Production, characterization, and cross-reactivity studies of monoclonal antibodies against the coccidiostat nicarbazin

A cELISA was developed for the coccidiostat nicarbazin. On the basis of previous computer-assisted molecular modeling studies, p-nitrosuccinanilic acid (PNA-S) was selected as a hapten to produce antibodies to 4,4'-dinitrocarbanilide (DNC), the active component of the coccidiostat nicarbazin. Synthesis is described for the hapten [p-nitro-cis-1,2-cyclohexanedicarboxanilic acid (PNA-C)] used in a BSA conjugate as a plate coating antigen. Monoclonal antibodies (Mabs) were isolated that compete with nicarbazin, having IgM(kappa) isotype. Because of the lack of water solubility of nicarbazin, N,N-dimethylformamide (DMF) (3%, v/v) and acetonitrile (ACN) (10%, v/v) were added to the assay buffer to achieve solubility of nicarbazin and related compounds. The Nic 6 Mabs had an IC(35) value for nicarbazin of 0.92 nmol/mL, with a limit of detection of 0.33 nmol/mL. Nic 6 exhibited high cross-reactivity for PNA-S and PNA-C, and 3-nitrophenol, 4-nitrophenol, and 1-(4-chlorophenyl)-3-(4-nitrophenyl) urea. However, Nic 6 had little or no cross-reactivity with 15 other related compounds.     

Rapid time-resolved fluoroimmunoassay for the screening of narasin and salinomycin residues in poultry and eggs

Anticoccidial drugs are extensively used in the poultry industry to control the infection of the single-cell protozoa of the genus Eimeria. The most commonly used coccidiostats in poultry are the polyether ionophores such as narasin and salinomycin. This paper presents a rapid and simple method for the screening of residues of these two coccidiostatic compounds in poultry and eggs. The method is based on time-resolved fluoroimmunoassay. Sample preparation of eggs consists only of one extraction and evaporation step, and a solid phase extraction step is needed only for the muscle sample preparation. Mean recoveries were 91.0% from muscle tissue and 81.1% from eggs for both narasin and salinomycin. The performance of the assay was evaluated only for narasin because salinomycin had a cross-reactivity of 100% in the assay, and the recoveries of the compounds were not significantly different (P >0.05). The limits of detection [mean + 3 x standard deviation (SD)] of narasin were 0.56 and 0.28 microg/kg, and the limits of quantification (mean + 9 x SD) were 1.80 and 0.57 microg/kg for muscle and eggs, respectively. The coefficients of variation (CV) of the interassay precision of the method, evaluated by five replicate analyses of muscle samples spiked with 2 microg/kg of narasin and egg samples spiked with 1 microg/kg of narasin, were 4.1 and 6.4%, respectively. The CVs of intra-assay precision tests, determined by 10 replicate analyses at the above-mentioned concentration levels, were 3.8 and 4.5%, respectively.     

Development and validation of a lateral flow device for the detection of nicarbazin contamination in poultry feeds

Concentrations of the coccidiostat nicarbazin as low as 2 mg/kg in feed can result in violative drug residues arising in poultry liver. A lateral flow device (LFD) was developed for the detection of contaminating concentrations of nicarbazin following solvent extraction of poultry feeds. Test results, as determined by both visual and instrumental measurement, are available within minutes. For 22 feed samples, nicarbazin-free and fortified at 2 mg/kg, the % relative inhibition ranged from 0 to 45% and from 53 to 85%, respectively. Nicarbazin contamination at the critical concentration (2 mg/kg) can be determined in all cases providing the sampling is representative. A wide range of feed samples taken at a mill that incorporated nicarbazin into poultry feed were analyzed. Data generated for these samples by both the LFDs and a mass spectrometric method were compared, and a significant correlation was achieved.     

Enzyme immunoassay for screening of sulfathiazole in honey

A simple enzyme immunoassay (EIA) was developed to screen honey samples for sulfathiazole (ST) adulteration. Honey samples required only a 30-fold dilution before use in the procedure. Because 96 well microtiter plates were used and only 100 microL of diluted honey sample was required per well, numerous replicates or samples could be tested simultaneously. The EIA was able to detect at least 0.3 ppm levels of ST in honey and also provide a rough quantitation of ST amounts.     

