Enzyme immunoassay for semicarbazide - The nitrofuran metabolite and food contaminant

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed-all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC beta) of 0.25 mu g kg(-1) when a threshold of 0.21 mu g kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 mu g kg(-1) fortified sample, n = 20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 mu g kg(-1). N-FZ-incurred muscles (12) containing SEM at 0.5-5.0 mu g kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver. (C) 2007 Elsevier BN. All rights reserved.

Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean+3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 mug kg(-1). Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 mug kg(-1). The detection capability (CCbeta), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 mug kg(-1). Incurred prawn samples (n=8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 mug kg(-1) were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 mug kg(-1) and a CCbeta of below 0.4 mug kg(-1).

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 µg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 µg/kg, respectively.

Monoclonal antibody-based ELISA for the quantification of nitrofuran metabolite 3-amino-2-oxazolidinone in tissues using a simplified sample preparation

A sensitive and specific monoclonal ELISA for the determination of tissue bound furazolidone metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedure enables the detection of AOZ in matrix supernatant after homogenisation, protease treatment, acid hydrolysis and derivatisation of AOZ released from the tissue by o-nitrobenzaldehyde. The formed p-nitrophenyl 3-amino-2-oxazolidinone (NPAOZ) is determined by ELISA calibrated with matrix-matched standards in the concentration range of 0.05-5.0 mu g l(-1). The assay was validated according to criteria set down by Commission Decision 2002/657/EC for the performance and validation of analytical methods for chemical residues. Detection capability, set on the basis of acceptance of no false negative results, was 0.4 mu g kg(-1) for shrimp, poultry, beef and pork muscle. This sensitivity approaches the established confirmatory LC-MS/MS able to quantify tissue-bound AOZ at levels as low as 0.3 mu g kg(-1). An excellent correlation of results obtained by ELISA and LC/MS-MS within the concentration range 0-32.1 mu g kg(-1) was found in the naturally contaminated shrimp samples (r = 0.999, n = 8). A similar con-elation was found for the incurred poultry samples within the concentration range of 0-10.5 mu g kg(-1) (r = 0.99, n = 8). (c) 2005 Elsevier B.V All rights reserved.

The indirect effects of biofuels and what to do about them:The case of grass biomethane and its impact on livestock

Grass biomethane surpasses the 60% greenhouse gas (GHG) savings relative to the fossil fuel replaced required by EU Directive 2009/28/EC. However, there are growing concerns that when the indirect effects of biofuels are taken into account, GHG savings may become negative. There has been no research to date into the indirect effects of grass biomethane; this paper aims to fill that knowledge gap. A causal-descriptive assessment is carried out and identifies the likely indirect effect of a grass biomethane industry in Ireland as a reduction in beef exports to the UK. Three main scenarios are then analyzed: an increase in indigenous UK beef production, an increase in beef imported to the UK from other countries (EU, New Zealand and Brazil), and a decrease in beef consumption leading to increased poultry consumption. The GHG emissions from each of these scenarios are determined and the resulting savings relative to fossil diesel vary between -636% and 102%. The significance of the findings is then discussed. It is the view of the authors that, while consideration of indirect effects is important, an Irish grass biomethane industry cannot be held accountable for the associated emissions. A global GHG accounting system is therefore proposed; however, the difficulty of implementing such a system is acknowledged, as is its probable ineffectualness. Such a system would not treat the source of the problem - rising consumption. The authors conclude that the most effective method of combating the indirect effects of biofuels is a reduction in general consumption. © 2011 Society of Chemical Industry and John Wiley & Sons, Ltd.

Welfare risk assessment:The benefits and common pitfalls

Risk is defined as a situation involving exposure to danger. Risk assessment by nature characterises the probability of a negative event occurring and quantifies the consequences of such an event. Risk assessment is increasingly being used in the field of animal welfare as a means of drawing comparisons between multiple welfare problems within and between species and identifying those that should be prioritised by policy-makers, either because they affect a large proportion of the population or because they have particularly severe consequences for those affected. The assessment of risk is typically based on three fundamental factors: intensity of consequences, duration affected by consequences and prevalence. However, it has been recognised that these factors alone do not give a complete picture of a hazard and its associated consequences. Rather, to get a complete picture, it is important to also consider information about the hazard itself: probability of exposure to the hazard and duration of exposure to the hazard. The method has been applied to a variety of farmed species (eg poultry, dairy cows, farmed fish), investigating housing, husbandry and slaughter procedures, as well as companion animals, where it has been used to compare inherited defects in pedigree dogs and horses. To what extent can we trust current risk assessment methods to get the priorities straight? How should we interpret the results produced by such assessments? Here, the potential difficulties and pitfalls of the welfare risk assessment method will be discussed: (i) the assumption that welfare hazards are independent; (ii) the problem of quantifying the model parameters; and (iii) assessing and incorporating variability and uncertainty into welfare risk assessments. © 2012 Universities Federation for Animal Welfare.

