Validation of new automated turbidimetric immunoassays for the measurement of haptoglobin and inter-α-trypsin inhibitor heavy chain H4 specific for the bovine species.

BACKGROUND: Good strategical programs are required for the early detection of disease even in the absence of evident clinical signs, which is crucial in satisfying animal welfare. Haptoglobin (Hp) and inter-α-trypsin inhibitor heavy chain H4 (ITIH4) are acute phase proteins and good biomarkers of early inflammation in cattle, with plasma levels that significantly increase after injury or infection. OBJECTIVES: We aimed to develop and validate two new immunoturbidimetric methods for Hp and ITIH4. METHODS: Species-specific antibodies were obtained and used to develop the immunoassays. For the Hp assay, antibodies were fixed to latex microparticles to enhance detection. The immunoassays were set up in an automated analyzer to carry out validation studies. Reference intervals were calculated using Reference Value Advisor. RESULTS: The Hp immunoturbidimetric method had a linear analytical range up to 0.40 mg/mL. The limit of detection (LoD) was 0.005 mg/mL, and the limit of quantification (LoQ) was 0.007 mg/mL. Total imprecision was less than 7%. Comparison with ELISA and single radial immunodiffusion (SRID) showed good correlation, whereas the comparison with the colorimetric method showed constant and proportional differences. The ITIH4 immunoassay showed linearity up to 5 mg/mL, and the LoD was 0.002 mg/mL. Total imprecision was less than 6%. Method comparison showed a good correlation with single radial immunodiffusion, both methods being equivalent. Bilirubin, triglycerides, and hemoglobin presented no interference in any of the assays. Reference intervals were 0.007-0.017 mg/mL for Hp and 0.2-0.7 mg/mL for ITIH4 in dairy cows 10 days before parturition. CONCLUSIONS: Immunoturbidimetric methods developed for Hp and ITIH4 can measure basal and increased levels of these proteins, showing adequate precision, accuracy, and robustness.

Abundance of plasma proteins in response to divergent ratios of dietary ω6:ω3 fatty acids in gestating and lactating sows using a quantitative proteomics approach.

This study aimed to investigate the characteristic proteomic pattern of plasma from sows supplemented with low dietary ω6:ω3 fatty acids (FAs) ratio during gestation and lactation. Two dietary treatments (n = 8 each) comprised either a control ratio of ω6:ω3 FAs (CR, 13:1 during gestation and 10:1 during lactation) or a low ratio (LR, 4:1 during gestation and lactation) by adding soybean oil or linseed oil, respectively. High-resolution mass spectrometry-based quantitative proteomics was applied on plasma (n = 5 each) at day 108 of gestation (G108) and at the end of lactation (L-End), and a total of 379 proteins and 202 master proteins were identified. Out of these, four differentially abundant proteins between LR and CR samples at G108 may relate to serine-type endopeptidase inhibitor activity. Differentially abundant proteins in L-End versus G108 (12 up-regulated and 10 down-regulated) were positively correlated with the events that regulate plasma lipoproteins, stimulus- and defence-responses. These findings demonstrate the benefit of increased dietary ω3 FAs in modifying proteins involved in protective mechanisms against increased stresses in key life cycle phases in pigs. In addition, proteome changes from late gestation to late lactation disclosed the underlying mechanism of pigs in response to reproduction-related stimuli. SIGNIFICANCE: This study aimed to provide a proteomics insight into the beneficial effects of maternal diet supplementation with a low ω6:ω3 fatty acids ratio, based on previously reported performance and zootechnical data. The results suggest that a low dietary ω6:ω3 fatty acids ratio could enhance the cellular defence mechanisms against increased stresses and in particular to oxidative stress in sows during gestation and lactation, as reflected in proteomic changes of haptoglobin (HP), alpha-1-antitrypsin (SERPINA1) and serum amyloid P-component (APCS). Furthermore, significantly changed proteome profiles in sow plasma between late gestation and lactation phases have been revealed for the first time. This finding identified the adaptation mechanisms of sows to changing physiological events during reproduction.

Measurement of ITIH4 and Hp levels in bitches with pyometra using newly developed ELISA methods.

