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ABSTRACT: The objective of this study was to evaluate and compare different sample treatments and storage conditions in order to determine optimal conditions for total and acylated canine ghrelin measurement using ELISA assays. Under the conditions assessed in this study, total ghrelin concentration was stable when stored at room temperature or under cooled conditions (4°C) for approximately six hours regardless of the type of anticoagulant used. When samples were stored at under -20°C, three freeze/thaw cycles during storage did not alter total ghrelin concentration if serum or plasma treated with EDTA or with EDTA-aprotinin is used. When samples were stored at under -80°C, no significant differences were observed after three freeze/thaw cycles in total ghrelin concentrations in serum or plasma treated with EDTA, EDTA-aprotinin or heparin. While acylated ghrelin showed to be very instable, its analysis should be performed within an hour of sample collection or stored at -80°C preferably in serum in order to acquire accurate data. Addition of HCl did not improve total or acylated ghrelin stability. Furthermore, acidification of plasma samples decreased the stability of total ghrelin and impeded determination of acylated ghrelin with the ELISA used in this study.
The Veterinary record. 01/2013;
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ABSTRACT: The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA359-379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 × 10-3 μg/ml and a limit of quantification of 24.5 × 10-3 μg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r = 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P < 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.
animal 11/2012; · 1.74 Impact Factor
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ABSTRACT: This study assesses the utility of saliva samples to monitor the time course of the acute-phase response to different viruses in pigs under field conditions by using time-resolved immunofluorometric assays (TR-IFMA). A total of 30 pigs from three different farms, located in Southeast Spain, were used. Farm 1 had outbreaks of porcine circovirus type 2, farm 2 had infections with porcine reproductive and respiratory syndrome virus and farm 3 had concomitant infections with both viruses. Serology was used to determine the time of seroconversion of pigs to two different pathogens. The levels of two acute-phase proteins (APPs), C-reactive protein (CRP) and haptoglobin (Hp), were measured in saliva and serum samples and compared with pig's serology. Kinetic curves of both APPs across the study obtained in saliva samples were similar to those of serum, with R of 0.68 and 0.78 for CRP and Hp, respectively. The median CRP and Hp concentrations in saliva were higher around the theorized time of infection, according to previous experimental studies, and at seroconversion of animals. CRP increments were apparent 1 week before the increments obtained in Hp. These findings indicate that salivary APP concentrations, by using TR-IFMA, can be used in longitudinal studies as non-invasive early indicators of health status.
animal 02/2012; 6(2):321-6. · 1.74 Impact Factor
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ABSTRACT: The objectives of this study were to perform the optimization and validation of three commercially available immunoassays for the measurement of IgA, IgG, and IgM (Igs) in porcine saliva samples and to determinate if their concentrations may be used to distinguish healthy from diseased animals. Intra and inter assay coefficients of variation were lower than 15% in all cases. All methods showed good linearity and recovery; and detection limits were low enough to detect Igs levels in healthy and diseased animals. The clinical validation showed an increase statistically significant (P<0.05) in the group of diseased animals versus healthy pigs. Therefore, these assays may be used in porcine saliva samples, in addition, the measurement of Igs in saliva could be a practical tool, simple and minimally invasive, to evaluate the humoral immune status of pigs.
Research in Veterinary Science 10/2011; 93(2):682-7. · 1.65 Impact Factor
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ABSTRACT: Eight colostrum-deprived calves aged 8-12 weeks were inoculated intranasally with a non-cytopathic strain of bovine viral diarrhoea virus (BVDV) genotype-1 and the effects on the hepatic immune response were studied. Two calves were sacrificed at each of 3, 6, 9 and 14 days post-inoculation (dpi) and two uninoculated animals were used as negative controls. BVDV was detected in hepatic macrophages and monocytes from 3 to 14dpi and in Küpffer cells (KCs) from 6 to 14dpi. Increases in the numbers of MAC387(+) KCs and monocytes, but not interstitial macrophages, differentiated by morphological features, were evident in the liver following inoculation with BVDV. There was a substantial increase in the number of monocytes positive for tumour necrosis factor (TNF)-α, but only small increases in the numbers of TNF-α(+) KCs and interstitial macrophages and interleukin (IL)-6(+) monocytes, KCs and interstitial macrophages. There was an increase in the number of interstitial CD3(+) T lymphocytes in the liver, but no substantial changes in the numbers of circulating CD3(+) T lymphocytes, interstitial or circulating CD4(+) or CD8(+) T lymphocytes, or CD79αcy(+) B lymphocytes. Serum haptoglobin and serum amyloid A increased transiently at 12dpi. Upregulation of some pro-inflammatory cytokines by hepatic macrophages is evident in subclinical acute BVDV type 1 infection in calves.
