine, Turin University, Turin, Italy. Email: email@example.com Introduction Swine loading and transport both involve exposure to social stress (mixing of unfamiliar animals) and physical stress (rough handling). Severe and persistent stress, or a series of stressors, can lead at first to a pre-pathological state, and eventually to a diseased state which itself leads to decreased reproduction, changed metabolism and the development of abnormal behaviour such as tail and ear biting or cannibalism. Tail biting is one of the most important welfare reducing factors in modern pig production. The acute phase response consists in the release, stimulated by proinflammatory cytokines, of acute phase proteins (APP) into the bloodstream, in response to a challenge. Positive APP in pigs include haptoglobin (Hp), C-reactive protein (CRP), major acute phase protein (pig-MAP) and serum amyloid A (SAA). Their induction is associated with a decrease in synthesis of negative APP such as albumin (Alb), the most abundant constitutive plasma protein, retinol binding protein (RBP), α1-acid glycoprotein and transthyretin. The combination of results from positive and negative APP in an acute phase index can increase their diagnostic potential. The aim of this work was to evaluate the role of some positive and negative APP in farming pigs after a long distance transportation and a subsequent aggression episode. Materials and Methods 20 Landrace x Large White 4-months old gilts were imported from Denmark to Italy (a 48 h journey) and then monitored for 28 days. The animals were located in a 6 x 3.5 meter pen with food and water ad libitum. Blood samples were collected from the jugular vein immediately upon arrival and 28 days later (T1, T28). On day 25 the pigs developed an aggressive behaviour consisting in tail biting episodes and resulting in physical injuries (skin lesions, wounded and bleeding tails). Serum samples were obtained by centrifugation (3000 rpm, 10 min) and stored at –80°C. Serum haptoglobin and SAA concentrations were measured with commercial assay kits (Tridelta Development, Greystones, Ireland), while pig-MAP concentration was determined with a sandwich ELISA test (PigCHAMP Pro Europa S.A., Segovia, Spain). Serum albumin was first separated by agarose gel electrophoresis (Paragon Serum Protein Electrophoresis Kit, Beckman Coulter) and then quantified by densitometry. Total proteins concentration was performed with a commercial assay kit (BCA Protein Assay Reagent Kit, Pierce, Rockford). Results were statistically analysed by the Spearman test, parametric methods (t-paired student test) and non parametric methods (Kruskal-Wallis and Wilcoxon tests) with the Analyse-it software. Results Results here below show acute phase proteins concentrations related to the total proteins concentration: APP values in Fig. 1 are expressed as the percentage of total proteins. Positive acute phase proteins, Hp and pig-MAP, concentrations were found to be significantly increased (p < 0,001) on T28 compared to their values on T1. SAA showed very low concentrations in T1 and higher levels on T28. Negative acute phase protein albumin showed a high and statistically significant decrease (p < 0,001) in T28 levels, compared to T1 levels. 85 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Fig.1 – pig-MAP, SAA, Hp and Alb boxplots. Discussion These results show that stress affects the behaviour and the welfare of farming animals. Furthermore, tail biting induces a strong acute phase reaction, demonstrating the great potential of acute phase proteins in veterinary medicine: APP are useful tools in monitoring inflammatory processes for diagnostic and prognostic purposes and also for analyzing various non-inflammatory conditions. An acute phase index, incorporating positive and negative acute phase proteins, can provide valuable information for assessing animal health and help to promote an optimal growth in the farm. 86 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 31 THE ACUTE PHASE RESPONSE TO SURGERY IN CLINICALLY NORMAL AND DISEASED HORSES Pollock PJ, Prendergast, M, Bellenger CR Department of Veterinary Surgery, University College Dublin, Dublin, Ireland Introduction A variety of inflammatory markers, coupled with changes in a number of haematological and biochemical parameters