Commission Regulation 2021/808 sets performance criteria for methods for analysing pharmacologically active substances in food-producing animals. Due to the vast numbers of veterinary drugs and sample matrices, conventional re-validation of existing methods to this regulation is costly and disruptive to the delivery of routine analyses. This paper presents an alternative approach to method validation based on adding fortified samples to routine screening batches that is cost-effective and fits seamlessly within routine analytical workflows. The relationships between added concentration and each of three quantities: (1) mean measurement result, (2) the uncertainty of the mean result, and (3) the within laboratory reproducibility standard deviation are directly estimated by a maximum likelihood fit of a statistical model to measurement results. Hence, the model provides estimates of trueness, relative standard deviation and measurement uncertainty at concentrations of interest that are within the range of concentrations used in the model fit. The CCα (decision limit for confirmation) and CCβ (detection capability for screening) are calculated from estimates of measurement uncertainty. Because this approach uses data collected during routine analyses across a large number of analytical batches, rather than relying on specifically constructed validation studies, the resulting estimates of trueness and within-laboratory reproducibility better reflect real-world method performance. Estimates of performance do not rely on patterns of replication at specific concentrations or the availability of a particular number of results. Instead, the sufficiency of validation data is judged by the relative standard errors of estimates of within lab-reproducibility at concentrations of interest gained by a parametric bootstrap of the fitted statistical model. R script (a programming language used for statistical analysis) which demonstrates model fitting, performance parameter estimation and assessment of validation quality via a parametric bootstrap is provided here along with further scripts that demonstrate the method in two examples.

Phyllosticta citricarpa is the causal agent of citrus black spot (CBS), a disease affecting most citrus species and cultivars, particularly lemons (Citrus limon) and sweet oranges (Citrus sinensis). The pathogen causes fruit blemishes and premature fruit drop, leading to substantial yield and quality losses. Phyllosticta citricarpa is of global economic importance and is regulated by several citrus-growing countries, including those in the European Union (EU). The aim of this study was to develop a novel approach for early detection of P. citricarpa in environmental samples and to assess the status of P. citricarpa in selected citrus-growing areas of the EU where this species had previously been reported from leaf litter. Between 2018 and 2021, citrus plant material, air and rainwater samples were collected and analysed for the presence of Phyllosticta spp. in orchards in Greece, Italy, and Malta. Molecular analyses using qPCR assays targeting P. citricarpa were employed for the analysis of air and rainwater samples. The performance of the testing protocol was evaluated prior to sample analysis, and 1000 conidia were determined to be the minimum number of spores required for reliable detection. No CBS symptoms were observed, and no evidence of P. citricarpa was found in collected citrus plant material, air, or rainwater samples. The only Phyllosticta species detected during this study was the endophyte P. capitalensis, isolated from citrus leaf litter in Greece. These findings support the results of official EU surveys, which indicate the current absence of P. citricarpa in the surveyed areas and demonstrate the value of integrating environmental monitoring with molecular diagnostics for plant health surveillance. The methodologies developed here offer a robust framework for early warning and rapid response, supporting efforts to prevent CBS outbreaks in Europe and can be adapted for other fungal pathogens.

Antimicrobial Resistance (AMR), i.e., the evolution of microbes to become resistant to chemicals used to control them, is a global public health concern that can make bacterial diseases untreatable. Inputs including antibiotics, metals, and biocides can create an environment in the agrifood chain that selects for AMR. Consumption of food represents a potential exposure route to AMR microbes and AMR genes (ARGs), which may be present in viable bacteria or on free DNA. Ready-to-eat (RTE) foods are of particular interest because they are eaten without further cooking, so AMR bacteria or ARGs that are present may be consumed intact. They also represent varied production systems (fresh produce, cooked meat, dairy, etc.). An evidence gap exists regarding the diversity and consumption of ARGs in RTE food, which this study begins to address. We sampled 1001 RTE products at retail sale in the UK, in proportion to their consumption by the UK population, using National Diet and Nutrition Survey data. Bacterial DNA content of sample extracts was assessed by 16S metabarcoding, and 256 samples were selected for metagenomic sequencing for identification of ARGs based on consumption and likely bacterial DNA content. A total of 477 unique ARGs were identified in the samples, including ARGs that may be involved in resistance to important antibiotics, such as colistin, fluoroquinolones, and carbapenems, although phenotypic AMR was not measured. Based on the incidence of ARGs in food types, ARGs are estimated to be present in a high proportion of average diets. ARGs were detected on almost all RTE food types tested (48 of 52), and some efflux pump genes are consumed in 97% of UK diets.

