Swine Urine Research Articles

Surface-Enhanced Raman Spectroscopy for Rapid Determination of β-Agonists in Swine Urine

Abstract Surface-enhanced Raman spectroscopy (SERS) combined with chemometric method was used for the determination of β-agonists including clenbuterol, salbutamol and ractopamine in standard solutions. For standard solutions, the characteristic bands of SERS spectra could be detected at concentrations as low as 2 μg L −1 for clenbuterol and salbutamol, and 0.1 mg L −1 for ractopmiane; while the R 2 of actual values vs. predicted values based on partial least squares (PLS) regression model for the three tested drugs ranged from 0.9134 to 0.9368. For real sample analysis, three β-agonists was artificially added to urine samples (1–20 mg L −1 ) collected from ten different swine, and then a rapid liquid-liquid extraction (LLE) method was used to extract the targets before SERS analyses. Ractopamine in swine urine could be detected at as low as 1 mg L −1 . For quantitative analysis, the R 2 of actual values vs. their values predicted by the PLS models was 0.8810. The LLE method followed by SERS detection took less than 30 min for analysis of β-agonists in swine urine, which indicates the method has the potential for the rapid determination of β-agonists in swine urine with SERS technology.

Synchrony of net nitrogen mineralization and maize nitrogen uptake following applications of composted and fresh swine manure in the Midwest U.S.

Understanding how the quality of organic soil amendments affects the synchrony of nitrogen (N) mineralization and plant N uptake is critical for optimal agronomic N management and environmental protection. Composting solid livestock manures prior to soil application has been promoted to increase N synchrony; however, few field tests of this concept have been documented. Two years of replicated field trials were conducted near Boone, Iowa to determine the effect of composted versus fresh solid swine manure (a mixture of crop residue and swine urine and feces produced in hoop structures) on Zea mays (maize) N uptake, in situ soil net N mineralization, and soil inorganic N dynamics. Soil applications of composted manure increased maize N accumulation by 25 % in 2000 and 21 % in 2001 compared with fresh manure applications (application rate of 340 kg N ha -1 ). Despite significant differences in net N mineralization between years, within year seasonal total in situ net N mineralization was similar for composted and fresh manure applications. Partial N budgets indicated that changes in soil N pools (net N mineralization and soil inorganic N) in the surface 20 cm accounted for 67 % of the total plant N accumulation in 2000 but only 16 % in 2001. Inter- annual variation in maize N accumulation could not be attributed to soil N availability. Overall, our results suggest that composting manures prior to soil appli- cation has no clear benefit for N synchrony in maize crops. Further work is required to determine the biotic and abiotic factors underlying this result.

A high-affinity anti-salbutamol monoclonal antibody: Key to a robust lateral-flow immunochromatographic assay

Among the components that make up a lateral-flow immunochromatographic assay (ICA), antibody is the key. In this paper, salbutamol (SAL) as a model analyte was meticulously designed to prepare immunogen and coating antigen in distinctly different ways. Four hybridoma cell lines were prepared and identified. Among them, C9 had highest affinity, best dose–response behavior, lowest limit of detection, and highest specificity and was chosen to be labeled with colloidal gold as the detector reagent and applied on the conjugate pad. Goat anti-mouse antibody and SAL–BSA conjugate were sprayed on a nitrocellulose membrane as test line and control line, respectively. Under the optimized conditions, the ICA strip was constructed based on a binding inhibition format. Color intensity on the test line was visually distinguishable from that of the negative sample within 5min, with the visual detection limit of 1ngml−1 in phosphate-buffered saline. Cross-reactions with other β-agonists were not found (<1%). The results from ICA were in a good agreement with those obtained by enzyme-linked immunosorbent assay. The developed ICA has potential as a useful on-site screening tool for SAL in swine urine.

