Integrating High throughput Sequencing into Survey Design Reveals Turnip Yellows Virus and Soybean Dwarf Virus in Pea (Pisum Sativum) in the United Kingdom.

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Variation in Inspection Efficacy by Member States of Wood Packaging Material Entering the European Union.

The use of wood packaging materials (WPMs) in international trade is recognized as a pathway for the movement of invasive pests and as the origin of most introductions of Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) in Europe and North America. Following several pest interceptions on WPM associated with stone imports from China, the European Union (EU) agreed to survey certain categories of imports based on the EU Combined Nomenclature Codes for imports, which are based on the international Harmonized System. Between April 2013 and March 2015, 72,263 relevant consignments were received from China in the EU and 26,008 were inspected. Harmful organisms were detected in 0.9% of the consignments, and 1.1% of the imports did not have markings compliant with the international standard for treating WPM, ISPM 15. There were significant differences between the detection rates of harmful organisms among EU member states. In member states that inspected at least 500 consignments, the rate of detection ranged from 6.9% in Austria and France to 0.0% in Spain and Poland. If this difference in detection rate is the result of differences in the methods and intensity of inspection in different member states then an approximate sevenfold increase in the interception of harmful organisms may be achieved if all states were to achieve detection rates achieved by Austria and France. The EU data from 1999 to 2014 indicated an increasing number of interceptions of Bostrichidae and Cerambycidae since 2010. This study demonstrates that there is an ongoing threat of non-native forest pests being imported on WPM.

Abundance of badgers (Meles meles) in England and Wales.

The European badger (Meles meles) is of considerable interest in the UK as it is both a protected species and the main wildlife reservoir for bovine tuberculosis infection in cattle. While there have been three national badger surveys in the 1980s, 1990s and 2011-13, using the number of badger main setts as a proxy for the abundance of badger social groups, none has combined contemporary data on social group size at landscape and national scales. We estimated social group size by genotyping hair samples collected at 120 main setts across England and Wales and employing a capture-mark-recapture method based on genotypes. The estimated mean social group size in England and Wales was 6.74 (±0.63) badgers. There was considerable variation in badger social group size among Land Class Groups (LCGs), with a low of 2.67 in LCG3 and a high of 7.92 in LCG4. Combining these results with the recent Badger Sett Survey of England and Wales, we estimate there are approximately 485,000 badgers (95% confidence intervals 391,000-581,000) in England and Wales. Although direct comparison with previous estimates is not ideal owing to methodological differences, our results are consistent with a marked increase in the badger population of England and Wales since the 1980s.

Variability of residue concentrations of ciprofloxacin in honey from treated hives.

Honey bees (Apis mellifera L.) were treated with a model veterinary drug compound (ciprofloxacin) in a 3-year study (2012-14) to investigate the variability of residue concentration in honey. Sucrose solution containing ciprofloxacin was administered to 45 hives (1 g of ciprofloxacin per hive) at the beginning of the honey flow in late May/mid-June 2012, 2013 and 2014. Buckfast honey bees (A. mellifera - hybrid) were used in years 2012 and 2013. Carniolan honey bees (A. mellifera carnica) were used instead of the Buckfast honey bees as a replacement due to unforeseen circumstances in the final year of the study (2014). Honey was collected over nine scheduled time points from May/June till late October each year. Up to five hives were removed and their honey analysed per time point. Honey samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine ciprofloxacin concentration. Statistical assessment of the data shows that the inter-hive variation of ciprofloxacin concentrations in 2012/13 is very different compared with that of 2014 with relative standard deviations (RSDs) of 138% and 61%, respectively. The average ciprofloxacin concentration for 2014 at the last time point was more than 10 times the concentration compared with samples from 2012/13 at the same time point. The difference between the 2012/13 data compared with the 2014 data is likely due to the different type of honey bees used in this study (2012/13 Buckfast versus 2014 Carniolan). Uncertainty estimates for honey with high ciprofloxacin concentration (upper 95th percentile) across all hives for 55-day withdrawal samples gave residual standard errors (RSEs) of 22%, 20% and 11% for 2012, 2013 and 2014, respectively. If the number of hives were to be reduced for future studies, RSEs were estimated to be 52% (2012), 54% (2013) and 26% (2014) for one hive per time point (nine total hives).

Carrot yellow leaf virus is associated with carrot internal necrosis.

Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots.

Density and abundance of badger social groups in England and Wales in 2011-2013.

In the United Kingdom, European badgers Meles meles are a protected species and an important wildlife reservoir of bovine tuberculosis. We conducted a survey of badger dens (main setts) in 1614 1 km squares across England and Wales, between November 2011 and March 2013. Using main setts as a proxy for badger social groups, the estimated mean density of badger social groups in England and Wales was 0.485 km(-2) (95% confidence interval 0.449-0.521) and the estimated abundance of social groups was 71,600 (66,400-76,900). In the 25 years since the first survey in 1985-88, the annual rate of increase in the estimated number of badger social groups was 2.6% (2.2-2.9%), equating to an 88% (70-105%) increase across England and Wales. In England, we estimate there has been an increase of 103% (83-123%) in badger social groups, while in Wales there has been little change (-25 to +49%).