Detection of residues of the coccidiostat diclazuril in poultry tissues by liquid chromatography-tandem mass spectrometry after withdrawal of medicated feed

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the quantitative determination of diclazuril in poultry tissues and feed is presented. A simple clean up with an organic solvent was carried out. A reversed-phase C(18) column was used for the high-performance liquid chromatography (HPLC) to separate the analyte with a gradient of acetonitrile and water as mobile phase. The precursor ions produced by electrospray negative ionization were selected for collisional dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC (Off. J. Eur. Communities 2002, L221, 8-36). For the detection of diclazuril in poultry meat, the decision limit was found to be 0.5 microg/kg. An animal experiment was set up in which 70 chickens were held for 6 weeks. From day 22 until day 32, they were fed feed containing 730 microg/kg diclazuril. From day 33 until day 42, every day six chickens were slaughtered, and breast, thigh, and liver were analyzed. Average steady-state concentrations of 94, 135, and 722 microg/kg in breast, thigh, and liver were obtained, respectively. Nine days after withdrawal of the medicated feed, diclazuril was still present in the different sample types.     

Enzyme immunoassay for screening sulfamethazine residues in swine blood

An enzyme immunoassay (EIA) was developed to screen for residues of sulfamethazine (SMT) and its metabolites in swine blood. Swine blood was treated with perchloric acid and centrifuged. The supernatant solution was neutralized with K2HPO4, centrifuged, and applied to a reverse phase C8 cartridge. The analytes were eluted with methanol-water (2 + 3), and the eluate was diluted and assayed. Average recoveries, using 14C-labeled compounds, were 73, 72, 61, and 62% for SMT, N4-glucosylSMT, N4-acetylSMT, and N4-desaminoSMT, respectively. Tubes coated with antibody were incubated with the eluate and an SMT-beta-galactosidase conjugate. Bound enzyme was detected with fluorogenic substrate. When blood was fortified with 0.1 ppm SMT or a molar equivalent of metabolite, the average relative response of the EIA was 100%, control blood; 61%, SMT; 66%, glucosylSMT; 60%, acetylSMT; and 77% desaminoSMT.     

Sensitive enzyme immunoassay of cephalexin residues in milk, hen tissues, and eggs

A sensitive enzyme immunoassay for cephalexin (CEX) was developed using the rabbit antiserum to CEX, beta-D-galactosidase-labeled CEX, and a double-antibody separation method. The immunogen of CEX was prepared by coupling the amino group of CEX to thiol groups introduced into bovine serum albumin by the use of N-(m-maleimidobenzoyloxy)succinimide as a cross-linker. Highly titered antiserum to CEX was produced in rabbits immunized with the immunogen. Enzyme labeling of CEX with beta-D-galactosidase was done by using N-(gamma-maleimidobutyryloxy)succinimide as the cross-linker. The limit of detection was 30 ng CEX/mL sample solution. Application of the method to CEX drug residues detected 30 ng/mL in milk, 60 ng/g in egg yolk, and 400 ng/g in hen tissue.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs

Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.     

Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study

A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.     

Rapid procedure for determination of nicarbazin residues in chicken tissues

A procedure is described for the quantitation of nicarbazin residues in chicken tissues. The method includes extraction of tissue with chloroform-ethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with methanol-pH 6.0 phosphate buffer (1 + 1). Extracts are separated on a 15 cm, 5 micron C18 column with methanol-pH 6.0 phosphate buffer (6.5 + 3.5) as the mobile phase. The dinitrocarbanilide portion of the complex is detected and quantitated with an electrochemical detector in the reductive mode. Recoveries, based on dinitrocarbanilide, were greater than 95% in liver, breast, and thigh muscle tissues fortified with 0.25-8.0 ppm nicarbazin.     

Determination of flubendazole and its metabolites in eggs and poultry muscle with liquid chromatography-tandem mass spectrometry