Anaphylaxis to pork kidney is related to IgE antibodies specific for galactose‐alpha‐1,3‐galactose

Carbohydrate-specific IgE antibodies present on nonprimate mammalian proteins were incriminated recently in delayed meat anaphylaxis. The aim of this study was to explore whether anaphylaxis to mammalian kidney is also associated with galactose-α-1,3-galactose (αGal)-specific IgE. Fourteen patients with anaphylaxis to pork or beef kidney underwent prick tests to meat and kidney. Some patients also underwent skin tests to Erbitux(®) (cetuximab). IgE antibodies to αGal, swine urine proteins, beef and pork meat, serum albumin proteins, cat, and rFel d 1 were measured by ImmunoCAP(®). The αGal levels were estimated in meats and kidney by ELISA inhibition assay. Cross-reactivity between αGal and pork kidney was studied with the ImmunoCAP(®) inhibition assay. Among the 14 patients, 12 presented with anaphylactic shock. Reactions occurred within 2 h from exposure in 67% of patients. Associated risk factors were observed in 10 cases, and alcohol was the main cofactor. Three patients underwent an oral challenge to pork kidney, and anaphylaxis occurred after ingestion of small quantities (1-2 g). Prick tests to kidney were positive in 54% of patients. All tested patients showed positive skin tests to Erbitux(®). All patients tested positive for IgE to αGal, with levels ranging from 0.4 to 294 kU/l. IgE binding to αGal was inhibited by raw pork kidney extract (mean, 77%; range, 55-87%), which showed a high amount of αGal determinants. Pork or beef kidney anaphylaxis is related to αGal IgE. Its peculiar severity could be due to an elevated content of αGal epitopes in kidney.

Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine

To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC(50)) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products.

Determination of Four Kinds of &amp;beta; -Agonists in Swine Urine by Molecularly Imprinted Solid Phase Extraction Followed Gas Chromatography Coupled Mass Spectrometry

Determination of Four Kinds of <i>&amp;beta;</i> -Agonists in Swine Urine by Molecularly Imprinted Solid Phase Extraction Followed Gas Chromatography Coupled Mass Spectrometry

Surface Enhanced Raman Spectroscopy for Rapid Determination of &amp;beta; -Agonists in Swine Urine

Surface Enhanced Raman Spectroscopy for Rapid Determination of <i>&amp;beta;</i> -Agonists in Swine Urine

Rapid Determination of Ractopamine in Swine Urine Using Surface-Enhanced Raman Spectroscopy

Ractopamine is approved for use in swine to improve carcass leanness in the United States, but banned in the European Union and China because ractopamine residue may pose health risks. This study investigated the possibility of applying surface-enhanced Raman spectroscopy (SERS) for analysis of ractopamine in swine urine. Ractopamine (0.1-10 μg mL(-1)) was added to urine samples collected from 20 swine to prepare a total of 240 samples. A simple centrifugation, a liquid-liquid extraction (LLE) method, and a more complicated method involving liquid-liquid extraction and solid-phase extraction (LLE-SPE) were used to extract ractopamine from urine samples. Principal component analysis (PCA) and partial least-squares (PLS) regression were used for spectral data analyses. Although no satisfactory result was obtained with the centrifugation method, ractopamine could be detected at levels of 0.8 and 0.4 μg mL(-1) with the LLE and LLE-SPE extraction methods, respectively. The R2 of the PLS model of actual ractopamine values versus predicted values was 0.74 for the LLE method and 0.73 for the LLE-SPE method. The SERS method with simple sample preparation has great potential for rapid analysis of ractopamine in swine urine.