have classically been used to diagnose, monitor and prognosticate disease in horses. Unfortunately these traditional markers respond fairly slowly to the presence of disease and inflammation and have wide normal ranges (Allen and Cold 1988; Pepys et al 1989). Serum amyloid A (SAA) is an acute phase protein common to human beings, cattle, sheep, mice, and several other species, including the horse. In several of these species, plasma concentration of SAA has been shown to increase 1000-fold following tissue injury, cellular necrosis, inflammation, and infection and decline rapidly in the recovery phase. Materials and Methods In this study, the serum concentration of serum amyloid A (SAA), haptoglobin and fibrinogen was measured in a group of 26 horses before, and at intervals after, elective (n=19) and non-elective surgery(n=8). A second group of normal horses was sampled to establish normal values. Plasma SAA concentration was measured using an ELISA and compared with a number of other parameters, including routine haematological and biochemical parameters, fibrinogen, and haptoglobin. Results There was a significant, rapid, and repeatable increase in SAA concentration in response to surgical trauma in both elective and non-elective horses. The control horses demonstrated a range of serum concentrations in aggreement with the normal range expected in the horse, 0- 0.2цg/mL. Following surgery a peak mean concentration of 16.41цg/mL for elective horses and 27.29цg/mL for non-elective horses was noted 24 hours after the end of the surgical procedure. In contrast, the serum concentration of haptoglobin and fibrinogen demonstrated a more gradual rise following surgery and failed to decline by the end of the sampling period, 72 hours after surgery. Conclusion In conclusion, this study demonstrated that serum amyloid A, when compared to traditional measured parameters of inflammation in horses, has a tight normal range and increases rapidly in response to surgical stimuli and infection. Furthermore, SAA concentrations return to normal rapidly in the recovery phase, and consequently measurement of SAA is useful in the diagnosis and monitoring of surgical disease in horses. The ELISA method for SAA measurement was simple to perform and gave useful results quickly. The measurement of SAA in normal and diseased horses should be useful in monitoring the recovery of horses with surgical disease and may be of use in determining prognosis. 87 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 32 ONE-STEP IMMUNOCROMATOGRAPHIC METHOD FOR THE DETECTION OF ELEVATED LEVELS OF PIG-MAP CONCENTRATION Barrios B1, Navarro A1, Piñeiro M2, Piñeiro C2, Alava MA3, Lampreave F3. 1 Operon S.A Camino del Plano 19, Cuarte de Huerva, Zaragoza, Spain2PigCHAMP Pro Europa S.A., Segovia, Spain3 Dpto de Bioquímica y Biología Molecular y Celular. Universidad de Zaragoza, Spain Introduction. Increases in basal pig APP concentration have been reported in response to experimental or natural infection, as well as under the effect of stressors like the change of facility, mixing of animals, or transport. Differences in APP serum levels have been found between high and low health status herds, and elevated levels of APP were associated with losses of productive performance. Several studies have demonstrated that APP serum concentration can also be used as a biomarker to assess the efficacy of therapeutic treatments or vaccination. Pig-MAP is one of the main APPs in pig. Its basal concentration is lower than 1 mg/mL, increasing up to 8-12 mg/mL during the acute phase response. In this study, a prototype qualitative membrane-based immunoassay for the detection of acute phase levels of Pig-MAP has been developed and evaluated in serum samples. Material and Methods. The immunocromatographic test (ICT) device is formed by a semi-rigid plastic strip on which a chromatographic membrane and absorbent materials are attached. In the lower part of the strip there is a conjugate pad that contains colored microspheres coated with an anti Pig- MAP monoclonal antibody. The reaction zone contains two parallel lines in which a cooperant anti pig- MAP monoclonal antibody, and a control antibody are fixed. In the upper end is an absorbent that allows the chromatographic flow. The strip also contains a blood separator