Poster Abstracts | ONE Conference 2022

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Title of work	Poster author	Author affiliation	Assigned session
Standardisation of the PCR-RFLP for the molecular detection of Toxocara cani and Toxocara cati	Maria Alejandra Vethencourt Ysea	University of Medical Sciences (UCIMED)	Infectious diseases, from emergence to pandemics: improving understanding and getting prepared	DETAILED INFO Maria Alejandra Vethencourt Ysea, University of Medical Sciences (UCIMED) Title of work Standardisation of the PCR-RFLP for the molecular detection of Toxocara cani and Toxocara cati Co-author(s) Valerio Idalia E-POSTER VIDEO POSTER DOWNLOAD POSTER ABSTRACT Introduction Visceral larva migrans (VLM) is a clinical condition that can be caused by immature stages of some helminths, mainly nematodes. Depending on its anatomical location, the infection can range from being asymptomatic to causing serious health problems that compromise the lives of infected patients. Parasites belonging to the genus Toxocara (Toxocara canis and T. cati) are considered to be one of the main causal agents of this clinical presentation, being reported with some frequency in paediatric patients around the world. Being a zoonosis, the reservoirs of the parasites are animals, mainly domestic dogs and cats, whose faeces which contain immature eggs reach the ground, where they reach maturity, entering human beings orally, through contaminated water and food or by geophagy. Diagnosis in humans is carried out with serological methods, however, cross-reactions with other ascarididae are not uncommon, so molecular methods stand out as a more sensitive and specific alternative for their diagnosis, in addition, they would investigate the presence of eggs of these helminths in the environment. Methodology DNA from helminths identified as Toxocara cani or T. cati, stored in the presence or absence of 70 % ethanol at -20 ° C, was extracted with the commercial Machery-Nagel kit, after overnight digestion at 56 ° C . The quality of the extracted DNA was evaluated in terms of concentration and integrity. PCR was standardised to amplify the region of rDNA comprising ITS-1, 5.8 S, ITS-2. The PCR was carried out with the primers NC2 and NC5, described by Zhu et al., 2000. The sensitivity of the PCR was evaluated in terms of the minimum amount of DNA that generated a visible amplified in agarose gel. The characterisation of the parasites was carried out by RFLP (length polymorphism obtained with restriction enzymes) by digesting the amplified with the endonucleases Hinf I, Rsa I and Hae III. The agarose gels were visualised with the aid of a UV transilluminator (Slimline Series; Spectroline), the image was captured with an image digitiser (Enduro TM GDS, Labnet international, Inc.). The RFLPs obtained will be analysed with the help of TotalLab 1D software, version 14.0. Results The DNA obtained was between 1.29 and 11.9 ng / uL, with a considerably high degree of degradation. Despite this, PCR amplification was achieved, both for adults samples stored in alcohol or not stored in alcohol, of an amplification of approximately 1180 base pairs (bp), corresponding to the literature. The standardised PCR managed to amplify up to 5 pg / µL of DNA. Through RFLP analysis, it was possible to molecularly differentiate T. canis from T. cati. The analysis of the partial sequence of the region to be amplified for T. cati (Genebank MX309925.1; of 974 bp) and T. cani (Genebank JN617989.1 of 968 bp) by means of RFLP in a Bioinformatic way (http: // www. bioinformatics.org/sms2/rest_digest.html) coincided with the obtained polymorphism. In this regard. For T. cati only cut points for Hae III enzyme (RFLP: 595, 180, 156, 72 and 24 bp) and for Rsa I (RFLP: 652, 278 and 103 bp) were obtained. For T. canis it was possible to obtain RFLP for the 3 enzymes. For Hinf I an RFLP of 550, 395, 94 and 54 bp was obtained; for Hae III an RFLP of 850, 198, 57 and 16 bp and for Rsa I an RFLP of 500 and 288 bp. Discussion The PCR-RFLP thus standardised can be used for the molecular detection of the species that causes VLM, in addition to serving as a molecular tool for the investigation of eggs of these helminths in soils, waters or vegetables that are consumed raw. On the other hand, this molecular technique could be useful to carry out a first approach in the investigation of Ascarididae that infect fish for human consumption.