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%.

Construction of measurement uncertainty profiles for quantitative analysis of genetically modified organisms based on interlaboratory validation data.

A method is presented for estimating the size of uncertainty associated with the measurement of products derived from genetically modified organisms (GMOs). The method is based on the uncertainty profile, which is an extension, for the estimation of uncertainty, of a recent graphical statistical tool called an accuracy profile that was developed for the validation of quantitative analytical methods. The application of uncertainty profiles as an aid to decision making and assessment of fitness for purpose is also presented. Results of the measurement of the quantity of GMOs in flour by PCR-based methods collected through a number of interlaboratory studies followed the log-normal distribution. Uncertainty profiles built using the results generally give an expected range for measurement results of 50-200% of reference concentrations for materials that contain at least 1% GMO. This range is consistent with European Network of GM Laboratories and the European Union (EU) Community Reference Laboratory validation criteria and can be used as a fitness for purpose criterion for measurement methods. The effect on the enforcement of EU labeling regulations is that, in general, an individual analytical result needs to be < 0.45% to demonstrate compliance, and > 1.8% to demonstrate noncompliance with a labeling threshold of 0.9%.

Generic approach to validation of small-molecule LC-MS/MS biomarker assays.

BACKGROUND: While the regulatory guidelines that describe the validation requirements for small molecules are very comprehensive, they are written primarily for xenobiotic drug molecules. However, the presence of endogenous analyte in control matrix presents an added analytical challenge that must be overcome if small-molecule biomarker assays are to be developed and characterized, especially where downregulation of analyte concentrations is expected. EXPERIMENTAL: A generic surrogate matrix calibration protocol has been successfully applied to the measurement of a number of small-molecule exploratory biomarkers using LC-MS/MS. The use of analyte-free matrix enables conventional calibration curves to be constructed across the anticipated range of sample concentrations. The evaluation of matrix effects is carried out using an experiment similar to the parallelism experiment used in ligand-binding assays. CONCLUSION: There is currently no published consensus approach to validation of small-molecule biomarker methods. This paper presents a generic approach to endogenous method validation for consideration as bioanalytical best practice for this type of assay.

Simultaneous determination of six major ergot alkaloids and their epimers in cereals and foodstuffs by LC-MS-MS.

This paper describes a new and rapid method for accurate quantification of the six ergot alkaloids, ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The six ergot alkaloids studied have been defined by the European Food Safety Authority (EFSA) as among the most common and physiologically active ones. In addition, the method enables the quantification of the corresponding six epimers (ergo-inines) of these ergot alkaloids. This is of considerable importance in terms of the differences in toxicity of the isomeric forms. The method involves extraction under alkaline conditions using a mixture of acetonitrile and ammonium carbonate buffer followed by a rapid clean-up using dispersive solid-phase extraction with PSA (primary secondary amine) and a short chromatographic LC-run (21 min) with subsequent MS-MS detection. The method was developed and validated using ten different cereal and food samples. The major strength of the new method compared with previously published techniques is the simplicity of the clean-up procedure and the short analysis time. The limits of quantification were 0.17 to 2.78 µg kg(-1) depending on the analyte and matrix. Recovery values for the 12 ergot alkaloids spiked into ten different matrices at levels of 5, 50, and 100 µg kg(-1) were between 69 and 105% for 85 of 90 recovery measurements made over six days. Measurement uncertainty values were highly satisfactory. At a concentration level of 5 µg kg(-1) the expanded measurement uncertainty ranged from ±0.56 to ±1.49 µg kg(-1), at a concentration level of 100 µg kg(-1) the expanded measurement uncertainty ranged from ±8.9 to ±20 µg kg(-1). Both LOQs and measurement uncertainties were dependent on the analyte but almost independent of the matrix. The method performance was satisfactory when tested in a mini-intercomparison study between three laboratories from three different countries.

Measurement uncertainty associated with sample processing of oranges and tomatoes for pesticide residue analysis.

The homogeneity of analytical samples and the stability of pesticides during the sample processing of oranges and tomatoes were evaluated. The mean concentrations of 14C-labeled chlorpyrifos in analytical portions (subsamples) after processing show that homogeneity is dependent on sample type as well as the processing procedure. The homogeneity of analytical samples of tomatoes processed cryogenically was much better than those processed at ambient temperature. For tomatoes, the minimum analytical portion masses required for between-analytical portion variation of < 0.3 Ho were 110 and 5 g for processing at ambient and cryogenic temperatures, respectively. Results for orange showed that analytical portion sizes of 5 g provided sufficient homogeneity from both sample processing procedures. Assessments of pesticide stability demonstrated that most were relatively stable during processing at either ambient or cryogenic temperatures. However, some pesticides, including dichlofluanid, chlorothalonil, tolylfluanid, and dicloran, appeared to suffer much greater losses (>20%) during processing at ambient temperature. For these analytes, loss is interpreted as chemical degradation.