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

Balteatide: A Novel Antimicrobial Decapeptide from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically-active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families, the dermatoxins, phylloxins, plasticins, distinctins and the medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus, the Gram-negative bacterium, Escherichia coli and the yeast, Candida albicans, and unusually for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

Session 4: CVD, diabetes and cancer - Evidence for the use of the Mediterranean diet in patients with CHD

Diet is associated with the development of CHD. The incidence of CHD is lower in southern European countries than in northern European countries and it has been proposed that this difference may be a result of diet. The traditional Mediterranean diet emphasises a high intake of fruits, vegetables, bread, other forms of cereals, potatoes, beans, nuts and seeds. It includes olive oil as a major fat source and dairy products, fish and poultry are consumed in low to moderate amounts. Many observational studies have shown that the Mediterranean diet is associated with reduced risk of CHD, and this result has been confirmed by meta-analysis, while a single randomised controlled trial, the Lyon Diet Heart study, has shown a reduction in CHD risk in subjects following the Mediterranean diet in the secondary prevention setting. However, it is uncertain whether the benefits of the Mediterranean diet are transferable to other non-Mediterranean populations and whether the effects of the Mediterranean diet will still be feasible in light of the changes in pharmacological therapy seen in patients with CHD since the Lyon Diet Heart study was conducted. Further randomised controlled trials are required and if the risk-reducing effect is confirmed then the best methods to effectively deliver this public health message worldwide need to be considered.

Ecology of testate amoebae in an Amazonian peatland and development of a transfer function for palaeohydrological reconstruction

Tropical peatlands represent globally important carbon sinks with a unique biodiversity and are currently threatened by climate change and human activities. It is now imperative that proxy methods are developed to understand the ecohydrological dynamics of these systems and for testing peatland development models. Testate amoebae have been used as environmental indicators in ecological and palaeoecological studies of peatlands, primarily in ombrotrophic Sphagnum-dominated peatlands in the mid- and high-latitudes. We present the first ecological analysis of testate amoebae in a tropical peatland, a nutrient-poor domed bog in western (Peruvian) Amazonia. Litter samples were collected from different hydrological microforms (hummock to pool) along a transect from the edge to the interior of the peatland. We recorded 47 taxa from 21 genera. The most common taxa are Cryptodifflugia oviformis, Euglypha rotunda type, Phryganella acropodia, Pseudodifflugia fulva type and Trinema lineare. One species found only in the southern hemisphere, Argynnia spicata, is present. Arcella spp., Centropyxis aculeata and Lesqueresia spiralis are indicators of pools containing standing water. Canonical correspondence analysis and non-metric multidimensional scaling illustrate that water table depth is a significant control on the distribution of testate amoebae, similar to the results from mid- and high-latitude peatlands. A transfer function model for water table based on weighted averaging partial least-squares (WAPLS) regression is presented and performs well under cross-validation (r 2apparent=0.76,RMSE=4.29;r2jack=0.68,RMSEP=5.18. The transfer function was applied to a 1-m peat core, and sample-specific reconstruction errors were generated using bootstrapping. The reconstruction generally suggests near-surface water tables over the last 3,000 years, with a shift to drier conditions at c. cal. 1218-1273 AD

Secular trends in reported portion size of food and beverages consumed by Irish adults

The present analysis aimed to investigate the changes in the reported portion sizes (PS) of foods and beverages commonly consumed by Irish adults (18-64 years) from the North South Ireland Food Consumption Survey (NSIFCS) (1997-2001) and the National Adult Nutrition Survey (NANS) (2008-10). Food PS, which are defined as the weight of food (g) consumed per eating occasion, were calculated for comparable foods and beverages in two nationally representative cross-sectional Irish food consumption surveys and were published in NSIFCS and NANS. Repeated measure mixed model analysis compared reported food PS at the total population level as well as subdivided by sex, age, BMI and social class. A total of thirteen commonly consumed foods were examined. The analysis demonstrated that PS significantly increased for five foods ('white sliced bread', 'brown/wholemeal breads', 'all meat, cooked', 'poultry, roasted' and 'milk'), significantly decreased for three ('potatoes', 'chips/wedges' and 'ham, sliced') and did not significantly change for five foods ('processed potato products', 'bacon/ham', 'cheese', 'yogurt' and 'butter/spreads') between the NSIFCS and the NANS. The present study demonstrates that there was considerable variation in the trends in reported food PS over this period.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Apparent metabolizable energy from microalgae spirulina maxima for broiler chickens.

2016

Broiler chicken ileal apparent digestibility of calcium and phosphorus from microalgae spirulina maxima.

2016