to be used with whole blood. To perform the test, 10 ul of serum samples were added to microtiter plate wells, and incubated for 5 minutes with a dilution buffer containing a precise amount of Pig-MAP antibodies to block the Pig- MAP concentration present in normal state. A strip was introduced in each well, and the immunocromatography performed during 10 minutes. The excess of Pig-MAP present in the sample was captured by the antibody coated-colored microspheres that migrate upwards on the strip and were retained by the antibody fixed in the capture line. This leads to the apparition of a visible pink line (being its intensity dependant on protein concentration). The system also contains blue colored microparticules, that are retained by the control antibody to give a blue control line. 162 serum samples were analysed by the ICT. The concentration of Pig-MAP in the samples was determined by a sandwich ELISA (PigCHAMP Pro Europa S.A., Segovia, Spain), based on the same monoclonal antibodies that the ICT. The results obtained with the ICT were classified as positive (from slighty to clearly positive pink line for Pig-MAP and a blue control line), negative (only the blue control line). Results. A cut off of 1.5 mg/ml was established for the normal serum. Serum samples were classified in two groups according to Pig-MAP concentration: lower than 1.5 mg/ml ("negative") and higher than 1.5 mg/ml (" positive"). Of the 162 samples analysed 109 were lower than 1.5 mg/ml and 53 higher than 1.5 mg/ml. The sensibility of the ICT (% of ITC positive of the positive serum) was 90.5 %. The specificity (% of ICT negative of the negative serum) was 94.4 %. The positive predictive value (% of real positive detected by ICT of the total ICT positive) was 88.8% and the negative predictive value (% of real negative detected by ICT of the total ICT negative) was 95.3 %. Discussion. Interest on animal APP and its use in animal production is increasing every year, and assays for the different species are currently under development. The ICT described here is an easy, fast and accurate method for the detection of animals suffering an acute phase response that results in an elevation of Pig-MAP concentration. The test showed a good sensibility (91%) and specificity (94%), and a very good negative predictive value (95.3%). The test can be applied to detection of "normal" or healthy animals and may be incorporated in the quality systems to evaluate the health status of pig herds at farms or slaughterhouses. 88 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 33 PORCINE HAPTOGLOBIN PURIFICATION THROUGH GEL FILTRATION Fuentes P1, Cabezas-Herrera J2, Cerón JJ1, Martínez JS1, Ramis G3, Parra MD1. 1 University of Murcia, Animal Medicine and Surgery Department, Murcia, Spain. email@example.com; 2 Laboratory of Molecular Biology. Virgen de la Arrixaca Hospital. Murcia. Spain; 3Department of Research, Development and Investigation, CEFUSA, Murcia, Spain Introduction: Haptoglobin (Hp) is an α2-glycoprotein which participes in haemoglobin transport. It is one of the moderated acute phase proteins in swine, showing increases during various natural and experimental induced infections, and it has been proposed as a tool for health status monitoring in pig production system. In this report we describe a simple protocol for porcine haptoglobin purification based on gel filtration by using a FPLC system in order to improve the speed and decrease the complexity of current procedures for haptoglobin purification. Material and Methods: Serum with high Hp concentration was obtained from 3 month old pigs with acute rectal prolapse. Specimens were collected by yugular venipuncture and serum was pooled and stored at –20ºC. Three ml of the serum rich in Hp were saturated by 50% ammonium sulphate. After a gentle stirring for 30 min at room temperature, the precipitate was discarded by centrifugation and the supernatant was dialyzed against NaCl 0.9% by using NAPTM 10 desalting columns (Amersham Biosciences). After dialysis, the solution was concentrated by Centriconâ (Amicon, Millipore, USA) and followed by an adition of ultrapure urea to 6M and then filtered through a 0.22µm pore-size filter (Millexâ-GV). The final solution was applied to a SuperdexTM 200 10/300 GL column (Amersham Biosciences, Uppsala, Sweden), equilibrated