RSS 1.0Food Safety
The objective of the Food Safety department is to provide the science to underpin a total chain risk based approach to food safety, focusing on microbial and chemical contaminants in the ‘farm to fork’ food chain.
Recent Submissions
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An investigation of the effect of rapid slurry chilling on blown pack spoilage of vacuum-packaged beef primalsThe aim of this study was to investigate if rapid slurry chilling would retard or prevent blown pack spoilage (BPS) of vacuum-packaged beef primals. Beef primals were inoculated with Clostridium estertheticum subspp. estertheticum (DSMZ 8809), C. estertheticum subspp. laramenise (DSMZ 14864) and C. gasigenes (DSMZ 12272), and vacuum-packaged with and without heat shrinkage (90°C for 3 s). These packs were then subjected to immediate chilling in an ice slurry or using conventional blast chilling systems and stored at 2°C for up to 100 days. The onset and progress of BPS was monitored using the following scale; 0‑no gas bubbles in drip; 1‑gas bubbles in drip; 2‑loss of vacuum; 3‑‘blown’; 4‑presence of sufficient gas inside the packs to produce pack distension and 5‑tightly stretched, ‘overblown’ packs/packs leaking. Rapid slurry chilling (as compared to conventional chilling) did not significantly affect (P > 0.05) the time to the onset or progress of BPS. It was therefore concluded that rapid chilling of vacuum-packaged beef primals, using an ice slurry system, may not be used as a control intervention to prevent or retard blown pack spoilage.
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Real-time PCR methods for the detection of blown pack spoilage causing Clostridium species; C. estertheticum, C. gasigenes and C. ruminantiumA set of real-time PCR methods for the detection of C. estertheticum, C. gasigenes and C. ruminantium, the causative agents of blown pack spoilage (BPS) in vacuum packaged beef, was developed. Robust validation of the sensitivity and specificity was carried out in the three matrices (beef meat drip, wet environmental swabs and dry environmental swabs) as encountered in our testing laboratory and against Clostridium strains (n = 76) and non-Clostridium strains (n = 36). It was possible to detect 4–5 spores per ml for C. estertheticum, 2 spores per ml for C. gasigenes and 8 spores per ml for C. ruminantium, without the need for enrichment of the samples. This high sensitivity is particularly important for the beef sector, not just for testing spoiled product but also in the early detection of contaminated beef and in validation of sporicidal cleaning procedures for critical pieces of equipment such as the vacuum packaging machine, which have the potential to contaminate large volumes of product.
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Development and validation of a quantitative method for 15 antiviral drugs in poultry muscle using liquid chromatography coupled to tandem mass spectrometryThe objective of this work was to develop a quantitative multi-residue method for analysing antiviral drug residues and their metabolites in poultry meat samples. Antiviral drugs are not licensed for the treatment of influenza in food producing animals. However, there have been some reports indicating their illegal use in poultry. In this study, a method was developed for the analysis of 15 antiviral drug residues in poultry muscle (chicken, duck, quail and turkey) using liquid chromatography coupled to tandem mass spectrometry. This included 13 drugs against influenza and associated metabolites, but also two drugs employed for the treatment of herpes (acyclovir and ganciclovir). The method required the development of a novel chromatographic separation using a hydrophilic interaction chromatographic (HILIC) BEH amide column, which was necessary to retain the highly polar compounds. The analytes were detected using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. A range of different sample preparation protocols suitable for polar compounds were evaluated. The most effective procedure was based on a simple acetonitrile-based protein precipitation step followed by a further dilution in a methanol/water solution. The confirmatory method was validated according to the EU 2021/808 guidelines on different species including chicken, duck, turkey and quail. The validation was performed using various calibration curves ranging from 0.1 µg kg−1to 200 µg kg−1, according to the analyte. Depending on the analyte sensitivity, decision limits achieved ranged from 0.12 µg kg−1 for arbidol to 34.7 µg kg−1 for ribavirin. Overall, the reproducibility precision values ranged from 2.8% to 22.7% and the recoveries from 84% to 127%. The method was applied to 120 commercial poultry samples from the Irish market, which were all found to be residue-free.