The optimization of a quantitative and sensitive LC-MS/MS method to determine flubendazole and its hydrolyzed and reduced metabolites in eggs and poultry muscle is described. The benzimidazole components were extracted from the two matrices with ethyl acetate after the sample mixtures had been made alkaline. The HPLC separation was performed on an RP C-18 column with gradient elution, using ammonium acetate and acetonitrile as mobile phase. The analytes were detected after atmospheric pressure electrospray ionization on a tandem quadrupole mass spectrometer in MS/MS mode. The components were measured by the MS/MS transition of the molecular ion to the most abundant daughter ion. The overall extraction recovery values for flubendazole, the hydrolyzed metabolite, and the reduced metabolite in eggs (fortification levels of 200, 400, and 800 microg kg(-1)) and muscle (fortification levels of 25, 50, and 100 microg kg(-1)) were, respectively, 77, 78, and 80% and 92, 95, and 90%. The trueness (fortification levels of 400 and 50 microg kg(-1), respectively, for eggs and muscle), expressed as a percentage of the added values for these analytes, was, respectively, 89, 100, and 86 and 110, 110, and 98%. The proposed MS detection method operating in the MS/MS mode is very selective and very sensitive. The limits of detection for flubendazole and its hydrolyzed and reduced metabolites in egg and muscle were, respectively, 0.19, 0.29, and 1.14 microg kg(-1) and 0.14, 0.75, and 0.31 microg kg(-1). The limits of quantification were, respectively, 1, 1, and 2 microg kg(-1) and 1, 1, and 1 microg kg(-1). The discussed method was applied to a pharmacokinetic study with turkeys. Residue concentrations in breast and thigh muscle of turkeys orally treated with flubendazole were quantified. Medicated feed containing 19.9 and 29.6 mg kg(-1) flubendazole was provided to the turkeys for seven consecutive days. For the trial with the recommended dose of 19.9 mg kg(-1), one day after the end of the treatment, the mean sum of the flubendazole plus hydrolyzed metabolite residue values in thigh and breast muscle declined to below the maximum residue limit (50 microg kg(-1)) and were, respectively, 36.6 and 54.1 microg kg(-1). The corresponding values with the higher dose of 29.6 mg kg(-1) were, respectively, 101.7 and 119.7 microg kg(-1).     

Modeling of throughfall chemistry and indirect measurement of dry deposition

Precipitation over a forested watershed is altered by interaction with plant surfaces which act as a filter for airborne gases and particles. This results in a major transfer to the forest floor of materials captured, washed, and leached from the forest canopy. Sequential samples of wetfall and sequential samples of throughfall under deciduous and coniferous trees were collected and chemically analyzed for major anions and cations. A simple washoff, mixing model based on leaf area index was used to simulate throughfall chemistry and to decouple foliar exudation from dry deposition. Model results gave excellent predictions of the measured sequential throughfall using estimated values of dry deposition. The model can also be used to calculate dry deposition, if the sequential throughfall data and wetfall data are used as input variables.     

Determination of furaltadone and nifursol residues in poultry eggs by liquid chromatography-electrospray ionization tandem mass spectrometry

The use of nitrofurans as veterinary drugs has been banned from intensive animal production in the European Union (EU) since 1993. The objective of the present study was to evaluate the accumulation and depletion of furaltadone and nifursol and their side-chain metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) and 3,5-dinitrosalicylic acid hydrazide (DNSAH) in eggs after administration of therapeutic and subtherapeutic doses of the drugs to laying hens during three consecutive weeks. LC-MS/MS, with positive and negative electrospray ionization methods, was used for the determination of parent compounds and metabolites in yolk and egg white and was validated according to criteria established by Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) of the analytical methodology for metabolites were 0.1 and 0.5 μg/kg for AMOZ and 0.3 and 0.9 μg/kg for DNSAH, respectively. For the parent compounds, CCα and CCβ were 0.9 and 2.0 μg/kg for furaltadone and 1.3 and 3.1 μg/kg for nifursol, respectively. The data obtained show that the parent compounds are much less persistent than their side-chain metabolites in either yolk or egg white. Between the studied metabolites, AMOZ is the most persistent and could be detected in either yolk or egg white three weeks following withdrawal from treatment.     

Effect of different vitamin D supplementations in poultry feed on vitamin D content of eggs and chicken meat

According to a new European Union regulation, vitamin D(3) can be partially or totally substituted with 25-hydroxyvitamin D(3) (25-OH-D(3)) in hens' feed. The purpose of this study was to clarify how this regulation has affected the vitamin D content of commercial eggs and chicken meat. Another aim was to investigate how effectively 25-OH-D(3) is transferred from the hens' diet to egg yolk by analyzing eggs from farms using known commercial feeds and by conducting an animal study. Vitamin D determinations were made by HPLC methods. The vitamin D(3) contents of two commercial egg yolk pools were 4.9 ± 0.14 and 4.0 ± 0.10 μg/100 g, and the 25-OH-D(3) contents were 1.3 ± 0.19 and 1.0 ± 0.07 μg/100 g. The chicken meat pools contained 0.2-0.3 μg of vitamin D(3)/100 g, whereas the content of 25-OH-D(3) was ≤0.2 μg/100 g. These results are comparable to earlier data. The animal and farm studies showed that 25-OH-D(3) was effectively transferred from the hens' diet to yolk. However, because the relative activity between 25-OH-D(3) and vitamin D(3) is unknown, it remains questionable whether the use of 25-OH-D(3) in hens' feed is beneficial to human vitamin D intake from eggs.     