Measurement of acute phase proteins (APPs) as biomarkers in canine medicine is in increasing demand. In the present study, the development and validation of two ELISA methods for the quantification of canine inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) are shown. The adequate imprecision and accuracy and wide analytical range make the developed methods appropriate to quantify ITIH4 and Hp in serum samples. The inter- and intra-assay CVs were lower than 10 %, and the assays maintained linearity under dilution and showed analytical equivalence with the method of radial immunodiffusion. The measurement of CRP, Hp and ITIH4 in sera from bitches affected by pyometra allowed us to determine that ITIH4 behaves as a moderate APP in dogs. The group of bitches affected by pyometra showed very high levels of CRP (147 ± 91 mg/L), corresponding to a strong inflammatory process, which resulted in a moderate increase in the concentrations of Hp (7 times) and ITIH4 (3 times) compared to the control group.

Development and validation of an ELISA for the quantification of bovine ITIH4 in serum and milk.

Inter alpha trypsin inhibitor heavy chain 4 (ITIH4) is a serum protein belonging to the Inter alpha trypsin inhibitor (ITI) family, which was previously characterized by our group as a new APP in cattle. This protein was firstly described in pigs where is known to be a major acute phase protein, also denominated Pig-MAP. Increases of ITIH4 of up to 12 times the pre-infection values were previously reported in the serum of heifers with experimentally induced summer mastitis. ITIH4 was detected in the milk of cows with mastitis by western blot, but the method previously used to quantify this protein, radial immunodiffusion, was not sensitive enough to quantify it in milk samples. In this study we developed an ELISA method which allows the quantification of bovine ITIH4 in serum and milk samples. Previously developed antibodies were used to perform the assay, including anti bovine ITIH4 polyclonal antibodies and a monoclonal antibody against pig ITIH4 that also recognizes the bovine homologous protein. The ELISA developed showed an adequate precision, with inter and intra- assay coefficients of variation lower than 10% for serum and milk samples. The assay keeps linearity under dilution for both serum and milk samples. A good agreement was observed between the values measured by ELISA and radial immunodiffusion in serum samples.

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species.

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 µg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.

Preparation of canine C-reactive protein serum reference material: A feasibility study.

BACKGROUND: The availability of a species-specific reference material is essential for the harmonization of results obtained in different laboratories by different methods. OBJECTIVES: We describe the preparation of a canine C-reactive protein (cCRP) serum reference material containing purified cCRP stabilized in a serum matrix. The material can be used by manufacturers to assign values to their calibrator and control materials. METHODS: The serum matrix was obtained using blood collected from healthy dogs, stabilized and submitted for a delipidation process. The reference material was prepared by diluting purified cCRP in the serum matrix containing 1.0 mol/L HEPES buffer, 3.0 mmol/L calcium chloride, 80,000 kUI/L aprotinin, and 1.0 mmol/L benzamidine hydrochloride monohydrate at a pH of 7.2, and dispensing (0.5 mL) the matrix into vials that were then frozen. RESULTS: The pilot batch of 200 vials was shown to be homogeneous and stable after a stability study at various temperatures and over a total time of 110 days. The prepared material was submitted to an assignment value study. Eight laboratories from different European countries participated by using the same reagents for an immunoturbidimetric method adapted for different analyzers. The obtained cCRP concentration in the reference material was 78.5 mg/L with an expanded uncertainty (k = 2) of 4.2 mg/L. CONCLUSIONS: Canine C-reactive protein serum reference material has been produced that allows harmonization of results obtained by different methods and different laboratories, thus reducing the possibility of errors and misunderstandings.

A new automated turbidimetric immunoassay for the measurement of canine C-reactive protein.

BACKGROUND: In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. OBJECTIVES: The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. METHODS: A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). RESULTS: The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. CONCLUSIONS: The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use.

Coherence of animal health, welfare and carcass quality in pork production chains.

Aim of the study was to measure the potential impact of animal health and welfare on the carcass quality. 99 pigs under equal housing and feeding conditions were involved in the study. Effects of the immune system on carcass composition, meat quality and performance data of slaughter pigs became measureable by quantification of acute phase proteins (APP), haptoglobin (Hp) and pig major acute phase protein (Pig-MAP). The results were not significantly affected by gender or breed. The calculated correlations between chosen animal health indicators and carcass quality parameters prove an influence of health and welfare on performance, carcass composition and meat quality traits. The acute phase proteins could also be valuable as a predictive indicator for risk assessment in meat inspection, as increased Hp concentrations in slaughter blood indicate a 16 times higher risk for organ abnormalities and Pig-MAP concentrations above 0.7mg/ml a 10 times higher risk.

The use of acute phase proteins for monitoring animal health and welfare in the pig production chain: the validation of an immunochromatographic method for the detection of elevated levels of pig-MAP.