How to strengthen the One Health education in the Veterinary Higher Education context – the approach implemented at Egas Moniz	Maria Gaivão Sepúlveda	Egas Moniz - Cooperativa de Ensino Superior (EM)	Infectious diseases, from emergence to pandemics: improving understanding and getting prepared	DETAILED INFO Maria Gaivão Sepúlveda, Egas Moniz - Cooperativa de Ensino Superior (EM) Title of work How to strengthen the One Health education in the Veterinary Higher Education context – the approach implemented at Egas Moniz Co-author(s) Alexandre Trindade, Daniel Murta, Lara Alves, Alexandra Sanfins, Manuel Pequito, Ricardo Assunção E-POSTER DOWNLOAD POSTER ABSTRACT Introduction As the human population continues to grow and our relationships with animals continue to strengthen, understanding the interdependencies of humans, animals and the environment becomes ever more critical. Veterinarians are essential to achieve One Health and to protect the health and safety of its three pillars – animals, humans and environment - and their role is easy to understand because animals both impact and are impacted by humans and the environment. Indicators show that One Health is an integral part of the curriculum in most veterinary degrees awarded in Europe. However, veterinarians and other professions are still being educated mainly in isolation, with minimal interdisciplinary or transdisciplinary interaction. At Egas Moniz, we strongly believe that the One Health approach in veterinary education has to mean more than a good knowledge of prevention and control of diseases, epidemiology, use of medicines, food safety, ethical responsibilities, animal health and welfare and sustainable farming. Our Integrated Master’s in Veterinary Medicine has been structured and implemented, awarding the responsibility for the education of 21st century veterinarians, where One Health concept is imprinted on its “DNA”. Methodology In general, veterinary medical education has been facing new challenges, especially in preparing veterinarians for the societal challenges of the twenty-first century. To ensure that One Health is well implemented and understood throughout teaching and research, the Integrated Master’s in Veterinary Medicine (IMVM) at Egas Moniz (EM) is developing and implementing a quantitative survey to map how well interdisciplinary teaching and research is understood and implemented for the One Health approach. A questionnaire-based approach is being planned and structured, comprising both multiple-choice and open questions to collect data on the integration of interdisciplinary collaboration in undergraduate and post-graduate education and research projects. The focus will be on: interaction with other educational establishments and professions; the potential for learning more about the way it is integrated in the different curricula; an understanding of the difficulties and challenges faced with the implementation of this novel teaching model; and the academics’ perception of the value of such a teaching model, by collecting views on the actual or expected benefits for the students, the profession and society. Results EM is a health institution dedicated to advancing the knowledge, learning and education of its students, at the service of improving health conditions for global society. Thus, health, in a broad sense, constitutes the core of our education programs and research. The IMVM-EM is also reflecting this vision, contributing to the multidisciplinary and transdisciplinary perspectives expected and desired in One Health education. At IMVM-EM, issues that in traditional curricula are covered in silos are instead approached transversally, supported by educational strategies as problem-based learning activities. Integrated approaches and selection of case studies that properly cover the One Health content are carefully selected by a team of professors with a multidisciplinary and complementary background. Contents and materials are produced with a focus on avoiding repetition and promoting the interlinks between courses. The questionnaire will provide quantitative information about the impact of these methodologies, identifying those that should be privileged and establishing those examples that should be mirrored, considering the appropriate adequacy. Discussion The World Organisation for Animal Health (OIE) has been a leader in recognising that an understanding of the principles of One Health should be at the core of veterinary education. The Association of American Veterinary Colleges addressed this issue when it published a Roadmap for Veterinary Medical Education in the 21st Century. In this roadmap, it recommended that all veterinary students achieve competency before graduation in three main areas: multispecies knowledge plus clinical competence in one or more species or disciplines; One Health competency related to the intersection of animal, human and environmental health; and the development of professional competencies. Knowing this, the concept of One Health has captured the enthusiasm of EM by seeking to improve animal and human health through stronger interdisciplinary collaboration and a holistic approach that demands an open collaboration encompassing multidisciplinary, interdisciplinary and transdisciplinary health education. It is evident that changing the culture to encompass One Health in daily veterinary teaching and practice requires a complete change in the way professionals approach all phases of biomedical practice.