and run with TSA buffer, pH 7.5 with a flow rate of 20ml/h at room temperature. Fractions were collected using a fraction collector (Amersham Biosciences). The purity of the Hp preparation obtained was assessed by SDS-PAGE using a 5% stacking gel and 12% resolving gel of acrylamide. Results: The chromatogram obtained by FPLC showed 3 peaks, the second one corresponded mainly to haptoglobin as it could be seen by SDS-PAGE. The content of this peak yielded two bands, the first one with a molecular size of 44 kDa, corresponding to the heavy chain of Hp (b), and the second one with 12.8 kDa, corresponding to the light chain of Hp (a). Minor impurities of 24 and 11.20 kDa were also observed. Discussion: Molecular size of the protein purified in this study agrees with previous reports and therefore the procedure describe here could be widely used for porcine Hp purification. The present work could provide a more simplified way of purifying Hp than the ones described by Shim et al., (1971) and Hiss et al., (2003), since we only use a chromatographic step and might be an alternative to the method described by Yang & Mao (1999), also based on an unique chromatographic step, in laboratories in which FPLC were available. The purified protein could be used for improvement of current Hp assays or for the development of new and highly sensitive assays such as time-resolved fluoroimmunoassays, in which pure Hp is needed for use as primary standard. References: Hiss S, Knura-Deszczka 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. Veterinary Immunology and Inmmunopathology 96: 73-82. Shim B, Yoon C, Oh S, Lee T, Kang Y. 1971. Studies on swine and canine serum haptoglobins. Biochimica et Biophysica Acta 243: 126-136 Yang SJ, Mao SJT. 1999. Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. Journal of Chromatography B, 731: 395-402. 89 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 34 MULTI-ANALYTE IMMUNOASSAY PROFILES OF PLASMA BIOMARKERS OF INFLAMMATORY CHANGE IN A RAT MODEL OF EARLY MESENTERIC VASCULAR INJURY O'Brien PJ, Chevalier S, Schenck E, Pawlowski V, Dagues N, Ledieu D. Safety Sciences Europe, Pfizer, Sandwich UK and Amboise, France. Introduction: Phosphodiesterase-inhibitors may induce mesenteric vascular injury in rats (1,2). We studied early plasma biomarkers of vascular injury using a novel, multi-analyte, immunoassay profile of 60 acute phase reactants, cytokines, chemokines, growth factors, and hormones using Luminex technology (Rules Based Medicine) that has recently become commercially available (Charles River Laboratories, Manston, Kent, UK). Although the immunoassays were developed for application in mice, they were stated by the vendor to have cross-reactivity for rats. Material and Methods: Rats (25 Spraque Dawley males, aged 7 weeks) were treated with a single dose of 0, 160, or 320 mg / kg of a potent phosphodiesterase inhibitor administered by oral gavage. After 16 hours, plasma was analysed from blood collected into tubes with EDTA anticoagulant. Results: Concentrations were below the lower limit of detection (mean + 3 SD for blank) for 39 of 60 parameters. Of the measurable analytes, no treatment related effects were detected for 9: growth hormone, interleukin-11, insulin, leptin, aspartate aminotransferase, myoglobin, immunoglobulin A, and monocyte chemoattractant proteins 1 and 3. Treatment produced mild to moderate effects on 12 other measurable analytes. It increased Profile of Inflammatory Changes fibrinogen (Ia), haptoglobin (Hp), interleukin-10 (IL-10), C-reactive protein (CRP), von Willebrand factor (vWF), and vascular epithelium MDC growth factor (VEGF), by 80, 72, 56, 45, 30, and 30% respectively, SCF and decreased macrophage-derived chemokine (MDC), stem cell factor Eotaxin (SCF), eotaxin, vascular cell adhesion molecule-1 (VCAM-1), and VCAM-1 M-CSF Analyte macrophage colony stimulating factor (M-CSF) by 50, 48, 45, 19, and 12% (1-way ANOVA; p < 0.05). There were differences (Student's VEGF unpaired, 2-tailed, t-test) between high and low groups for SCF, GCP-2 vWF / IL-8, M-CSF, eotaxin, and vWF; SCF, eotaxin, M-CSF, and vWF CRP were not affected at the low dose, whereas GCP-2 was increased at the low dose only. Granulocyte chemotactic protein-2 / interleukin 8 IL-10 (GCP-2 / IL-8) concentrations were