The serum concentration of acute phase proteins (APPs) increases in the presence of disease or stress, which makes APPs notable parameters for the global assessment of animal health and welfare. A rapid, immunochromatographic test (ICT) for the detection of elevated levels of pig Major Acute-phase Protein (pig-MAP), one of the main APPs in pigs, was evaluated in more than 1400 pig serum samples obtained from commercial farms. The ICT showed a good performance with a relative sensitivity (Sn) and specificity (Sp) of 94 and 97%, respectively, for a threshold of 1.5mg/mL (comparison with ELISA). Differences in the pig-MAP levels and the number of positive samples with the ICT were observed within the season of sampling, farms, and age groups at one farm, according to the presence of disease or lesions. The ICT was also evaluated in blood samples obtained at slaughter in association with the carcase inspection. The results from this study indicate that the ICT may be used for the evaluation of groups of pigs, after analysing one sub-sample of these pigs, and might be a useful tool in routine health and welfare monitoring programmes aimed to improve the quality of pig production.

Optimal combinations of acute phase proteins for detecting infectious disease in pigs.

The acute phase protein (APP) response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect any disease with an inflammatory component seven porcine APPs were analysed in serum sampled at regular intervals in six different experimental challenge groups of pigs, including three bacterial (Actinobacillus pleuropneumoniae, Streptococcus suis, Mycoplasma hyosynoviae), one parasitic (Toxoplasma gondii) and one viral (porcine respiratory and reproductive syndrome virus) infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP), haptoglobin (Hp), pig major acute phase protein (pigMAP) and serum amyloid A (SAA)) and three negative (albumin, transthyretin, and apolipoprotein A1 (apoA1)) were performed in the more than 400 serum samples constituting the serum panel. This was followed by advanced statistical treatment of the data using a multi-step procedure which included defining cut-off values and calculating detection probabilities for single APPs and for APP combinations. Combinations of APPs allowed the detection of disease more sensitively than any individual APP and the best three-protein combinations were CRP, apoA1, pigMAP and CRP, apoA1, Hp, respectively, closely followed by the two-protein combinations CRP, pigMAP and apoA1, pigMAP, respectively. For the practical use of such combinations, methodology is described for establishing individual APP threshold values, above which, for any APP in the combination, ongoing infection/inflammation is indicated.

Development and validation of an ELISA for the quantification of pig major acute phase protein (Pig-MAP).

Measurement of acute phase proteins (APPs) levels in blood is increasingly being used for monitoring health and welfare in farm animals. In this work a sandwich-type ELISA for the quantification of pig Major Acute phase Protein (Pig-MAP), one of the main APP in pigs, has been developed and validated. Two Pig-MAP specific monoclonal antibodies were developed in mouse. One of the monoclonal antibodies was fixed to microtiter plates and the other was coupled to horseradish peroxidase and used as detection antibody. To calibrate the assay dilutions of a standard pig serum of known Pig-MAP concentration were added to the plate in each assay. The assay showed good accuracy, kept linearity under dilution and recovery was proportional. The detection limit was 0.1 microg/mL. Precision was adequate with coefficients of variation lower than 8% for both inter and intra-assays. A good linear correlation between Pig-MAP concentration values obtained by ELISA and by radial immunodiffusion, used as reference method, was found (r = 0.978; beta = 1.02). Pig-MAP concentration was analysed in serum samples obtained from two pig herds of different health status (10 animals per age and herd, of 10, 12, 14, 18 weeks of age). Mean values obtained in the farm of low health status were higher than the obtained in the farm of high health status (p<0.001). In the farm of high health status, mean Pig-MAP concentration remained constant at the different ages analysed (mean values of 0.83+/-0.18 mg/mL) whereas in the farm of low health status differences between age groups were found. In this farm (low health status) mean values for the total of pigs analysed were of 1.68+/-0.74 mg/mL.

Pig-MAP and haptoglobin concentration reference values in swine from commercial farms.

Pig-MAP (Major Acute-phase Protein) and haptoglobin concentrations were determined in pigs from commercial farms, and reference intervals obtained for different productive stages. Pig-MAP serum concentrations were lower in sows than in adult boars (mean values 0.81 vs. 1.23 mg/mL) and the opposite was observed for haptoglobin (1.47 vs. 0.94 mg/mL). No differences were found between parities, except for a minor decrease in haptoglobin concentration in the 4th parity. A linear correlation between pig-MAP and haptoglobin concentration was observed. In the period 4-12 weeks of life, pig-MAP mean concentrations were around 1mg/mL, being lower in the finishing period (0.7-0.8 mg/mL). Haptoglobin concentrations increased with time, from around 0.6 mg/mL at 4 weeks of age to 1.4 mg/mL at 12 weeks. Mean values of around 0.9 mg/mL were observed in the finishing period. A wider distribution of values was observed for haptoglobin than for pig-MAP concentrations. Differences between herds were observed, with the highest values obtained in a herd with signs of respiratory disease.