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Effect of applying crust-freezing after skin-packaging on the natural microflora of Atlantic salmon (Salmo salar) during storage at low temperaturesThe aim of the present study was to evaluate the effect of crust-freezing (CF) on fresh salmon fillets in skin-packaging during storage at −2.0°C. After CF, all treated samples and untreated controls were stored in a refrigerated cabinet for 20 d. Sampling was carried out at days 0, 2, 6, 8, 10, 14 and 20 in order to analyse total volatile basic nitrogen (TVB-N) and levels of mesophilic and psychrophilic viable counts (MVC and PVC). Enterobacteriaceae (ENT), lactic acid bacteria (LAB), H2S-producing bacteria (SPB) and Pseudomonas spp. (PSE). No significant differences in TVB-N were found between samples except for those taken on day 20 where TVB-N levels of CF samples were lower than controls. Our results suggest that ENT might be the limiting microbial group to determine the end of shelf-life. Thus, if this group is used as an indicator of acceptability, the shelf-life of salmon can be extended from 8 to 20 d when skin-packed and then treated with CF.
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A preliminary study of Salmonella spp., Listeria monocytogenes, Escherichia coli O157, Enterococcus faecalis and Clostridium spp. in Irish cattleAlthough Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. present a significant food safety and/or spoilage issue for the beef sector, there are limited data on their prevalence in Irish cattle. The objectives of this preliminary study were to investigate the distribution (percentage of farms positive) of Salmonella spp., E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. and the overall prevalence (%) of these bacteria in cattle on a small cohort of Irish beef farms. A total of 121 fresh bovine faecal samples were obtained on 10 randomly selected beef farms in the Northeast of Ireland and tested for the target pathogens using standard culture-based methods. Presumptive positives were confirmed using previously published polymerase chain reaction (PCR) methods. Salmonella were not detected in any of the samples. E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. were present on 50%, 40%, 100% and 100% of farms, respectively, with overall (all farms) prevalence rates in cattle of 9%, 8.2%, 61.9% and 87.6%, respectively. This study suggests that E. coli O157 may be more prevalent than previously thought and L. monocytogenes, E. faecalis and Clostridium spp. are widespread in Irish beef animals.
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Viromes of one year old infants reveal the impact of birth mode on microbiome diversityEstablishing a diverse gut microbiota after birth is being increasingly recognised as important for preventing illnesses later in life. It is well established that bacterial diversity rapidly increases post-partum; however, few studies have examined the infant gut virome/phageome during this developmental period. We performed a metagenomic analysis of 20 infant faecal viromes at one year of age to determine whether spontaneous vaginal delivery (SVD) or caesarean section (CS) influenced viral composition. We find that birth mode results in distinctly different viral communities, with SVD infants having greater viral and bacteriophage diversity. We demonstrate that CrAssphage is acquired early in life, both in this cohort and two others, although no difference in birth mode is detected. A previous study has shown that bacterial OTU’s (operational taxonomic units) identified in the same infants could not discriminate between birth mode at 12 months of age. Therefore, our results indicate that vertical transmission of viral communities from mother to child may play a role in shaping the early life microbiome, and that birth mode should be considered when studying the early life gut virome.
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The response of foodborne pathogens to osmotic and desiccation stresses in the food chainIn combination with other strategies, hyperosmolarity and desiccation are frequently used by the food processing industry as a means to prevent bacterial proliferation, and particularly that of foodborne pathogens, in food products. However, it is increasingly observed that bacteria, including human pathogens, encode mechanisms to survive and withstand these stresses. This review provides an overview of the mechanisms employed by Salmonella spp., Shiga toxin producing E. coli, Cronobacter spp., Listeria monocytogenes and Campylobacter spp. to tolerate osmotic and desiccation stresses and identifies gaps in knowledge which need to be addressed to ensure the safety of low water activity and desiccated food products.
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Ranking hazards pertaining to human health concerns from land application of anaerobic digestateAnaerobic digestion (AD) has been identified as one of the cleanest producers of green energy. AD typically uses organic materials as feedstock and, through a series of biological processes, produces methane. Farmyard manure and slurry (FYM&S) are important AD feedstock and are typically mixed with agricultural waste, grass and/or food wastes. The feedstock may contain many different pathogens which can survive the AD process and hence also possibly be present in the final digestate. In this study, a semi-quantitative screening tool was developed to rank pathogens of potential health concern emerging from AD digestate. A scoring system was used to categorise likely inactivation during AD, hazard pathways and finally, severity as determined from reported human mortality rates, number of global human-deaths and infections per 100,000 populations. Five different conditions including mesophilic and thermophilic AD and three different pasteurisation conditions were assessed in terms of specific pathogen inactivation. In addition, a number of scenarios were assessed to consider foodborne incidence data from Ireland and Europe and to investigate the impact of raw FYM&S application (without AD and pasteurisation). A sensitivity analysis revealed that the score for the mortality rate (S3) was the most sensitive parameter (rank coefficient 0.49) to influence the final score S; followed by thermal inactivation score (S1, 0.25) and potential contamination pathways (S2, 0.16). Across all the scenarios considered, the screening tool prioritised Cryptosporidium parvum, Salmonella spp., norovirus, Streptococcus pyogenes, enteropathogenic E. coli (EPEC), Mycobacterium spp., Salmonella typhi (followed by S. paratyphi), Clostridium spp., Listeria monocytogenes and Campylobacter coli as the highest-ranking pathogens of human health concern resulting from AD digestate in Ireland. This tool prioritises potentially harmful pathogens which can emerge from AD digestate and highlights where regulation and intervention may be required.