The Veterinary Journal 04/2011; 190(2):e110-6. · 2.24 Impact Factor
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ABSTRACT: Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS-PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.
The Veterinary Journal 03/2011; 187(3):356-62. · 2.24 Impact Factor
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ABSTRACT: The objective of this study was to develop a novel competitive ELISA to measure the acute phase protein serum amyloid A (SAA) in pigs using species-specific reagents. Polyclonal antibodies were produced in rabbits immunised with recombinant porcine SAA (rSAA) expressed in Escherichia coli. Both the rSAA and polyclonal antibodies were used to develop a novel competitive assay that was analytically and clinically validated. This assay had within- and between-run coefficients of variation of 8.6% and 25%, respectively, and demonstrated a high level of accuracy as determined by linearity-under-dilution (correlation coefficient, r=0.965). The analytical and functional limits of detection were 3.3 and 105.02mg/L, and the upper and lower quantification limits were 66.9 and 2.8mg/L, respectively. Statistically significant differences (P<0.0001) were found between the concentrations of SAA in healthy and diseased pigs. This novel assay precisely and sensitively measures SAA levels in pigs and will facilitate the more accurate assessment and study of the acute phase response in this species.
The Veterinary Journal 03/2011; 190(2):e7-11. · 2.24 Impact Factor
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ABSTRACT: The aim of this study was to validate two commercially available ELISA assays for total ghrelin measurement in dogs: one canine-specific and one originally designed for measuring human ghrelin. The two assays showed intra-assay coefficient of variations (CVs) lower than 10%, while the inter-assay CVs exceeded the 15% limit. Sample dilutions resulted in linear regression equations with correlation coefficients close to 1. In order to compare methods and verify ability of the ghrelin assays to differentiate between low and high levels, ghrelin concentrations were measured in plasma samples obtained before and at different times after glucose administration in five Beagle dogs. A statistically significant changes in ghrelin after glucose administration was recorded only with assay B. In conclusion, the human ELISA validated in this study showed a good intra-assay precision, accuracy, and when applied to the glucose injection study, was better in distinguishing high and low canine ghrelin levels than the canine ELISA assay.
J Anim Physiol a Anim Nutr 12/2010; 96(1):1-8. · 0.86 Impact Factor
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ABSTRACT: This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.
Comparative immunology, microbiology and infectious diseases 12/2009; 33(6):e51-8. · 2.99 Impact Factor
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ABSTRACT: The purpose of this study was to study the use of saliva samples as alternative to serum for acute phase protein (APP) quantifications in pigs at field conditions. To this end, haptoglobin (Hp) and C-reactive protein (CRP) concentrations were examined in 100 animals at different ages. Sixty pigs were from a farm with chronic PRRS virus infection and 40 from a specific pathogen free (SPF) farm. A serological study was performed to obtain an overview of the immune status of animals and to evaluate possible concomitant infections in animals with PRRS infection. The results reported in this study showed that both saliva and serum samples had higher APP concentrations in PRRS pigs aged 8-9, 17-18 and 24-25 weeks in conventional herds than SPF pigs of the same age (p<0.05). In addition, increases in APP were obtained with age independently of the health status of the animals. According to the ROC analyses performed, saliva could be a better specimen than serum to quantify Hp and CRP levels in field conditions and may contribute to a more efficient detection of diseased animals at farm level.
Veterinary Immunology and Immunopathology 07/2009; 132(2-4):218-23. · 2.08 Impact Factor
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ABSTRACT: Study objectives were to investigate whether C-reactive protein (CRP) in pig saliva could be quantified using an adapted, time-resolved immunofluorometry assay (TR-IFMA), and to determine whether the assay could distinguish healthy from diseased animals. The test method had intra- and inter-assay coefficients of variation of 5.75% and 9.73%, respectively, the limit of detection was 0.47ng/mL and the coefficient of determination was 0.98. Analysis of CRP concentrations in paired serum and saliva samples from 50 pigs gave a positive correlation (r=0.702, P<0.01) and the salivary CRP concentration was able to distinguish healthy from diseased animals in 62 samples from pigs with naturally occurring or experimentally-induced inflammation. The results suggest that this minimally invasive, straightforward and sensitive assay may be useful in pig health and welfare monitoring.