Pig major acute-phase protein and apolipoprotein A-I responses correlate with the clinical course of experimentally induced African Swine Fever and Aujeszky's disease.

In the present work, we studied the acute phase protein response after experimental virus infection in pigs. The animals were experimentally infected with African Swine Fever (ASF) or Aujeszky's disease (AD) viruses. The clinical course of ASF infection correlated with increasingly high levels of pig Major Acute-phase Protein (pig-MAP) (mean value of 6 mg/mL on day 6 post infection (p.i.), from 6 to 9 times higher than day 0) and sharp apolipoprotein A-I (apo A-I) decrease (mean value of 0.5 mg/mL, from 4 to 10 times lower than day 0 on day 4 p.i.). AD-clinical signs appeared at day 3 p.i., both in vaccinated (moderate clinical signs) and non-vaccinated pigs (severe outcome within 48 h p.i.). Pig-MAP and apo A-I profiles also followed clinical signs (changing from 0.70 mg/mL to around 3 mg/mL and from around 3 mg/mL to 0.96 mg/mL, respectively in non-vaccinated animals), with minor changes in concentration in the vaccinated group. Haptoglobin levels significantly increased in ASF and AD infected animals (mean maximum values of 2.77 and 3.96 mg/mL, respectively). Minor differences for the C-Reactive Protein in the case of ASF were observed, whereas its concentration increased more than 7 times in AD-infection. The albumin level was not modified in either case. The correlation of clinical signs to our data suggests the potential use of pig-MAP and apo A-I in monitoring infections in swine.

Characterisation of the pig acute phase protein response to road transport.

The acute phase protein (APP) response was evaluated after prolonged transportation of pigs under commercial conditions. Elevated serum APP concentrations were observed in two groups of boars immediately after their arrival at a destination farm compared with within-animal control samples obtained one month later. The effect was more pronounced in the first group of pigs conveyed under average transport conditions (Transport 1, 24 h), although the second group was transported for a longer time period (Transport 2, 48 h) but in superior transport conditions. In a second trial, pigs were sampled before transport, on arrival at an abattoir (following 12 h transport), and at the slaughter-line (after 6 h lairage). Significant increases in major acute phase protein (Pig-MAP), haptoglobin, serum amyloid A, C-reactive protein, and a decrease in apolipoprotein A-I, were observed at slaughter. The results demonstrate that shipment of pigs by road can result in an APP response that is probably related to the stress of transport.

Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS).

The objective of this study was to determine the serum concentration levels of selected acute phase proteins (APP), haptoglobin (HPT) and pig-major acute phase protein (pig-MAP), in postweaning multisystemic wasting syndrome (PMWS) affected pigs and PCV2-subclinically infected pigs. In a first study, a group of 15 eight-week-old conventional pigs from a PMWS affected farm were bled and a complete necropsy, histopathology and in situ hybridisation to detect PCV2 were performed. Based on the results, pigs were classified as suffering from PMWS (n = 10) or healthy animals (n = 5). In a second study, a group of 45 pigs from another PMWS affected farm were selected and bled at 3, 7, 12 and 28 weeks of age. The assessment of PCV2 infection status in these pigs was retrospectively done by PCV2 PCR in serum samples. Selected APP were measured in the serum of all studied pigs by means of radial immunodiffusion. Mean HPT and pig-MAP levels were significantly increased (p = 0.004 and p = 0.0006 respectively) in PMWS-affected pigs when compared to levels found in healthy pigs (2.52 +/- 0.88 mg/mL vs. 1.06 +/- 0.73 mg/mL for HPT and 3.81 +/- 1.53 mg/mL vs. 0.76 +/- 0.34 mg/mL for pig-MAP). In the second study, no significant difference in mean HPT and pig-MAP values were observed between PCV2 PCR positive and negative pigs of any age. However, both APP increased significantly with age in PCV2 PCR negative pigs. Altogether, the present results suggest that APP levels are significantly increased in pigs that develop PMWS, but not in animals with a PCV2 subclinical infection.