Novel Approach towards Food Safety in Milk: Detection of Antimicrobials and Non-Targeted Metabolite Residues as a pivotal approach to preventingantibiotic resistance diffusion	Maria Nobile	University of Milan, La Statale	Tackling antimicrobial resistance in food producing environments	DETAILED INFO Maria Nobile, University of Milan, La Statale Title of work Novel Approach towards Food Safety in Milk: Detection of Antimicrobials and Non-Targeted Metabolite Residues as a pivotal approach to preventingantibiotic resistance diffusion Co-author(s) Sara Panseri, Luca Maria Chiesa, Federica Di Cesare, Maria Nobile, Roberto Villa, Radmila Pavlovic, Francesco Arioli E-POSTER DOWNLOAD POSTER ABSTRACT Introduction For over 60 years, antibiotics have been widely used in animal husbandry for the prevention and treatment ofcommon pathologies. Moreover, the misuse of antibiotics to increase growth performance and feed efficiency can lead to the presence of antibiotic residues in milk. Concerns over antibiotic residues in food of animal origin arise due to the potential threat of direct toxicity to consumers, but mainly because low dosages of antibiotics could result in the alteration and possible development of resistant strains of bacteria. Regarding the above-mentioned main uses, the presence of residues in milk may be due to the miscellaneous use of antibiotics, either directly or indirectly (e.g., from farming and production environments), which represents a real threat to consumer health. This study aims to verify the absence of administered antimicrobials after therapeutic treatments, taking into consideration the withdrawal period. The targeted search for the previously administered antibiotics was implemented through a non-targeted search for their metabolites, which could still be pharmacologically active andinterfere in the cheese-making process. Methodology 141 raw bovine milk samples were collected from local farms located in the Piedmont Region of northern Italy, where most of the milk produced is used in Grana Padano PDO cheese production. The samples were all selected from dairy cows previously treated with different antimicrobial drugs due to medical conditions. The collection of milk was performed in accordance with the withdrawal period of all the drugs administered and, in this particular case, at the 7th milking. Confirmatory analyses were performed in duplicate according to the method described in our previous work. In brief, 1 mL of raw bovine milk, spiked at 2 ng mL−1 with the IS, extracted with 5 mL of McIlvaine buffer (pH 4.0) and 100 μL, 20% w/v of Trichloroacetic acid and then defatted with hexane, was purified by HLB SPE (Hydrophilic–Lipophilic Balance for Solid Phase Extraction). Analyses were performed by an HPLC system (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Thermo Q-Exactive Orbitrap (Thermo Fisher Scientific, San Jose, CA, USA). All the mass spectrometry (MS) parameters for the full-scan acquisition (FS) were combined with the data-independent acquisition (DIA) for the MS2 response. Results 41 samples (29 %) showed the residual presence of a treatment, despite the fact that the withdrawal period was amply respected, with the sample collection taking place after the seventh milking. Moreover, in 9 % of the total samples, some compounds not indicated in the treatment protocol of the animal were detected, often also during screening tests. In particular, a major unexpected finding was that MRLs were exceeded in eight samples (20 % of the positive samples, or 6 % of the total samples). Antimicrobial metabolites were also found in all samples with the presence of enrofloxacin and lincomycin, three metabolites (ciprofloxacin, des-ciprofloxacin and des-enrofloxacin) for the detected quinolone and lincomycin sulfoxide for lincosamide. Enrofloxacin, according to the literature, is extensively metabolised into ciprofloxacin and other minor metabolites, with the former still retaining antimicrobial activity. Two new metabolites that had not been reported in the literature were also tentatively identified and presented for their chemical structure. Discussion Regarding the new metabolites, the ion with m/z 334.1198 including its MS2 spectrum can be attributed to a compound resulting from the break down of the piperazine ring. This compound, entitled 1-cyclopropyl-6-fluoro-7-((2-(methylamino)-2-oxoethyl amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid) according to IUPAC nomenclature, is abbreviated as ENRO-N-methylacetamide. The second metabolite (5-(1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-2-hydroxy-4-oxo-1,2,3,4-tetrahydroquinoline-3-carboxamido)-2-oxopentanoic acid), named ENRO-ornithine (m/z = 491.2311), proved to be essential as it was similar to enrofloxacin itself. Some metabolites of Enrofloxacin are cleared from plasma to milk, for example lysine, but ornithine conjugate has not been reported so far. In particular, ciprofloxacin, des-ciprofloxacin, des-enrofloxacin, ENRO-N-methylacetamide and ENRO-ornithine were found in one sample treated with enrofloxacin, in which the parent drug was found at a higher concentration (25.50 ng mL−1). The detection of ciprofloxacin and ENRO-ornithine in one sample not declared for enrofloxacin treatment, in which the parent drug was detected at 0.20 ng mL−1 represents an important finding.