Statistical modelling as an aid to the design of retail sampling plans for mycotoxins in food.

A study has been carried out to assess appropriate statistical models for use in evaluating retail sampling plans for the determination of mycotoxins in food. A compound gamma model was found to be a suitable fit. A simulation model based on the compound gamma model was used to produce operating characteristic curves for a range of parameters relevant to retail sampling. The model was also used to estimate the minimum number of increments necessary to minimize the overall measurement uncertainty. Simulation results showed that measurements based on retail samples (for which the maximum number of increments is constrained by cost) may produce fit-for-purpose results for the measurement of ochratoxin A in dried fruit, but are unlikely to do so for the measurement of aflatoxin B1 in pistachio nuts. In order to produce a more accurate simulation, further work is required to determine the degree of heterogeneity associated with batches of food products. With appropriate parameterization in terms of physical and biological characteristics, the systems developed in this study could be applied to other analyte/matrix combinations.

Single laboratory validation of a method for the determination of hydroxymethylfurfural in honey by using solid-phase extraction cleanup and liquid chromatography.

A method is described for the determination of hydroxymethylfurfural (HMF) in honey. The method, which is based on solid-phase extraction cleanup followed by liquid chromatography (LC) with UV absorbance detection, was tested on a variety of different honey types: liquid, set, blended, filtered, crystalline, and comb honey. A sample of honey fortified with a known amount of HMF acted as an in-house reference material. LC with diode-array detection showed that the HMF peak did not contain any peaks of coeluting interfering species. Stability studies showed that honey samples should not be repeatedly frozen and thawed because the temperature changes caused a gradual increase in the HMF concentration. It was also shown that aqueous HMF standard solutions should be kept in the dark at 4 degrees C to avoid degradation of the HMF. The method was internally validated, and the measurement uncertainty was estimated to be +/-9.0 at 40 mg/kg, the legal limit. A comparison of the relative standard uncertainty with the Horwitz relative standard deviation showed that the method was suitable for its purpose and should be validated by a collaborative trial.

Method validation of resistive heating--gas chromatography with flame photometric detection for the rapid screening of organophosphorus pesticides in fruit and vegetables.

A rapid method for the screening of organophosphorus (OP) pesticides in fruit and vegetables is reported. Sample extracts were analysed using resistive heating-gas chromatography (RH-GC) with flame photometric detection (FPD). A CarboFrit insert in the GC liner allowed injection of crude extracts onto the GC system. Separation of up to 20 pesticides was achieved in 4.3 min with excellent retention time stability. Signal-to-noise ratios of 5:1 or better were obtained for the majority of the pesticides at the lowest calibrated level (LCL), 0.01 microg ml(-1), with excellent linearity over the range 0.01-0.5 microg ml(-1) (0.004-0.2 mg kg(-1) equivalent). Average recoveries between 70 and 116% were obtained for pesticides spiked at 0.01 and 0.1 mg kg(-1) with associated R.S.D. values < or =20% in the majority of cases. Estimates of relative reproducibility standard deviation (R.S.D.(R)), made by combining observed R.S.D. values with estimates of uncertainty associated with mean recovery allowed the determination of HORRAT values which confirmed that the method is capable of producing results which are fit for purpose. The validated method was then used to screen peaches, grapes and sweet peppers for a total of 37 pesticides. Incurred residue results obtained using RH-GC-FPD were in good agreement with the results from analysis of the same samples using MS confirmation.

Application of uncertainty analysis in assessing dietary exposure.

Conventional approaches for assessing dietary exposure to contaminants and additives in food are deterministic, using point estimates for consumption and contamination. In reality, both consumption and contamination are variable. Furthermore our knowledge of them is uncertain, e.g. due to measurement uncertainty. Conventional approaches attempt to allow for this by using worst-case assumptions or safety factors, but these are often subjective and may result either in overestimation or underestimation of the true range of exposures. Probabilistic approaches take account of variability and uncertainty by using distributions rather than point estimates for consumption and contamination. The outputs are distributions for exposure, which provide a more complete and balanced description of risk for the decision-maker. These approaches also facilitate the use of sensitivity analysis to identify those factors that impact most on exposure, and to identify areas of uncertainty where additional data will improve exposure estimates. This paper reviews examples of the application of these methods to the assessment of dietary exposure to food contaminants, including dioxins in seafood, where it was found that the greatest uncertainties relate to toxicity rather than exposure. Further work required to implement probabilistic approaches for dietary exposure assessment is discussed.