Modified dispersive liquid–liquid microextraction followed by high-performance liquid chromatography for the determination of clenbuterol in swine urine

A modified dispersive liquid–liquid microextraction (DLLME) technique combined with an HPLC-UV procedure was developed for the extraction and determination of clenbuterol in swine urine. The modification involved the selection of methyl tert-butyl ether (MTBE) as the dispersive solvent, which had a low solubility in aqueous samples, playing the part of dispersion with the help of violent shaking. MTBE improved the partition of clenbuterol into the extractant, and helped the formation of phase separation. Various factors affecting the extraction efficiency including selection of the organic extractant and the dispersive solvent, the volume of extractant and dispersive solvent, salt concentration, NaOH concentration and centrifugation time were evaluated and optimised. Under the optimal conditions, precision, linearity (correlation coefficient, r 2 = 0.996 over the concentration range of 10–1000 ng ml−1), detection limit (2.4 ng ml−1) and enrichment factor of 55 were obtained. The modification to the DLLME made it suitable for analytes with pronounced solubility, especially when the compounds are highly polar and thus more difficult to extract effectively by DLLME. The procedure was suitable for the fast screening of clenbuterol residue in swine urine.

A carbon nanotube-based high-sensitivity electrochemical immunosensor for rapid and portable detection of clenbuterol

Carbon nanotubes have shown their unique advantages of mechanical, chemical and electronic properties in bioanalysis. We herein report a new method to efficiently and reproducibly prepare multi-walled carbon nanotubes (MWNTs)–protein sensing layers for electrochemical immunosensors. This method employs centrifugation to prepare a conjugate of MWNTs and goat anti mouse-immunoglobulin G (IgG) (secondary antibody). The conjugates were then deposited on screen-printed electrodes to form a nanostructured layer (MWNT-I layer). CLB monoclonal antibody was assembled through its binding to the secondary antibody. The MWNT-I layer-based electrodes were used for rapid and sensitive amperometric immunosensing detection of clenbuterol (CLB) in swine urine samples. Horseradish peroxidase-coupled CLB (CLB–HRP) competed with free CLB in the samples to bind the monoclonal antibody. It has shown significantly higher sensitivity and better reproducibility than the chemical conjugation method. This MWNT-based immunosensor is highly sensitive, leading to a limit of detection of 0.1ng/mL within a rapid assay time of 16min. Its sensitivity is at least 1 order of magnitude higher than that of a normal immunosensor (without MWNTs). The sensing device is portable with disposable screen-printed electrode, satisfactorily meeting the requirements for field detection of food security-related species.

Development of a Sensitive Monoclonal Antibody-Based ELISA for the Detection of Sulfamethazine in Cow Milk, Honey, and Swine Urine

A specific monoclonal antibody (MAb) against sulfamethazine was produced with hybridoma technology. This assay shows very high sensitivity with IC50 of 0.4 ng/mL and LOD of 0.05 ng/mL when it was run in 0.02 mol/L PBS (pH 7.5). This MAb has shown high specificity to sulfamethazine, with little cross reactivity for sulfamerazine (5.27%) and sulfadimethoxypyrimidine (1.12%) and very low cross reactivity values for the other test compounds (≤0.1%). Sulfamethazine was spiked in milk, honey, and swine urine. After a simple extraction procedure the extracts at appropriate dilution were analyzed by ELISA. Satisfactory results were obtained by this assay, with average recoveries of 95-116% and coefficients of variation (CVs) of 5-9%. These results suggests that the MAb-based ELISA will be a feasible quantitative/screening method for sulfamethazine residue in real samples.

Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene ( phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene ( ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase ( GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6–25 h assay period with detection and determination limits and EC 50 values for 17 β-estradiol of 2.8 ng L − 1 , 5.9 ng L − 1 , 33.2 ng L − 1 (nAES-P) and 3.1 ng L − 1 , 6.7 ng L − 1 and 39.4 ng L − 1 (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.