AdriAquaNet - Enhancing Innovation and Sustainability in Adriatic Aquaculture	Marco Galeotti	University of Udine	Advancing animal welfare to meet sustainability targets	DETAILED INFO Marco Galeotti, University of Udine Title of work AdriAquaNet - Enhancing Innovation and Sustainability in Adriatic Aquaculture Co-author(s) Sabina Passamonti, Emilio Tibaldi, Snježana Zrnčić, Jelka Pleadin E-POSTER DOWNLOAD POSTER ABSTRACT Introduction Farmed fish is a growing source of protein-rich food. While aquaculture grows globally, knowledge of the welfare of farmed fish is still lagging behind. Yet, animal welfare is at the heart of food supply sustainability; fish diseases stem from poor environmental conditions and antimicrobial resistance, which together result in poor food quality and lead to consumer mistrust. We must, as a matter of urgency, make a systematic and long-term commitment to defining farmed fish welfare parameters. To address this issue in the Adriatic Sea region, a community of Croatian and Italian researchers and aquaculture SMEs have joined forces in the AdriAquaNet project, which is funded by the ‘Interreg Italy-Croatia 2014-2020’ cross-border cooperation programme. The project is addressing the innovation needs of the ‘fish farm’, the ‘fish doctor’, and ‘the fish market’- the three main segments of the aquaculture value chain. Fish farms in the Adriatic Sea supply high-quality products for global export or to meet the intensive local demand during the summer touristic waves. Having a well-managed aquaculture sector is clearly essential for the local economy and maintaining consumer trust. Methodology The AdriAquaNet community brings together six research organisations and three fish farms. Through their collaboration, they are implementing R&I for three interconnected aquaculture sustainability goals: fish farm management, farmed fish welfare and health, and farmed fish marketing. The core approach for improving farmed fish welfare consists in integrating technological innovations into fish farms specialised in sea bass and sea breams, such as: a) the use of new feeds with alternative protein sources (insects) and added probiotics that protect against gut diseases; b) the use of new vaccines and vaccination protocols against pathogens; and c) the use of new natural drugs. The impact of these innovations on fish welfare has been assessed by gathering the data needed to both frame the intervention mode of action (i.e., gene expression, histology, haematology, gut microbiota, and biochemistry) and define end-points for monitoring animal welfare (i.e., Operational Welfare Indicators, growth, nutrient composition, sustainability index). From the One-health perspective, other datasets have been gathered on both environmental quality (climate and ecotoxicology indicators) and the nutritive parameters of food. Results Our data series show that the fish on the new diets with probiotics have the same sustainability index and nutritive properties as those fed conventional feed. However, the probiotic supplementchanged the intestine gene expression pattern towards a low inflammation state and improved resistance to gut pathogens, giving us reason to expect increased innate resistance to gut pathogens. Fish immunisation against pathogens by means of two autologous vaccines has been standardised and a manual for implementation will soon be delivered. Following a large-scale screening of several dozens of extracts and purified natural compounds, new bioactive entities have been identified that may undergo further testing as natural drugs for enhancing fish health and resistance against pathogens. A manual for the implementation of a new bivalent method based on the use of Operational Welfare Indicators in fish farms is due to be released by the end of the project. An important result of the project is the establishment of a close collaboration between researchers and fish farm managers, and their direct engagement in testing fish welfare innovations in their business. Discussion The goal of improving the environmental, nutritional and economic sustainability of Adriatic aquaculture by focusing on new science and technology is strategic for both Italy and Croatia. This has been made clear not only through the strategic R&I innovation plans of both EU Member States but also through the results of a consumer survey, which we gathered as part of this project. Citizens in our region appear to have a deep-rooted concern for the One-health concept and expect to be assured that the fish market offers them foodthat is produced with a great deal of respect for the environment and animals.