Liquid chromatographic determination of amprolium in poultry feed and premixes using postcolumn chemistry with fluorometric detection

Two extraction and liquid chromatographic procedures are presented which separate amprolium from compounds in poultry feed or premixes that could interfere with its fluorometric determination. The procedures are based on earlier work on the determination of thiamine in food samples. Amprolium is extracted from feed with a hexane-aqueous sulfosalicylic acid mix, separated on a C18 column, and detected fluorometrically after postcolumn derivatization. For premixes, water extraction is used. Values for the amprolium content of poultry feed obtained with these procedures are in good agreement with those obtained with AOAC official methods. It is suggested that these methods with suitable modifications may be of use for routine analysis of amprolium in feeds. The overall methods are rapid and appear to give reasonable results.     

An expression for wind erosion rate from dry and dense soil surface

Wind erosion is a physical process predominated by airborne grains rather than the wind itself. The soil deformation is either elastic–plastic or fully plastic during its collisions with solid grains, corresponding to dense and loose soils, respectively. Only the fully plastic deformation was previously taken into account in the physically-based wind erosion models. The impact erosion of dry and dense soil surface can be well quantified by the contact mechanics and strength theories. In this study, with the help of Owen's uniform saltation model, a simple expression for wind erosion rate is derived based upon the eroded volume per impact when soil deforms in the elastic–plastic manner. The new expression, a simple analytical function of several conventional variables and parameters of wind and soil, describes the effects of saltation flux and soil susceptibility. Its validity is verified by some field and laboratory experiments.     

Detection of beef and poultry by serological field screening tests (ORBIT and PROFIT): collaborative study

Ten laboratories each analyzed 30 raw meat and raw meat product samples in a collaborative study of the ORBIT (overnight rapid bovine identification test) and PROFIT (poultry rapid overnight field identification test) serological field screening tests for the detection of beef and poultry. Versatility of the tests was shown in the analysis of whole tissue, ground, or emulsified raw meat products. Both tests were demonstrated to be reliable and were capable of detecting adulterants present at the 10% level. The method has been adopted official first action.     

Simultaneous liquid chromatographic screening of five coccidiostats in chicken liver

A reverse-phase liquid chromatographic (LC) method is described for simultaneously determining 5 coccidiostats--aklomide, dinsed, ethopabate, nitromide, and zoalene in chicken liver. The method entails blender extraction of 10 g liver with ethyl acetate, column chromatography through Sephadex LH-20 and neutral alumina, and LC analysis on a C18 column with UV detection at 260 nm. The drugs were eluted from Sephadex with methanol-benzene (10 + 90), from alumina with methanol-dichloromethane (10 + 90), and from C18 with acetonitrile-water (linear gradient: 25% acetonitrile for 10 min, increasing to 55% over 15 min; flow rate 1 mL/min). Liquid chromatography was completed in 40 min and calculations were based on peak height measurements. Average recoveries of the coccidiostats from fortified liver ranged from 72 to 97%, except for dinsed, which showed a relatively constant average recovery of 57%. The detection limit for the standards was 2.5 ng on column. Levels as low as 50 ng/g were detected in fortified liver samples.     

Influence of mulch and poultry manure application on soil temperature,evapotranspiration and water use efficiency of dry season cultivated okra

Sustainable vegetable production especially during the dry season requires adequate conservation of soil water. This study was conducted to evaluate the sole and interactive effects of mulching (M) and poultry manure (PM) application on soil temperature (ST), crop evapotranspiration (ETc) and water use efficiency (WUE) of okra. The experiment was a Randomized Complete Block Design (RCBD) with three replicates. The treatments were M at 0 and 6 t ha⁻¹ and PM at 0, 10 and 20 t ha⁻¹. Soil temperature was measured using digital thermometer while ETc was determined by water depletion method using a Time Domain Reflectometer. Irrigation at field capacity was applied manually at 2-day intervals. Independent application of mulch significantly lowered ST while joint application of 20 t ha⁻¹ PM (PM20) and M significantly (p ≤ 0.05) reduced ST at 5 cm and 10 cm soil depth compared with the unmulched plots in both seasons. Application of 10 t ha⁻¹ PM (PM10) without M recorded the highest ETc (43.7 mm), while joint application of PM20 and M reduced ETc by about 93% compared with PM10 only. Okra used water most efficiently when PM20 was applied under mulched plot. There was 62.2% increase in WUE under mulched plots compared with the control while the residual effect of PM10 and M significantly increased WUE by 65.5%. It was evident that M alongside application of PM is a good strategy for regulating ST, moderating ETc and increasing okra WUE, especially during dry season farming.