BACKGROUND: Zymoseptoria tritici causes Septoria tritici blotch (STB), which is the biggest threat to wheat in the UK. Azole fungicides have been used since the 1980s to control STB, but resistance to these chemicals is now widespread. The main resistance mechanism is based on the accumulation of CYP51 mutations, with 33 mutations reported. Hence, farmers need an accurate estimate of the haplotype composition of Z. tritici populations to develop effective fungicide treatments and resistance management. RESULTS: Isolates from Z. tritici lesions were collected from three fields across three commercial farms using two sampling approaches. Analysis of the isolate sequences revealed that the number of distinct haplotypes and the haplotype composition of the most dominant haplotypes varied only between and not within farms. Conventional W-shaped and point sampling both found the same percentage of distinct haplotypes and frequencies of the six most dominant haplotypes. CONCLUSION: The results from this survey suggest that farm-resistance-management strategies should be based on farm-specific rather than national data, and that sampling within a single field is sufficient. W-shaped sampling is often recommended in sampling approaches, but this survey finds no evidence of this approach being more appropriate for detecting a greater percentage of distinct haplotypes which may aid the discovery of potential new resistance threats. (c) 2024 Fera Science Ltd. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Early detection of pests at a low population prevalence is a key pillar of surveillance for plant health. A novel pest is expected to undergo exponential growth when it first occurs in a host population. This allows for the use of mathematical rules of thumb for the number of samples required to detect presence before it reaches a target prevalence given the diagnostic performance of the tests used. Previous work assumes that pest presence is diagnosed by applying a single diagnostic test to each sample. However, diagnostic decisions are often made based on outcomes of multiple tests applied to the same sample in a consistent test program; for example, an assessment of symptoms followed by a PCR test applied to samples for which symptoms were observed. Each test can have different testing costs, as well as distinct and independent diagnostic performance and uncertainty values. A framework to optimize early detection surveys by minimizing overall costs for test programs that apply up to three diagnostic tests has been developed. The framework assesses the consequences of the test order and logical rule by which diagnoses are determined on costs across a range of pest prevalence. Explicit definitions of uncertainty in key parameters are incorporated to assess the consequences of uncertainty about their true value. Use of the framework is exemplified with two hypothetical case studies exploring the potential impact of selecting a suboptimal test program (based on Xylella fastidiosa) and investment in test improvements (based on lateral flow devices applied to Phytophthora pluvialis). [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