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Investigating the Dietary Habits of Male Irish Farmers to Prevent Mortality and MorbidityExcess mortality and morbidity among Irish farmers from non-communicable diseases (NCDs) has been linked to a range of occupational risk factors. Obesity is a key risk factor underpinning this excess burden and unhealthy eating habits are linked to overweight/obesity and to disease occurrence. This study investigated the dietary habits of a sub-group of Irish male farmers and explored how these might potentially impact on health outcomes. Cross-sectional survey research was undertaken using self-reported quantitative data, based on convenience sampling and a 24-h food re-call survey. Data were analysed using frequency and chi-square analysis. Where possible, findings were compared to national survey data for Irish males. Findings revealed that a high proportion of farmers were overweight or obese and that dietary habits consisted of low intake of fruit, vegetables, and dairy and a high intake of meat, fried and processed foods, salt, and sugary and/or salty snacks. Younger farmers reported a significantly higher intake of processed meats; however, no associations were found between age, lifestyle behaviours, and dietary habits. The findings provide a greater understanding of how dietary habits potentially contribute to poorer health outcomes among farmers and underline the need for health promotion interventions, including healthy eating campaigns, aimed at farmers.
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Emerging Technologies for Aerial Decontamination of Food Storage Environments to Eliminate Microbial Cross-ContaminationAir is recognized as an important source of microbial contamination in food production facilities and has the potential to contaminate the food product causing food safety and spoilage issues for the food industry. Potential for aerial microbial contamination of food can be a particular issue during storage in cold rooms when the food is not packaged and is exposed to contaminated air over a prolonged period. Thus, there are potential benefits for the food industry for an aerial decontamination in cold storage facilities. In this paper, aerial decontamination approaches are reviewed and challenges encountered for their applications are discussed. It is considered that current systems may not be completely e ective and environmentally friendly, therefore, it is of great significance to consider the development of nonresidual and verified decontamination technologies for the food industry and, in particular, for the cold storage rooms.
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Genetic characterisation of a subset of Campylobacter jejuni isolates from clinical and poultry sources in IrelandCampylobacter spp. is a significant and prevalent public health hazard globally. Campylobacter jejuni is the most frequently recovered species from human cases and poultry are considered the most important reservoir for its transmission to humans. In this study, 30 Campylobacter jejuni isolates were selected from clinical (n = 15) and broiler (n = 15) sources from a larger cohort, based on source, virulence, and antimicrobial resistance profiles. The objective of this study was to further characterise the genomes of these isolates including MLST types, population structure, pan-genome, as well as virulence and antimicrobial resistance determinants. A total of 18 sequence types and 12 clonal complexes were identified. The most common clonal complex was ST-45, which was found in both clinical and broiler samples. We characterised the biological functions that were associated with the core and accessory genomes of the isolates in this study. No significant difference in the prevalence of virulence or antimicrobial resistance determinants was observed between clinical and broiler isolates, although genes associated with severe illness such as neuABC, wlaN and cstIII were only detected in clinical isolates. The ubiquity of virulence factors associated with motility, invasion and cytolethal distending toxin (CDT) synthesis in both clinical and broiler C. jejuni genomes and genetic similarities between groups of broiler and clinical C. jejuni reaffirm that C. jejuni from poultry remains a significant threat to public health.