Rapid Alert Supply Network Extractor (RASNEX) tool to mine unstructured supply chain information from food and feed contamination notifications	Marc Lorenzen	German Federal Institute for Risk Assessment (BfR)	Augmenting human minds: artificial intelligence and big data in risk assessment	DETAILED INFO Marc Lorenzen, German Federal Institute for Risk Assessment (BfR) Title of work Rapid Alert Supply Network Extractor (RASNEX) tool to mine unstructured supply chain information from food and feed contamination notifications Co-author(s) Marc Christian Lorenzen, Daria Savvateeva, Robert Pieper, Carsten Fauhl-Hassek, Jorge Numata E-POSTER DOWNLOAD POSTER ABSTRACT Introduction Competent authorities of member countries use the Rapid Alert System for Food and Feed (RASFF) to report information on 1) any direct or indirect human health risk 2) any serious risk to animal health or the environment arising from food or feed. In the case of foodborne disease outbreaks, it is of utmost importance to rapidly identify the involved supply chain actors and withdraw the causative products from the market, since the impact on human and/or animal health and the economy is growing with the increasing lengths of such outbreaks. The mapping of relevant supply flows allows for the identification of additional nodes or sources of contaminated goods. A major challenge for risk managers is to gain and maintain an overview of any currently ongoing contamination events, as relevant information is continuously updated and is often provided through the RASFF system in non-standardised formats. Recent food related incidents in Europe illustrated that there is a need for a software system to support investigations on supply chains as well as exposure assessments in crisis situations. To meet these needs, the Rapid Alert Supply Network Extractor (RASNEX) [1], an open-source tool, was developed. Methodology RASNEX is programmed in the Konstanz Information Miner (KNIME) [2, 3] framework, since it is centred on data mining and data science, and makes use of FoodChain-Lab [4], which was developed by the German Federal Institute for Risk Assessment (BfR). RASNEX is available free of charge at https://doi.org/10.5281/zenodo.4322555.Parts of the data processing KNIME nodes are code snippets written in Python 3.7. Successful usage of the RASNEX tool requires the installation of the KNIME Analytics Platform integrated with Python 3 and of the FoodChain-Lab extension. RASFF notifications, providing information about contamination events, constitute the source of the workflow and are to be chosen according to the related inquiry. RASNEX may be employed for specific notifications as well as for multiple RASFF notifications related to a certain good (e.g. chicken eggs, pet food, etc.). Results RASNEX can extract all the actors involved in an ongoing or previous chemical contamination event or biological agent outbreak from the structured parts of RASFF notifications and compile the extracted information in sheets, thereby enabling the use of this data for further analysis with other tools (e.g. Excel, RACE [5], FoodChain-Lab and others). RASNEX generates a graphical mapping of all of the actors of the supply network provided and extracted by the software, who are involved in the contamination events of interest. In addition, the tool screens the “additional information” section and the follow-up-documents for the involved parties and products that were not reported in the main section of the RASFF notifications. Discussion RASNEX is a user-friendly tool that performs an automated data cleaning from the RASFF notifications thus facilitating and tremendously accelerating the extraction of the information that is relevant at both national and European level. In this first step, RASNEX hasfocused on the extraction of data on the main and related products from the major sections of an RASFF notification. RASNEX is being continuously improved to also enable the extraction of data from the additional information section (free text fields) and attached documents (e.g. PDF files, Excel spreadsheets in various forms). In the course of a collaborative project between BfR and the European Food Safety Agency (EFSA), RASNEX is being further developed to address this limitation. This further development hints at the future modifications to the RASFF system that will facilitate future data exchange and interpretation. References: 1. Lorenzen et al., doi:10.1371/journal.pone.0254301 2. Berthold et al., doi:10.1145/1656274.1656280 3. Berthold et al., doi: 10.1007/978-3-540-78246-9_38 4. Weiser et al., doi: 10.1371/journal.pone.0151977 5. Fürst et al., doi: 10.2903/sp.efsa.2019.EN-1625