During a project investigating pea viruses in the United Kingdom and developing a workflow for high-throughput sequencing in crop surveillance, pea necrotic yellow dwarf virus (PNYDV) was identified at one site, along with pea enation mosaic virus-1, pea enation mosaic virus-2, pea enation mosaic virus satellite RNA, pea seedborne mosaic virus, and turnip yellows virus. Following an initial finding of small PNYDV contigs by an RNA sequencing protocol on MiSeq (Illumina), confirmation and prevalence testing by PCR and Sanger sequencing determined the average PNYDV prevalence to be 3%. This is the first report of PNYDV, and a nanovirus, in the United Kingdom. Initial characterization of the octopartite genome by PCR revealed that component DNA N had a truncated sequence that differed from those previously reported. This led to confirmation testing using rolling circle amplification (RCA) into MiSeq and then MinION (Oxford Nanopore). Sequences were obtained by both RCA-MiSeq and RCA-MinION, with MinION producing more complete genomes than RCA-MiSeq. Data obtained from sequencing were used to provide an initial estimation of the genomic formula of PNYDV. Previous work has looked into comparing each method; however, this study shows how these methods can be used together. The truncated sequence for DNA N was also found by RCA-MiSeq and RCA-MinION and confirmed by specific primers, but further work is required to understand the impact of this truncation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Although the importance of test validation in a plant health context is widely recognized, it is arguably not yet used to its full potential. This concept note describes the basic concepts underlying test performance and validation, as well as their current interpretation and use by key stakeholder groups. It is argued that, due to a number of communication pitfalls and associated limitations, current approaches are too fragmented and could lead to suboptimal results. Recommendations on how to improve the collection and use of validation data are based on work conducted in the EU VALITEST project (2018 to 2021). A key output of the project is its contribution to the standardization of test performance studies from sample selection and testing to interpreting and communicating test results. In addition, a prototype mathematical framework has been developed, which enables users to more easily consider and communicate trade-offs in test selection and development. The framework provides the basis for a more holistic approach, aiming to streamline collaboration between stakeholder groups. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Since the first report of the virus in 2014, tomato brown rugose fruit virus (ToBRFV) has spread widely through Europe, the Americas and Asia. Within Europe there is currently a requirement for annual surveillance for the virus. However, little is known about the relative impact of sampling strategy with respect to timing of infection and the detection of virus from different plant parts. To test reliably for ToBRFV in crops of unknown infection status, this issue needed to be addressed. To do this, two different approaches were followed: (1) inoculation experiments were conducted at two institutes to look at the relative effects of time of infection, plant parts, cropping season and cultivar on detection of the virus; and (2) sampling and testing various plant parts were carried out during active outbreaks from two tomato production sites in the Netherlands to look at the effect of sampling plant parts on detection of the virus. In inoculation experiments, the greatest impact on detection was timing of infection, with plants infected early in the growth cycle showing a predictable development of infection. In plants infected later, infection was detectable in sepals (calyx) earlier than in older leaves. In the studies carried out on commercial crops during ToBRFV outbreaks, the highest virus concentrations were obtained from testing sepals and young leaves. Thus, in a young crop where sepals and fruit are not yet developed, sampling should focus on the young leaves; in a mature crop it may be better to sample sepals and/or fruit.

Appropriate statistical analysis of the validation data for diagnostic tests facilitates the evaluation of the performance criteria and increases the confidence in the conclusions drawn from these data. A comprehensive approach to analysing and reporting data from validation studies and inter-laboratory comparisons such as test performance studies is described. The proposed methods, including statistical analyses, presentation and interpretation of the data, are illustrated using a real dataset generated during a test performance study conducted in the framework of the European project, VALITEST. This analytical approach uses, wherever possible and whenever applicable, statistical analyses recommended by international standards illustrating their application to plant health diagnostic tests. The present work is addressed to plant health diagnosticians and researchers interested and/or involved in the validation of plant diagnostic tests, and also aims to convey the necessary information to those without a statistical background. Detailed statistical explanations are provided in the Appendices.

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.

Estimates of quantities needed to plan invasive species control, such as population size, are always uncertain; this is an issue that can become a problem when mishandled in ecological science and its communication. The complexities of incorporating uncertainty into sophisticated decision-support tools may be a barrier to their use by decision makers, leading to decisions being made without due regard to uncertainty and risking misplaced certainty of predicted outcomes. We summarise ways in which uncertainty has been incorporated into and used to advise decisions on the management of invasive non-native species and other problem species, and offer a simple conceptual model for accommodating and using uncertainty at the planning stage. We also demonstrate how frequently uncertainty has been misused and miscommunicated in the wildlife management literature. We contend that uncertainty in estimates of natural quantities must be acknowledged, can inform decisions and can be made to derive decisions, and should not be ignored if invasive species policy is to be delivered effectively. Uncertainty must be communicated thoroughly and correctly by scientists if decision makers are to understand its consequences for planning and resourcing control programmes.