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Biofortification of Chicken Eggs with Vitamin K—Nutritional and Quality ImprovementsNational nutrition surveys have shown that over half of all adults in Ireland, the United Kingdom (UK), and the United States of America (USA) have low vitamin K intakes. Thus, dietary strategies to improve vitamin K intakes are needed, and vitamin K biofortification of food may be one food-based approach. The primary aim of our study was to establish whether increasing the vitamin K3 content of hen feed can increase the vitamin K content of eggs, and the secondary aims were to examine the effects on hen performance parameters, as well as egg and eggshell quality parameters. A 12 week hen feeding trial was conducted in which Hyline chickens were randomized into four treatment groups (n = 32/group) and fed diets containing vitamin K3 (as menadione nicotinamide bisulfite) at 3 (control), 12.9, 23.7, and 45.7 mg/kg feed. Vitamin K1, menaquinone (MK)-4, MK-7, and MK-9 were measured in raw whole eggs via a liquid chromatography tandem mass spectrometry method. MK-4 was the most abundant form of vitamin K (91–98%) found in all eggs. Increasing the vitamin K3 content of hen feed over the control level significantly (p < 0.001) enhanced the MK-4 content of eggs (mean range: 46–51 µg/100 g, representing ~42–56% of US Adequate Intake values). Vitamin K biofortification also led to significant (p < 0.05) increases in the yellowness of egg yolk and in eggshell weight and thickness, but no other changes in egg quality or hen performance parameters. In conclusion, high-quality vitamin K-biofortified eggs can be produced with at least double the total vitamin K content compared to that in commercially available eggs.
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Assessment of the Effectiveness of Pre-harvest Meat Safety Interventions to Control Foodborne Pathogens in Broilers: a Systematic ReviewPurpose of Review Ensuring broilers’ meat safety is a priority to policy makers, producers, and consumers. This systematic review aims to update the recent knowledge on pre-harvest interventions to control main foodborne pathogens in broilers and to assess their effectiveness. Recent Findings A total of 815 studies were retrieved from PubMed® andWeb of Science for 13 pathogens. In total, 51 studies regarding Campylobacter spp., Salmonella spp., VTEC, ESBL-AmpC Escherichia coli, and Clostridium perfringens were included in this review. Summary Research mostly focused on Salmonella spp. and Campylobacter spp. Biosecurity and management interventions had mixed outcomes, while the effectiveness of feed additives, though intensively researched, remains controversial. Research on other pathogens (i.e. ESBL-AmpC E. coli/Salmonella, and Toxoplasma gondii) was scarce, with publications focusing on epidemiology and/or on source-attribution studies. This is also true regarding research on Listeria monocytogenes, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, and Staphylococcus aureus as these are frequently controlled by post-harvest interventions. Overall, studies on recent developments of novel pathogen-specific immunisation strategies are lacking.
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Effect of space allowance and mixing on growth performance and body lesions of grower-finisher pigs in pens with a single wet-dry feederBackground: Low space allowance (SA) and mixing may result in reduced growth performance (GP) and animal welfare issues because of adverse social behaviours directed to pen mates. This could be exacerbated in pens with single space feeders owing to social facilitation of feeding behaviour. The present study aimed to investigate the effect of SA and mixing on GP and body lesions (BL) in pens with one single space wet-dry feeder. Results: Two experiments were conducted on grower-finisher pigs from 10 to 21 weeks of age. In Exp1, pigs (N = 216) were assigned to three SA; 0.96m2/pig (n = 6 pens; 10 pigs/pen; SA96), 0.84m2/pig (n = 6; 12 pigs/pen; SA84) and 0.72m2/pig (n = 6; 14 pigs/pen; SA72), in a randomized design. In Exp2, pigs (N = 230) were used in a 2 × 2 factorial randomized design considering SA and mixing as treatments. Pigs were assigned to two SA; 0.96m2/pig (n = 10 pens; 10 pigs/pen; SA96) and 0.78 m2/pig (n = 10; 13 pigs/pen; SA78) and were either mixed or not at the entry to the finishing facility. GP was not affected by SA (P > 0.05) in either experiment. In Exp2, non-mixed pigs were 5.4 kg heavier (P < 0.001), gained 74 g more per day (P = 0.004), consumed 101.8 g more of feed per day (P = 0.007) and tended to have higher feed efficiency (P = 0.079) than mixed pigs from 11 to 21 weeks of age. Number of BL was affected by SA in both experiments. In Exp1, SA72 pigs had 74.4 and 97.4% more BL than SA96 and SA84 pigs at 20 weeks of age respectively (P < 0.01). In Exp2, SA78 pigs had 48.6, 43.6 and 101.3% more BL than SA96 pigs at 12, 16 and 21 weeks of age respectively (P < 0.05). Mixing did not affect the number of BL from 12 to 21 weeks of age in Exp2 (P > 0.05). Conclusion: Mixing had a considerable effect on growth performance thus, strategies to avoid or mitigate mixing should be considered. Although space allowance had no effect on growth performance, high number of body lesions in the lower space allowance indicates that space allowances equal or below 0.78 m2/pig are detrimental to the welfare of pigs despite following the EU legislation. Keywords: Animal welfare, Group size, Pig, Regrouping, Stocking density, Swine
