Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists), This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples, The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELISA results. Four calves were orally treated with RCT (0.1 mg kg(-1) body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli), High concentrations of RCT residues were found throughout the medication period (44-473 ng ml(-1); LC-MS-MS data) and remained present for several days following removal of the drug from the diet, RCT residues were no longer detectable 2 weeks after withdrawal, Good agreement (r(2) = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for both sets of analyses, The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal, However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

Detection of monensin residues in poultry liver using an enzyme immunoassay

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for the detection and quantification of monensin residues in liver has been developed, Samples (3 g) were extracted with acetonitrile-water and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised against a monensin-transferrin conjugate, The limit of detection (mean + 3s) calculated from the analysis of 12 known negative samples was 2.91 ng g(-1). Intra- and inter-assay RSD were determined as 8.5 and 10.6%, respectively, using a liver sample fortified with 20 ng g(-1) monensin, A pharmacokinetic study in which 70 six week old broilers were fed monensin at a rate of 120 mg kg(-1) in their feed for 14 d resulted in mean monensin liver residues of 102 ng g(-1). However these had fallen below the limit of detection of the assay within the 3 d withdrawal period recommended by the manufacturer.

PATHOLOGY AND RESIDUES IN VEAL CALVES TREATED EXPERIMENTALLY WITH CLENBUTEROL

Six veal calves were medicated with clenbuterol at 20 mu g kg bodyweightl day(-1) for 42 days before they were slaughtered, to evaluate the lesions and residues in target organs. Compared with six unmedicated calves the most noticeable changes were tracheal dilatation, decreased uterine weight, slight mucous hypersecretion in the uterus and vagina and depletion of liver glycogen. The highest concentrations of clenbuterol (62 to 128 ng/g(-1)) were recorded in the choroid/retina, and the aqueous humour had the lowest concentration (0.5 to 2.4 ng ml(-1)). The residue concentrations were higher than the maximum residue level set for clenbuterol (0.5 ng g(-1))

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

TISSUE RESIDUES IN PIGS FED ON MEAL CONTAMINATED WITH SULFADIMIDINE DURING MIXING

Two equal batches of pig feed were prepared sequentially in a half-ton mixer. The first was prepared to contain 100-mu-g/g sulphadimidine and the second had not added sulphadimidine. The medicated batch was shown to have 97.2-mu-g/g and the flush 2-mu-g/g. From the conditions of the experiment it was suggested that sulphadimidine was preferentially retained in the mixer.

Interaction strengths in food webs: issues and opportunities

1. Recent efforts to understand how the patterning of interaction strength affects both structure and dynamics in food webs have highlighted several obstacles to productive synthesis. Issues arise with respect to goals and driving questions, methods and approaches, and placing results in the context of broader ecological theory.

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

Diclofenac residues in carcasses of domestic ungulates available to vultures in India

Gyps vulture populations across the Indian subcontinent are declining rapidly and evidence indicates that veterinary use of the non-steroidal anti-inflammatory drug (NSAID) diclofenac is the major cause. Exposure of vultures to diclofenac is likely to arise from the consumption of livestock carcasses that have been treated shortly before death, however, detailed information regarding the prevalence and residual levels of diclofenac in carcasses available to vultures in India remains unreported. Here, we present data on diclofenac residues in 1848 liver samples taken from carcasses of dead livestock sampled at 67 sites in 12 states within India, between May 2004 and July 2005. Diclofenac residues were detected in carcasses in all states except Orisa, where only one site was sampled. The overall prevalence of detectable diclofenac (>10 microg kg(-1)) across all states was 10.1% and varied significantly among states, with up to 22.3% prevalence determined in Bihar. The geometric mean concentration of diclofenac found in samples in which the drug was detected was 352 microg kg(-1). The prevalence of carcasses containing diclofenac is similar to that previously proposed to be required to have caused the observed Gyps vulture declines in India. On the 11th of May 2006, the Drug Controller General (India) ordered the withdrawal of all licenses granted for the manufacture of diclofenac for veterinary use within India. However, if Gyps vultures are to be protected, potentially substantial existing stocks now need to be quickly and effectively removed from the Indian veterinary market.

Arsenic residues in predatory birds from an area of Britain with naturally and anthropogenically elevated arsenic levels

Birds of prey forage over large areas and so might be expected to accumulate contaminants which are elevated but heterogeneously distributed in the general environment. The aim of this study was to test the hypothesis that arsenic levels in raptors from a region with elevated environmental arsenic concentrations were higher than those in birds from an uncontaminated part of Britain. Arsenic concentrations in the liver, kidney and muscle of kestrels, Falco tinnunculus, sparrowhawks, Accipiter nisus, and barn owls, Tyto alba, from south-west (SW) England, an area with naturally and anthropogenically (through mining) elevated environmental arsenic concentrations, were compared with those in birds from SW Scotland, where no such geochemical anomaly exists. Arsenic residues in kestrels from SW England were approximately three times greater than those in birds from SW Scotland for the three tissue types analysed. This was not the case for the other species in which arsenic residues were similar in birds from both regions. It is suggested that differences between species in both diet and arsenic metabolism could explain why kestrels have elevated arsenic tissue burdens in response to general environmental contamination but sparrowhawks and barn owls do not.