A choice test bioassay was devised to screen compounds as potential semiochemicals (e.g., kairomones or allomones that mediate aggregation, attraction or repellence) for the obligate parasitic mite, Psoroptes ovis. The choice test used filter paper discs in a 1:4 test:control ratio and was found to be a reliable, effective and efficient method. Four mammalian lipid components were assessed as potential attractants—linoleic acid, arachidonic acid, methyl myristate and squalene—, and the insect/tick repellent DEET for potential repellence. Linoleic acid was significantly attractive to P. ovis adult females and has the potential to act as an attractant. Identification of P. ovis semiochemicals, especially attractants, would be beneficial in the development of novel control methods and tools for this species. This is essential considering the increase in resistance to the limited prophylactic chemical treatments in the UK, and the high prevalence of scab infections.

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask- and large-scale composting tests using green waste. After an exposure for up to 10 days at 20 degrees C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3-day exposure to 40 degrees C or a 1-day exposure to 45 degrees C although pseudosclerotia were more tolerant than mycelium to an exposure to 35 degrees C. Primordial apothecia of H. fraxineus emerged from 62%-100% of infected ash rachises collected from two infected sites and stored at 4 degrees C for 0-5 months; exposure to compost for up to 10 days at 20 degrees C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45 degrees C for 1 day or at 35 degrees C or 40 degrees C for 3 days in flasks or at 40 degrees C for 1 day or at 30 degrees C for 5 days in a large-scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50 degrees C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35 degrees C-45 degrees C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real-time PCR. However, exposure of rachises to compost at >53 degrees C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.

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.

This paper presents the inter-laboratory studies for the successful validation of qualitative real-time Polymerase Chain Reaction methods for the detection of Genetically Modified Organisms in food and feed. Two singleplex TaqMan methods were developed for the detection of the tE9 terminator present in several genetically modified events and the detection of the endogenous lectin gene from pea; the tE9 terminator donor organism. In addition, a TaqMan duplex method was validated, which allows the simultaneous detection of the pat and bar elements.The paper explains the different acceptance parameters used during the in-house validation, the method transferability study and finally the inter-laboratory validation, how this validation was conducted and the problems encountered. Statistical methods for the validation of the qualitative methods are also presented. This information can be used by laboratories for the verification of these methods during their implementation and for the validation of new methods.

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.

The aphid‐transmitted viruses Potato virus Y (PVY) and Potato virus A (PVA) commonly affect seed potatoes in the UK. The transmission efficiency for aphid species is used to calculate a potential transmission risk and is expressed as a relative efficiency factor (REF). These REFs have not previously been calculated for UK strains of viruses or aphid clones. Using a previously published method, REFs have been calculated for the aphid species and viruses commonly occurring in UK potatoes. The efficiency of transmission of Myzus persicae is nominally set to a REF of 1 and REFs for other species are calculated relative to this. These data represent the first set of REFs calculated for PVA transmission. Macrosiphum euphorbiae (REF 0.91) was almost as efficient as M. persicae at PVA transmission. The data were further analysed to compare transmission rates of PVY and PVA using a binomial (logit) generalized mixed model to take into account the potential influence of variation in virus titre between leaves. This approach found that there is little variation between the efficiency of transmission between clones of each aphid species or between strains within a virus species. This is a first report that Aphis fabae, Metopolophium dirhodum, Sitobion avenae, Acyrthosiphon pisum and Cavariella aegopodii have the ability to vector PVA. This study also represents a first report that C. aegopodii has the ability to vector PVY and confirms the potential of S. avenae, A. fabae, M. euphorbiae and Rhopalosiphum padi as important PVY vectors.

Pest and pathogens pose a major threat to food security and the natural environment, and these threats are moving around the globe. Trade, travel and transport have major roles to play in this and thus to improve plant biosecurity, we need enhanced phytosanitary inspection systems. In order to achieve this, there is a role for the more effective use of diagnostic technology, such as field-based testing or the use of next generation sequencing technology. In this review we examine the opportunities and challenges posed by using new technology within a plant biosecurity context, but in contrast to previous reviews, here we focus on practical challenges associated with deployment and routine use, rather than specific technical issues. These key challenges include the need to accelerate the development and deployment of new technologies into the field, the accelerated discovery of new pathogens and the need for new risk assessment approaches, and improvements to our understanding of how best to deploy and use new diagnostic tools for maximum impact. Throughout we focus on how interdisciplinary approaches are important to help us improve our understanding and achieve our goals.

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).