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An investigation of anticoccidial veterinary drugs as emerging organic contaminants in groundwaterIntensification of the food production system to meet increased global demand for food has led to veterinary pharmaceuticals becoming a critical component in animal husbandry. Anticoccidials are a group of veterinary products used to control coccidiosis in food-producing animals, with primary prophylactic use in poultry production. Excretion in manure and subsequent land-spreading provides a potential pathway to groundwater. Information on the fate and occurrence of these compounds in groundwater is scant, therefore these substances are potential emerging organic contaminants of concern. A study was carried out to investigate the occurrence of anticoccidial compounds in groundwater throughout the Republic of Ireland. Twenty-six anticoccidials (6 ionophores and 20 synthetic anticoccidials) were analysed at 109 sites (63 boreholes and 46 springs) during November and December 2018. Sites were categorised and selected based on the following source and pathway factors: (a) the presence/absence of poultry activity (b) predominant aquifer category and (c) predominant groundwater vulnerability, within the zone of contribution (ZOC) for each site. Seven anticoccidials, including four ionophores (lasalocid, monensin, narasin and salinomycin) and three synthetic anticoccidials (amprolium, diclazuril and nicarbazin), were detected at 24% of sites at concentrations ranging from 1 to 386 ng L−1. Monensin and amprolium were the two most frequently detected compounds, detected at 15% and 7% of sites, respectively. Multivariate statistical analysis has shown that source factors are the most significant drivers of the occurrence of anticoccidials, with no definitive relationships between occurrence and pathway factors. The study found that the detection of anticoccidial compounds is 6.5 times more likely when poultry activity is present within the ZOC of a sampling point, compared to the absence of poultry activity. This work presents the first detections of these contaminants in Irish groundwater and it contributes to broadening our understanding of the environmental occurrence and fate of anticoccidial veterinary products.
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Recipe for a Healthy Gut: Intake of Unpasteurised Milk Is Associated with Increased Lactobacillus Abundance in the Human Gut MicrobiomeIntroduction: The gut microbiota plays a role in gut–brain communication and can influence psychological functioning. Diet is one of the major determinants of gut microbiota composition. The impact of unpasteurised dairy products on the microbiota is unknown. In this observational study, we investigated the effect of a dietary change involving intake of unpasteurised dairy on gut microbiome composition and psychological status in participants undertaking a residential 12-week cookery course on an organic farm. Methods: Twenty-four participants completed the study. The majority of food consumed during their stay originated from the organic farm itself and included unpasteurised milk and dairy products. At the beginning and end of the course, participants provided faecal samples and completed self-report questionnaires on a variety of parameters including mood, anxiety and sleep. Nutrient intake was monitored with a food frequency questionnaire. Gut microbiota analysis was performed with 16S rRNA gene sequencing. Additionally, faecal short chain fatty acids (SCFAs) were measured. Results: Relative abundance of the genus Lactobacillus increased significantly between pre- and post-course time points. This increase was associated with participants intake of unpasteurised milk and dairy products. An increase in the faecal SCFA, valerate, was observed along with an increase in the functional richness of the microbiome profile, as determined by measuring the predictive neuroactive potential using a gut–brain module approach. Conclusions: While concerns in relation to safety need to be considered, intake of unpasteurised milk and dairy products appear to be associated with the growth of the probiotic bacterial genus, Lactobacillus, in the human gut. More research is needed on the effect of dietary changes on gut microbiome composition, in particular in relation to the promotion of bacterial genera, such as Lactobacillus, which are recognised as being beneficial for a range of physical and mental health outcomes.
