Characterization of Beef Coming from Different European Countries through Stable Isotope (H, C, N, and S) Ratio Analysis.

The need to guarantee the geographical origin of food samples has become imperative in recent years due to the increasing amount of food fraud. Stable isotope ratio analysis permits the characterization and origin control of foodstuffs, thanks to its capability to discriminate between products having different geographical origins and derived from different production systems. The Framework 6 EU-project "TRACE" generated hydrogen (2H/1H), carbon (13C/12C), nitrogen (15N/14N), and sulphur (34S/32S) isotope ratio data from 227 authentic beef samples. These samples were collected from a total of 13 sites in eight countries. The stable isotope analysis was completed by combining IRMS with a thermal conversion elemental analyzer (TC/EA) for the analysis of δ(2H) and an elemental analyzer (EA) for the determination of δ(13C), δ(15N), and δ(34S). The results show the potential of this technique to detect clustering of samples due to specific environmental conditions in the areas where the beef cattle were reared. Stable isotope measurements highlighted statistical differences between coastal and inland regions, production sites at different latitudes, regions with different geology, and different farming systems related to the diet the animals were consuming (primarily C3- or C4-based or a mixed one).

Determining the geographical origin of Asian longhorn beetle (Anoplophora glabripennis) specimens using stable isotope and trace element analyses.

BACKGROUND: An outbreak of EU-quarantine-listed pest Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambicidae), the Asian longhorn beetle, in Kent (UK) resulted in environmentally and financially costly eradication action being taken. In this study the potential of using multi-element stable isotope or trace element analyses to determine the geographical origin of individual specimens has been investigated. RESULTS: The isotope ratios of A. glabripennis individuals for hydrogen varied within and across five locations. Carbon isotope ratios fell within the expected values for C3 plants (trees using the photosynthetic pathway common for moderate climates). Nitrogen isotope ratios indicated separation of UK laboratory from US (New York, Ohio, Massachusetts) beetles, while sulphur isotope ratios distinguished beetles from New York against the other four locations. Three trace elements (TEs) separated UK laboratory-reared beetles from US beetles (Ohio and New York) with ∼ 68% confidence. CONCLUSIONS: Stable isotope and TE analyses show potential to differentiate between newly arrived A. glabripennis individuals and those from previously undetected in-country populations, which would be of immediate practical benefit in making appropriate strategic decisions on surveillance and eradication. Analyses of additional samples from (i) the same populations, (ii) different locations and (iii) a variety of host trees will enhance the overall picture. © 2016 Crown copyright. Pest Management Science © 2016 Society of Chemical Industry.

Detection of antibiotic residues and association of cefquinome residues with the occurrence of Extended-Spectrum ß-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011.

Waste milk samples from 103 farms in England and Wales were examined for the presence of ß-lactam antibiotics and ESBL-producing Enterobacteriaceae. Approximately 10 months after the initial sampling, further waste milk, environmental and faecal samples from farms shown to be positive for CTX-M Escherichia coli were investigated further. Isolates with an ESBL phenotype were tested by PCR for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes. Isolates positive for blaCTX-M were sequenced to determine CTX-M type. Representative isolates were further examined by PFGE, plasmid replicon typing and serotyping. Of particular interest, 21.4% of waste milk samples contained residues of the cephalosporin cefquinome, which was significantly associated with CTX-M bacteria. Such bacteria occurred in 5.8% of the waste milk samples (including 3.9% CTX-M E. coli). CTX-M types identified were 1, 14, 14b and 15, but none of the E. coli were serotype O25, the serotype of the human pandemic strain.

An inter-laboratory validation of a multiplex dipstick assay for four classes of antibiotics in honey.

In this paper, we report the inter-laboratory validation (ILV) of a recently developed indirect competitive multiplex dipstick (Bee4sensor®) which is capable of the simultaneous detection of residues of some of the most frequently detected antibiotic residues in honey: sulfonamides, tylosin, fluoroquinolones and chloramphenicol. The multi-sensor dipstick can be interpreted via visual observation or by an instrumental measurement of four test lines. Statistical analysis of the ILV data demonstrated that the multi-sensor can reliably detect the presence of sulfathiazole at 25 µg kg(-1) and tylosin at 10 µg kg(-1), which fully meet the 'recommended concentrations' of the EU. Ciprofloxacin and chloramphenicol can be detected at 25 and 5 µg kg(-1) in honey, respectively. Whilst the concentration for chloramphenicol is above the EU minimum required performance limit of 0.3 µg kg(-1), this part of the multiplex test may still be of use to both the industry and enforcement authorities, to provide an early warning of contaminated honey. The estimated false-negative and false-positive rates for this easy-to-use and robust assay were less than 5%.

Can the unauthorised use of ceftiofur be detected in poultry?

Ceftiofur is a third-generation cephalosporin antibiotic used to treat cattle and swine for bacterial infection of the respiratory tract. It is not authorised for use in poultry within the European Union. Due to the complexity of the chemistry and metabolism of ceftiofur, maximum residue limits (MRLs) are based on desfuroylceftiofur (DFC) equivalents after chemical conversion of all compounds that have an intact ß-lactam ring. In practice the DFC is usually stabilised as the acetamide (desfuroylceftiofur acetamide - DFCA) for analysis. Because of recent evidence of off-label use in the European Union, a policy need emerged to develop a cost-effective method for the detection of ceftiofur residues in poultry tissues. One-day-old chicks were each dosed subcutaneously with ceftiofur and samples taken from day 1 to day 44 post-dosing. Residues of ceftiofur parent compound were detected in whole chicks, wing feathers and faeces. On the basis of this finding it was decided to evaluate ceftiofur parent, as the marker, instead of proceeding with the time-consuming conversion to DFCA. Expected metabolites, DFC and desfuroylceftiofur cysteine disulfide (DCCD), were not detected in whole chicks, muscle or liver, but DFC was found in wing feathers. These results indicate that determination of ceftiofur parent compound in either whole chicks or possibly wing feathers and faeces may allow the detection of the misuse of ceftiofur.