below the least detectable Hp concentration for all controls, but above this limit for 6 low dose and 2 Ia high dose rats. 0 50 0 0 -5 10 Conclusion: Cross-species reactivity from mouse to rat occurred in % Difference from Controls only 35% of immunoassays in the mouse multi-analyte profile. However, the 21 assays of the profile that were able to measure analyte in rat plasma were effective in identifying a major effect of treatment with phosphodiesterase inhibitor. All treatment-related effects could be attributed to a mild, acute inflammatory response, characterized by increased release of acute phase proteins (Ia, Hp, CRP, vWF) and altered concentrations of cytokines and chemokines (eg IL-10, eotaxin, GCP-2, and MDC) that are modulatory of the inflammatory response. VEGF, an angiogenesis factor induced by inflammation was also slightly affected. Reference: 1. Robertson DG, Reily MD, Albassam M, Dethloff LA. 2001. Metabonomic assessment of vascular injury in rats. Cardiovasc Toxicol. 1:7-19 2. Slim RM, Song Y, Albassam M, Dethloff LA. 2003. Apoptosis and nitrative stress associated with phosphodiesterase inhibitor-induced mesenteric vascular injury in rats. Toxicol Pathol. 31:638-45. 90 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 35 ACUTE PHASE PROTEIN CONCENTRATIONS IN FINISHING PIGS UNDER TWO DIFFERENT MANAGEMENT SYSTEMS Scott, K.1, Campbell, F.2, Chennells, D.3, Hunt, B.4, Armstrong, D.5, Taylor, L.5, Gill, B.5 Edwards, S.1 1 School of Agriculture, Food and Rural Development, University of Newcastle, Newcastle upon Tyne, NE1 7RU; 2Glasgow University Veterinary School, Glasgow, G61 1QH; 3Acorn House Veterinary Surgery, Bedford, MK41 7HN; 4VLA, Bury St. Edmunds, IP33 2RX.; 5MLC, Milton Keynes, MK6 1AX. Email: email@example.com Introduction Finishing systems for pigs must be economically efficient, provide good animal welfare and deliver safe product. In this study the concentrations of two serum Acute Phase Proteins (APP) in blood were used as one of a number of different parameters to assess the health and welfare implications for pigs finished in two contrasting housing systems. Materials and Methods Three separate studies were carried out between April 2002 and February 2004, each using a total of 1056 Large White x Landrace pigs from the same source. Intakes of 128 pigs were allocated at 34 kg liveweight between 4 pens within a single room alternately in either a fully-slatted (FS) or straw-based (ST) building. In addition to the housing comparison, each study involved a two treatment nutritional comparison of an aspect of liquid feeding, according to a factorial design. A blood sample was taken from a sample of 3 male and 3 female 'focal' pigs per pen at the start, at the mid-point (60 kg) and at slaughter (104 kg) for determination of the serum concentrations of two APP, C-Reactive protein (CRP) and Haptoglobin (Hp). Data were analysed using ANOVA with pen mean as the statistical unit. Results There were no significant effects of nutritional treatments on the concentration of APP in any of the 3 studies. Levels of CRP at entry increased with each study suggesting deteriorating health status in the source population. There was a highly significant correlation (r=0.55-0.64; P<0.001) between mean pen value for Hp and CRP at the same sampling time, but the correlation between levels of the same APP at different time points was non-significant or only weakly significant (r<0.3). Levels of APP were lower at slaughter than at entry (P<0.001). Pigs in the FS system had significantly higher levels of Hp than pigs in the ST system at both the mid-point and at slaughter (Table 1), with a similar tendency in CRP. This was despite a higher frequency of veterinary treatment for respiratory and enteric disease in the overall population in the ST system in all studies, although tail biting was more prevalent in the FS house. Overall growth rate in the ST system was significantly better than in FS in only one of the 3 studies. Table 1: Effects of housing system (H) and study number (S) and their interaction (I) on concentrations of C-Reactive protein (µg/ml) and Haptoglobin (mg/ml) in pig serum. Fully-Slatted Straw-Based Range S.E.M. P Study Number 1 2 3 1 2 3 H S I At entry C-Reactive protein 138 218 251 136 198 281 44-560 23.3 *** Haptoglobin 0.7 1.2 