The Veterinary Journal 05/2008; 181(3):261-5. · 2.24 Impact Factor
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ABSTRACT: A time-resolved immunofluorometric assay was evaluated for measurement of C-reactive protein in meat juice from diaphragmatic muscle collected from slaughtered pigs. Analytical and clinical validation of the method was performed by using meat juice samples, obtained by freezing and thawing muscle pieces. The intra- and inter-assay coefficients of variation ranged from 2.2-5.8% to 7.9-14.3%, respectively. The limit of detection was 0.00038 microg/ml. The method measured the CRP concentrations in a linear manner with a good accuracy (r=0.99). CRP concentrations in serum were highly correlated with those in diaphragmatic meat juice (r=0.90; p<0.001). CRP concentrations were significantly higher in clinically affected pigs compared to non-diseased pigs. The assay described here provides a sensitive method for measuring CRP concentrations in meat juice, which can represent a suitable alternative to serum or blood samples and simplifies the process of sampling collection at slaughter.
Veterinary Immunology and Immunopathology 05/2008; 122(3-4):250-5. · 2.08 Impact Factor
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ABSTRACT: Ceruloplasmin (Cp) determination could provide an objective measure of the health status of an animal and could be used as marker of animal health and welfare (Skinner, 2001) (Skinner, J. G., 2001. Special report. International standardization of acute phase proteins. Veterinary Clinical Pathology 30, 2-7.) but only manual methods have been reported to determine Cp concentrations in pigs (McCosker, 1961; Toussaint et al., 1995; Eckersall et al., 1996) (McCosker, P. J., 1961. Paraphenylenediamine oxidase activity and copper-levels in mammalian plasmas. Nature. 190, 887-889; Toussaint, M. J. M., Van Ederen, A. M., Gruys, E., 1995. Implication of clinical pathology in assessment of animal health and in animal production and meat inspection. Comparative Haematology International 5, 149-157; Eckersall, P. D., Saini, P. K., McComb, C., 1996. The acute phase response of acid soluble glycoprotein, alpha(1)-acid glycoprotein, ceruloplasmin, haptoglobin and C-reactive protein, in the pig. Veterinary Immunology and Immunopathology 51, 377-385). In the present study two automated methods based on the use of two different substrates for the determination of serum ceruloplasmin in pigs were developed, evaluated and compared. Both methods showed a good precision and detection limits, with no signs of inaccuracy and could be applied to biochemical autoanalyzers usually found in clinical laboratories using only minimal amounts of serum. Additionally the behaviour of Cp in experimental and clinical situations was studied showing an increase of around two fold after turpentine administration and significantly higher values in cases of pigs with inflammatory conditions when compared with healthy pigs.
Research in Veterinary Science 09/2007; 83(1):12-9. · 1.65 Impact Factor
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ABSTRACT: The aim of this study was to validate commercially available methods for porcine haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and major acute phase protein (Pig-MAP) determinations. Intra and inter assay coefficients of variation (CVs) were lower than 20% in all cases with exception of inter assay CVs for CRP and Pig-MAP assays with samples of low acute phase proteins concentration, and for SAA assay at any acute phase proteins concentration. All methods showed good linearity and detection limits were low enough to detect APPs levels in healthy animals. Hp and SAA were very affected by haemolysis. Lipaemia influenced mainly on SAA determination. Over 15-fold increase was observed in CRP and SAA concentrations after artificially induced inflammation by a single subcutaneous dose of turpentine, whereas Hp and Pig-MAP increased less than 5-fold.
Research in Veterinary Science 09/2007; 83(1):133-9. · 1.65 Impact Factor
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ABSTRACT: Haptoglobin is a positive acute-phase protein with a valuable role as a marker of inflammation in both human and veterinary medicine. The aim of this study was to validate a commercially available immunoturbidimetric method designed for human haptoglobin determination (Izasa SA, Barcelona, Spain) for its use in canine samples. Cross-reactivity between anti-human haptoglobin antiserum and canine haptoglobin was found when agarose gel immunodiffusion and ELISA tests were performed. The use of canine pooled serum with haptoglobin concentration of 6.3 g/L as standard provided higher analytical range than commercially available standards. Intra-assay and inter-assay coefficients of variation were 2.49% and 4.60%, respectively. A linear regression model between immunoturbidimetric results and a previously validated spectrophotometric method (Tridelta Development Limited, Ireland) yielded a slope at 95% confidence interval of 0.94 (0.86, 1.02) and y-intercept at 95% confidence interval of 0.11 (-0.59, 0.82). No significant differences were produced by anticoagulants, lipaemia and bilirubinaemia, although haemolysis significantly decreased haptoglobin. A significant increase of haptoglobin concentration was detected in inflammatory conditions such as pyometra and leishmaniasis, in neoplastic conditions, and after glucocorticoid administration. Canine serum haptoglobin concentration can be reliably measured using the commercially available Izasa immunoturbidimetric method developed for human haptoglobin determination. This method is precise and accurate, provides a wider analytical range than previous reported methods, and can be easily automated and used for routine haptoglobin determination in canine samples.