Development of rapid immunoassays for the detection of ractopamine in swine urine

The monoclonal antibodies (mAbs) against ractopamine (Rac) were prepared and their properties identified by indirect competitive enzyme-linked immunoabsorbant assay (ELISA). The IC50 of mAbs was 2.7 ng ml−1 towards Rac or 9.3 ng ml−1 towards Rac-glucuronides and no cross-reactivity (CR) towards other competitors except dobutamine (CR: 3.76%). Based on the mAbs, the Rac-kit (kit) and Rac-strip (strip) were developed to detect Rac residues in swine urine. The strip and kit assay could be performed within 5–10 min and 2 h, respectively, allowing the analysis of urine samples without the need for sample clean-up. The detection limits were 1 ng ml−1 for kit and 3 ng ml−1 with the unaided eye, and 0.2 ng ml−1 with the Strip Reader for strip. The correlation coefficients (R 2) were 0.988 for kit in the range 0–128.0 ng ml−1, and 0.987 for strip in the range 0–10.8 ng ml−1. Comparing the gas chromatography-mass spectrometry (GC-MS) with the kit or strip in swine urine spiked with Rac standards, the differences ranged from 1.4% to 4.5% for kit and 1.0% to 4.7% for strip. However, the differences were greater than 54% for the kit and 55% for the strip test for the analysis of urine from swine treated with Rac. The results obtained from GC-MS using hydrolysed urine samples were generally in good agreement with those obtained from strip or kit using non-hydrolysed urine samples.

Identification of the major metabolites of quinocetone in swine urine using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Quinocetone (QCT), 3-methyl-2-cinnamoylquinoxaline-1,4-dioxide, is a quinoxaline-N,N-dioxide used in veterinary medicine as a feed additive. QCT is broadly used in China to promote animal growth, but few studies have been performed to reveal the metabolism of QCT in animals until now. In the present study, the metabolites of QCT in swine urine were investigated using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS). Multiple scans of metabolites in MS and MS/MS modes and accurate mass measurements were performed simultaneously through data-dependent acquisition. Most measured mass errors were less than +/-5 mDa for both protonated molecules and product ions using external mass calibration. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS(2) spectra and known structure of QCT. As expected, extensive metabolism was observed in swine urine. Thirty-one metabolites were identified in swine urine, most of which were reported for the first time. The results reveal that the N-O group reduction at position 1 and the hydroxylation reaction occurring at the methyl group, the side chain or on the benzene ring are the main metabolic pathways of quinocetone in swine urine. There was abundant production of 1-desoxyquinocetone and hydroxylation metabolites of 1-desoxyquinocetone. The proposed metabolic pathway of quinocetone in vivo can be expected to play a key role in food safety evaluations.

Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine

A protein conjugate of streptomycin (streptomycin-bovine serum albumin (BSA) conjugate) was prepared and used as immunogen to produce monoclonal antibodies (MAb). One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentration (IC(50)) value of 4.65 ng/ml and the IC(20) value of 0.21 ng/ml in phosphate buffered saline (PBS). At optimum conditions, an indirect competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic assay (CGIA) were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/ml were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum detection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC(50)) of the test strip under an optical density scanner were calculated to be 0.1 +/- 0.01 ng mL(-1) and 0.1 +/- 0.01 ng mL(-1), 0.56 +/- 0.08 ng mL(-1), and 0.71 +/- 0.06 ng mL(-1), respectively, the cut-off levels with the naked eye of 1 ng mL(-1) and 1 ng mL(-1) for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.

Confirmatory method for the determination of nandrolone and trenbolone in urine samples using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

A confirmatory method for the simultaneous determination of nandrolone (α and β) and trenbolone (α and β) in urine samples by liquid chromatography electrospray mass spectrometry (LC–MS-MS) was developed. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–positive ion electrospray tandem mass spectrometry using deuterium labelled internal standards. The analytical procedure was applicable to bovine and swine urine samples. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results obtained showed that the method was suitable for statutory residues testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. The decision limits (CCα) obtained, were between 0.54 and 0.60 μg L −1; the recovery was above 64% for all the analytes. Repeatability was between 1.6% and 5.7% and within-laboratory reproducibility between 1.6% and 6.0% for all the steroids.