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An investigation of the ecological niches and seasonal nature of Clostridium estertheticum and Clostridium gasigenes in the Irish beef farm environmentBlown pack spoilage (BPS) of vacuum packaged beef is caused by psychrotolerant and psychrophilic Clostridium species (PPC), primarily Clostridium estertheticum and Clostridium gasigenes. The aim of this study was to investigate the environmental niches and impact of season on these BPS Clostridium spp. on Irish beef farms. On each of 5 different beef farms, faecal (10), soil (5), silage (5), bedding straw (5), drinking water (5), puddle/ditch water (5) and air (5) samples were collected during Spring, Summer, Autumn and Winter and tested for C. estertheticum and C. gasigenes using culture (direct plating and enrichment) and molecular, (conventional PCR and quantitative PCR (qPCR)), based techniques. C. estertheticum and C. gasigenes were detected in all sample types, with qPCR detection rates ranging from 4% to 50% and at concentrations of up to 1.5 log10 cfu g‐1 and 3.5 log10 cfu g‐1, respectively. The impact of season was not clear as the results were mixed depending on the detection method used. It was concluded that BPS causing C. estertheticum and C. gasigenes are widely distributed in the beef farm environment.
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An investigation of anticoccidial veterinary drugs as emerging organic contaminants in groundwaterIntensification of the food production system to meet increased global demand for food has led to veterinary pharmaceuticals becoming a critical component in animal husbandry. Anticoccidials are a group of veterinary products used to control coccidiosis in food-producing animals, with primary prophylactic use in poultry production. Excretion in manure and subsequent land-spreading provides a potential pathway to groundwater. Information on the fate and occurrence of these compounds in groundwater is scant, therefore these substances are potential emerging organic contaminants of concern. A study was carried out to investigate the occurrence of anticoccidial compounds in groundwater throughout the Republic of Ireland. Twenty-six anticoccidials (6 ionophores and 20 synthetic anticoccidials) were analysed at 109 sites (63 boreholes and 46 springs) during November and December 2018. Sites were categorised and selected based on the following source and pathway factors: (a) the presence/absence of poultry activity (b) predominant aquifer category and (c) predominant groundwater vulnerability, within the zone of contribution (ZOC) for each site. Seven anticoccidials, including four ionophores (lasalocid, monensin, narasin and salinomycin) and three synthetic anticoccidials (amprolium, diclazuril and nicarbazin), were detected at 24% of sites at concentrations ranging from 1 to 386 ng L−1. Monensin and amprolium were the two most frequently detected compounds, detected at 15% and 7% of sites, respectively. Multivariate statistical analysis has shown that source factors are the most significant drivers of the occurrence of anticoccidials, with no definitive relationships between occurrence and pathway factors. The study found that the detection of anticoccidial compounds is 6.5 times more likely when poultry activity is present within the ZOC of a sampling point, compared to the absence of poultry activity. This work presents the first detections of these contaminants in Irish groundwater and it contributes to broadening our understanding of the environmental occurrence and fate of anticoccidial veterinary products.
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Conserved redox-dependent DNA binding of ROXY glutaredoxins with TGA transcription factorsThe Arabidopsis thaliana CC‐type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC‐type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CC‐type GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC‐type GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CC‐type GRXs in bryophytes and mediated by a conserved ROXY C‐terminus. Redox EMSA analyses show a redox‐sensitive binding of MpTGA to the cis‐regulatory as‐1‐like element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CC‐type GRXs in modulating the activities of TGA TFs. Super‐resolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redox‐sensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats.
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Development and validation of a quantitative confirmatory method for 30 β-lactam antibiotics in bovine muscle using liquid chromatography coupled to tandem mass spectrometryA method was developed for the confirmatory and quantitative analysis of 30 β-lactam antibiotic residues in bovine muscle. The method includes 12 penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, mecillinam, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin), 12 cephalosporins (cefacetrile, cefadroxil, cephalexin, cefalonium, cefazolin, cefoperazone, cefotaxime, cefquinome, cefuroxime, desacetyl cephapirin, desfuroylceftiofur cysteine disulfide, desfuroylceftiofur dimer), five carbapenems (biapenem, doripenem, ertapenem, imipenem, meropenem) and faropenem. Samples were extracted using a simple solvent extraction with acetonitrile:water (80:20, v/v) and C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) detection. Chromatography was performed on a reversed phase CSH C18 column, using a binary gradient separation comprising of 0.01% formic acid and 0.2 mM ammonium acetate in water (mobile phase A) and 0.01% formic acid in acetonitrile (mobile phase B). The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 69% and 143% and precision ranged between 2.0% and 29.9% under within-laboratory reproducibility conditions. The developed method uses minimal sample preparation and 30 test samples can be analysed by a single analyst in a single day. To the best of our knowledge, this is the first method for carbapenems in foodstuff that does not require derivatisation.