1.0 0.6 1.0 1.1 0.3-2.1 0.1 At mid-point C-Reactive protein 165 238 189 126 219 160 45-529 37.8 0.08 Haptoglobin 1.4 1.5 1.1 1.0 1.2 0.9 0.6-2.3 0.1 ** * At slaughter C-Reactive protein 99 182 192 89 125 155 7.3-572 24.0 0.08 ** Haptoglobin 0.7 0.7 0.7 0.4 0.6 0.5 0.1-1.4 0.1 ** Conclusion Levels of APP were elevated in finishing pigs maintained in a fully-slatted relative to a straw-based system, but this did not appear to be related to clinical health indicators or growth rate differences at a population level. Investigation of the health and performance indicators associated with elevated APP in individual pigs is currently in progress. Reduction in levels of APP between entry and slaughter suggested the health and welfare status of pigs improved over the course of the studies in both systems. Acknowledgements This work was funded by Defra under project AW0130. 91 5th International Colloquium on Animal Acute Phase Proteins – Dublin – March 2005 Poster 36 GLYCOSYLATION PATTERNS OF SERUM PROTEINS DURING EXPERIMENTAL INFECTION OF MICE WITH Toxoplasma gondii Heegaard PMH, Lind P Danish Institute for Food and Veterinary Research, Department of Veterinary Diagnostics and Research, Copenhagen, Denmark. E-mail: email@example.com Introduction The acute phase protein (APP) response is accompanied by profound and transient changes in the glycosylation patterns of serum glycoproteins. These changes generally last for the duration of the acute phase protein response with the biggest changes generally observed at the peak APP response. A mouse model of aseptic inflammation for the study of such changes was previously established by us and it was shown that the glycosylation changes occurred not only in positive acute phase glycoproteins but also in non-reacting and negatively reacting glycoproteins . Interestingly, the pattern of change was constant for all glycoproteins investigated. Here we wanted to study the glycosylation changes during infection with the parasite Toxoplasma gondii. Materials & Methods Mice were inoculated i.p. with strain SSI119 of T. gondii and bled and killed at different times thereafter in groups of four until 27 days p.i. A control group received PBS. Crossed immunoelectrophoresis of serum samples with lectin- (con A-) interaction was used for quantitation of glycoforms of alpha-1-esterase and alpha-1-protease inhibitor. We measured the precipitate areas of the different glycoform subfractions which were classified according to lectin-binding as either non- reacting (0), weakly reacting (1) or strongly reacting (2). A summary measure of lectin reactivity ((0+1)/2) was then depicted as a function of day p.i. Results Alpha-1-esterase (a negative acute phase protein) and alpha-1-protease inhibitor (non-reacting) both exhibited a change in glycosylation patterns towards a greater proportion of lectin (con A) non-reactive glycoform subfractions during the acute phase of the T. gondii infection, corresponding to earlier findings in an aseptic inflammation model in mice. The glycosylation changes reflected accurately the onset and duration of systemic acute disease and traditional acute phase protein responses in the model. Furthermore, when comparing different doses of T. gondii inocula, the glycosylation response was found to correlate, with the higher dose leading to earlier glycosylation changes that the lower dose. With the high dose inoculation these changes occurred earlier than the serum TNF-alpha response. This is the first demonstration of the kinetics and range of glycosylation changes during an experimental infection with Toxoplasma gondii in mice. Discussion As a qualitative measure that is general, sensitive and independent of the acute phase nature of the glycoproteins, determination of glycoform patterns or lectin reactivity indexes holds promise as a rapid, robust and simple method for the early detection of an ongoing acute phase reaction in a serum sample. It remains to be established if such reactions also occur in production animals like pigs and cattle and if the reactions in these species are also generalised and not dependent on glycoprotein type. 92 The 5th International Colloquium On Animal Acute Phase Proteins Gratefully Acknowledges the Support of Our Principal Sponsors
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