Development of immunoassays for the detection of sulfamethazine in swine urine

The use of sulfonamides, such as sulfamethazine (SM2), in pig production is recognized as a public health risk as it inevitably results in sulfamethazine residues in pork. This study is aimed at establishing rapid, simple, reliable methods, with both sensitivity and specificity, for detecting sulfamethazine residues. For this purpose, monoclonal antibodies against sulfamethazine were prepared and characterized. No cross-reaction of the monoclonal antibodies was identified with other sulfonamides or analytes. Based on the competitive immunoassay principle, an indirect competitive ELISA kit (SM2 kit) and a rapid detection strip for detecting sulfamethazine residues were developed using monoclonal antibodies and the colloidal gold technique. The indirect competitive ELISA kit and the strip assay could be performed within 2 h and 5–10 min, respectively. The results showed that the detection limits were 1 ng ml−1 for the indirect competitive ELISA kit and 8 ng ml−1 with the unaided eye and 1 ng ml−1 with the strip reader for the rapid strip assay. Comparing the HPLC method with the SM2-kit or the strip in pig urine spiked with standards of SM2, the difference was <4.6% for SM2-kit and 4.3% for the strip. The two methods are suitable for the rapid screening of sulfamethazine residues in swine urine. Experimental data revealed that the two methods, especially the strip, proved to be sensitive, specific, rapid and easy to use for the quantitative, semi-quantitative or qualitative detection of SM2 residues in swine urine.

A new development of measurement of 19-Nortestosterone by combining immunochromatographic strip assay and ImageJ software

A rapid immunochromatographic assay was developed to detect 17β-19-Nortestosterone (19-NT). The 19-NT-OVA conjugate was immobilised to nitrocellulose membrane as a test line and goat anti-rabbit IgG as a control line. Anti-19-NT polyclonal antibody labelled with colloidal gold particles acted as detector reagent. We tested the sensitivity of the strip using spiked swine urine, and analysed the intensity of test line using ImageJ software. The sensitivity by naked eye measurement was determined to be 200 ng/ml and lower limit using ImageJ was distinguished as 5 ng/ml. The total assay time was less than 20 min, suitable for screening and quantifying the residue of 19-NT in swine urine.

Use of FT-IR to Identify Enhanced Biomass Production and Biochemical Pool Shifts in the Marine Microalgae, Chlorella ovalis, Cultured in Media Composed of Different Ratios of Deep Seawater and Fermented Animal Wastewater

Growth rates, photosystem II photosynthesis, and the levels of chlorophyll a and secondary metabolites of Chlorella ovalis were estimated to determine if they were enhanced by the addition of swine urine (BM) or cow compost water (EP) that had been fermented by soil bacteria to deep seawater (DSW) in an attempt to develop media that enabled batch mass culture at lower costs. Growth of C. ovalis in f/2, f/2-EDTA+BM60%, DSW+BM30%, and DSW+EP60% was enhanced and maintained in the log phase of growth for 16 days. The cell densities of C. ovalis in DSW+EP60% (4.1x10(6) Cells/ml) were higher than those of f/2 (2.9x10(6) Cells/ml), f/2-E+BM60% (3.7x10(6) Cells/ml), and DSW+BM30% (2.7x10(6) Cells/ml). The growth rate was also more favorable for C. ovalis cultured in DSW+EP60% (0.15 day(-1)) than that of C. ovalis cultured in the control medium (f/2) (0.12 day(-1)). Furthermore, the chlorophyll a concentration of C. ovalis cultured in DSW+EP60% (4.56 mg/l) was more than 2-fold greater than that of C. ovalis cultured in f/2 (2.35 mg/l). Moreover, the maximal quantum yields of photosystem II at 470 nm (Fv/Fm) were significantly higher in organisms cultured at f/2-E+BM60% (0.53) and DSW+EP60% (0.52) than in the other treatment groups. Finally, Fourier transformation infrared (FT-IR) spectroscopy revealed that C. ovalis grown in DSW+EP60% had more typical peaks and various biochemical pool shifts than those grown in other types of media. Taken together, the results of this study indicate that the use of DSW+EP60% to culture C. ovalis can reduce maintenance expenses and promote higher yields.