Veterinary Research Communications 02/2007; 31(1):23-36. · 0.82 Impact Factor
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ABSTRACT: Five acute phase proteins (APPs) [C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), pig-MAP and albumin] were measured in pigs with naturally occurring infections by porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae, and in animals with tail and ear bites, arthritis and other acute inflammatory processes. Healthy specific pathogen-free (SPF) pigs were used as controls. In PRRSV-infected pigs, all APPs with the exception of pig-MAP exhibited significant changes compared with controls. In animals affected with ADV only Hp presented changes of statistical significance, whereas pigs with PCV2 showed marked modifications in all APPs tested. Animals affected with Mycoplasmosis showed concentrations of all positive APPs significantly above levels obtained in SPF pigs, though albumin concentrations did not differ from controls. Finally, all APPs studied showed substantial changes in pigs with acute inflammation. The results indicated that an acute phase response was developed in the different diseases studied, this response being higher in animals with clinical signs and concurrent bacterial processes. Haptoglobin would be the APP that better reflects pathological states; however, to get more complete and valuable information it might be advisable to perform APPs profiles including another APP, such as CRP or SAA.
Journal of Veterinary Medicine Series B 01/2007; 53(10):488-93. · 1.48 Impact Factor
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ABSTRACT: The serum concentrations of haptoglobin, caeruloplasmin, C-reactive protein and serum amyloid A were measured in three groups of seven healthy dogs. Group 1 received a single dose of 1.1 mg/kg methylprednisolone acetate, administered subcutaneously; group 2 received 1 mg/kg per day of prednisone administered orally for three weeks; and group 3 received 2.2 mg/kg per day of prednisone administered orally for seven days. Before the administration of the glucocorticoids the serum concentrations of all the acute phase proteins were within the authors' laboratory reference ranges. After the administration of the drugs there were significant increases in the concentration of haptoglobin in all three groups, the increases being larger in groups 2 and 3. In contrast, the concentrations of C-reactive protein, caeruloplasmin and serum amyloid A were not affected.
The Veterinary record 07/2004; 154(26):814-7. · 1.25 Impact Factor
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ABSTRACT: The possible interference of haemolysis, lipaemia, bilirubinaemia and fibrinogen on capillary zone electrophoresis of canine samples were studied. Solutions of haemoglobin, lipid and bilirubin were prepared and mixed with serum aliquots to make up samples containing different concentrations of the putative interferent substance. In addition, samples of serum and plasma were assayed to assess the influence of fibrinogen. Haemolysis and lipids produced a change in electropherogram morphology giving an interference peak located in the beta-2 region when haemoglobin was increased, and in the alpha-2 region when lipids were increased. A rise in concentration of these interferents caused an increase in the beta and alpha-2 fractions respectively, and a decrease in the other fractions. Bilirubin did not alter morphology but gave an increase in the albumin and alpha-1 and a decrease in the alpha-2 and beta-2 fractions. No differences were found between serum and plasma samples, and fibrinogen did not produce any additional peak.
The Veterinary Journal 12/2002; 164(3):261-8. · 2.24 Impact Factor
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ABSTRACT: The concentrations of haptoglobin, C-reactive protein and ceruloplasmin were measured in symptomatic and asymptomatic dogs naturally infected by Leishmania infantum, and in healthy uninfected dogs to determine the potential value of these proteins for the diagnosis and prognosis of leishmaniasis. The concentrations of the acute phase proteins were significantly higher in the dogs with leishmaniasis than in the control dogs, and the concentration of C-reactive protein was significantly higher in the symptomatic dogs than in the asymptomatic dogs. There were no correlations between the acute phase proteins and the gamma globulins, the albumin/globulin ratio or the titre of anti-leishmanial antibodies.
The Veterinary record 03/2002; 150(8):241-4. · 1.25 Impact Factor
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ABSTRACT: Whole blood has been compared with erythrocytes and plasma for spectrophotometric cholinesterase determination in the dog. Cholinesterase activity was characterized using two substrates: acetylthiocholine and butyrylthiocholine. Acetylcholinesterase was the only form of cholinesterase present on erythrocytes and hydrolysed only acetylthiocholine. Butyrylcholinesterase (pseudocholinesterase) was predominant in plasma, hydrolysing mainly butyrylthiocholine. Based on these results, a method based on the use of two substrates (acetylthiocholine for monitoring acetylcholinesterase and butyrylthiocholine for determining butyrylcholinesterase) in the same whole blood sample is recommended for canine cholinesterase analysis. This way of monitoring both enzymes can be easily automated, yielding good within (CVs < 5%) and between-run (CVs < 7%) precision.
The Veterinary Journal 11/2000; 160(3):242-9. · 2.24 Impact Factor