Investigation into the experimental protocols required to determine maximum residue limits (MRLs) in honey.

There is current debate within the EU, and internationally, on how withdrawal periods and maximum residue limits (MRLs) may be set for honey production. Whilst comprehensive EU guidelines exist for calculating the withdrawal times of veterinary medicines in most food-producing species, the analytical variables to be studied for bees/honey are not well defined. The objective of this study was therefore to investigate and understand the factors, for example sampling variability, that is important in the development of a harmonized protocol that can be used to generate the robust scientific data necessary to assist risk assessors in proposing MRLs for honey. Ten bee colonies were treated in the spring with a model compound (ciprofloxacin). One hive was used to study intra-hive variation in residue concentrations and the other nine were used in an inter-hive study over a 41-week sampling period. All samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The highest mean concentration from nine hives used in the inter-hive study was 4627 µg/kg eight days (D8) after treatment. The concentration of ciprofloxacin declined to an average concentration of 1756 µg/kg at D30 and 733 µg/kg at D283 (over-winter sample). A generalized additive model was used to fit a smooth curve for trend estimation. For some individual hives the concentration of ciprofloxacin increased slightly at the later sampling time-points. Consequently it was not possible to interpolate, with confidence, a finite withdrawal period for ciprofloxacin at theoretical MRLs between 25 and 500 µg/kg. The observed variation in concentration of ciprofloxacin between hives indicates that the validity of the EU guideline for bees/honey, which requires five samples from five hives to calculate a withdrawal period, may require revision.

Determining the source of house flies (Musca domestica) using stable isotope analysis.

BACKGROUND: Intensive livestock units frequently produce flies in large numbers that, on migration, cause nuisance to the occupants of neighbouring dwellings. The resolution of such problems is often reliant on the unequivocal identification of the origin of the flies, particularly when several potential sources exist. This study evaluated stable isotope analysis as a method for differentiating adult houseflies (Musca domestica) on the basis of their dietary history so as to determine their likely source. RESULTS: Flies were reared in the laboratory on several substrates, including chicken and cattle manure, laboratory diet and household vegetable waste. Different fly parts (wings, heads and legs) and whole flies were analysed immediately after eclosion and after 10 days. The δ(13) C and δ(15) N values for adults that had developed on each diet type were highly distinct. Both isotopic ratios altered markedly after maintaining the flies for 10 days on a diet of cane sugar solution. CONCLUSIONS: Stable isotope analysis readily differentiated flies that had developed on a range of substrates. The technique, therefore, shows potential to be employed to determine the likely source of various nuisance insects, and to contribute to the abatement of such problems.

The impact of EGFR stimulation and inhibition on BPDE induced DNA fragmentation in oral/oropharyngeal mucosa in vitro.

Still, the vast majority of head and neck squamous cell carcinoma (HNSCC) can be linked to the "traditional" risk factors tobacco smoke and alcohol consumption. These tumors are believed to be the results of multiple years of carcinogenic impact on upper aerodigestive tract mucosa. The frequent observation, that one patient suffers from several synchronous cancers, multiple local recurrences, and second primary tumors led to the concept of field cancerization, first introduced by Slaughter and colleagues in 1953. As underlying molecular events, genetic instability, loss of heterozygosity, amplification, deletion, up- and down-regulation of oncogenes and/or tumor suppressor genes were revealed. One of the best studied oncogenic features of head and neck carcinogenesis are high expression levels of epidermal growth factor receptor (EGFR). Enhanced expression of the receptor was detected in histologically normal mucosa from HNSCC patients and increasing levels during the progress from hyperplasia to dysplastic lesion and invasive carcinoma were demonstrated. Whereas nearly all of our knowledge about EGFR biology in HNSCC is based on preclinical and clinical studies investigating receptor inhibitors, little is known about cause and function of EGFR in premalignant mucosa. In this study we show, that EGFR stimulation significantly decreases carcinogen induced DNA damage in normal mucosa from HNSCC patients and that this effect is completely abrogated adding an anti-EGFR antibody before stimulation, while there was no effect in non-tumor controls. The effect of EGFR inhibition was contrary. In non-tumor controls, blocking the receptor with an antibody significantly decreased DNA damage, whereas in cases no effect was seen. Our results indicate an important role of the receptor during chemical carcinogenesis. On the basis of this study we suppose, that increasing EGFR levels during head and neck carcinogenesis can be interpreted as a physiological response to permanent carcinogen impact on the mucosa.

Isotopic and elemental data for tracing the origin of European olive oils.

H, C, and O stable isotope ratios and the elemental profile of 267 olive oils and 314 surface waters collected from 8 European sites are presented and discussed. The aim of the study was to investigate if olive oils produced in areas with different climatic and geological characteristics could be discriminated on the basis of isotopic and elemental data. The stable isotope ratios of H, C, and O of olive oils and the ratios of H and O of the relevant surface waters correlated to the climatic (mainly temperature) and geographical (mainly latitude and distance from the coast) characteristics of the provenance sites. It was possible to characterize the geological origin of the olive oils by using the content of 14 elements (Mg, K, Ca, V, Mn, Zn, Rb, Sr, Cs, La, Ce, Sm, Eu, U). By combining the 3 isotopic ratios with the 14 elements and applying a multivariate discriminant analysis, a good discrimination between olive oils from 8 European sites was achieved, with 95% of the samples correctly classified into the production site.

Application of stable carbon isotope analysis to the detection of testosterone administration to cattle.

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.