A Representative Approach to Investigate In Vitro Species Differences in Crop Protection Active Ingredient Metabolism	Marc Lamshoeft	CropLife Europe	New approach methodologies: moving beyond animal testing	DETAILED INFO Marc Lamshoeft, CropLife Europe Title of work A Representative Approach to Investigate In Vitro Species Differences in Crop Protection Active Ingredient Metabolism Co-author(s) Leonie Madita Gellrich, Matthew Himmelstein, Thomas Holmes, Felix M. Kluxen, Marie Kohler, Gopinath Nallani, Udo Rabe, Paul Whalley E-POSTER DOWNLOAD POSTER ABSTRACT Introduction The scientific opinion of the European Food Safety Authority (EFSA) on the proposed design of in vitro comparative metabolism (IVCM) studies to evaluate pesticide active substances was reviewed by CropLife Europe (CLE) member experts. The study requirement originates from Commission Regulation (EU) No 283/2013. The IVCM study objective ‘is to evaluate whether all metabolites formed in the human in vitro test system as a surrogate of the in vivo situation are also present at a comparable level in animal species tested in toxicological studies, and, therefore, if their potential toxicity has been appropriately covered by animal studies’. EFSA proposes a study design which uses hepatocytes for identification of Unique Human Metabolites (UHM) and Disproportionate Human Metabolites (DHM), investigates 5 different species, and proposes a 3×3×3 sampling matrix per each species, with the assumption that the capabilities of in vitro test system sufficiently mimic the in vivo situation. In addition to the detailed description of experimental set-up, the opinion expands to include a description of toxicity testing and risk assessment and extensive discussion on future trends. Methodology The member experts identified seven major areas of concern 1) Legal Requirements – The regulation 283/2013 has no reference to DHM; 2) DHM assessment – the proposed design may not be suitable to address a DHM situation due to the inherent limitations of the in vitro system, and (if required) should consider existing knowledge of in vivo metabolism and apply a weight of evidence approach; 3) Definition of an identification threshold is needed – 5 % of applied radioactive dose; 4) Experimental set-up – The proposed extensive 3 × 3 × 3 approach may not be suitable; 5) Long-term incubations for slow-metabolising compounds – Option to waive based on in vivo metabolism information; 6) Timing of comparative in vitro studies – Needs to allow flexibility in timing of the studies for different registration strategies, and 7) Recommendations for the future – Topics (e.g. reactive metabolites, isoform specific metabolism, stereoisomers, and PBK modelling) should be considered for a separate opinion. Results Industry experience exists for IVCM studies in microsomes & hepatocytes to identify UHMs following Whalley et al 2017. A representative approach to investigate in vitro species differences in metabolism is proposed. Stepwise, a pilot experiment using cryopreserved hepatocytes from humans & another appropriate species (e.g. rat) at 1 or 2 incubations (1-20 µM, ~105 dpm) & multiple timepoints (e.g. 5, 30, 60, 120, 240 min) is conducted. Additional endpoints include cell (Trypan) & positive control ECOD for viability. The pilot phase is key to establishing analytical (14C & LC/MS) methods. Based on the pilot results, a definitive experiment with multiple species (human, rat, mouse, rabbit & dog) is designed using a single incubation concentration, 2 time points to cover changes in early & total metabolite formation (e.g. 30 & 240 min). Pilot & definitive experiments always include assessment of non-specific binding, positive & negative controls, extraction efficiency & recovery. Detection limits will be case specific, but the goal is to identify human metabolites formed at ≥5 % of the radioactive dose whether or not they are detected in other species, with detection to as low as 1 %. Discussion This representative approach will be demonstrated by use of example data. A weight of evidence approach and understanding of the metabolic pathway is important to cross-species comparison since Phase I and Phase II activity is known to differ among species. A flexible experimental approach is needed to account for chemical specific properties and recognise the limitations of relying on in vitro system. Identification of a UHM after consideration of the aforementioned points will require further investigation and potential toxicological evaluation.
Untargeted mass spectrometry-based proteomics for species and tissue identification in mixed feed and food samples	Madhushri Shrikant Varunjikar	Institute of Marine Research (IMR)	Innovation in food and feed: keeping safety assessments fit for purpose	DETAILED INFO Madhushri Shrikant Varunjikar, Institute of Marine Research (IMR) Title of work Untargeted mass spectrometry-based proteomics for species and tissue identification in mixed feed and food samples Co-author(s) Ikram Belghit Kai K. Lie, Sonnich Meier, Magnus Palmblad, Josef D. Rasinger E-POSTER DOWNLOAD POSTER ABSTRACT Introduction Due to globally rising demands for food and feed, novel protein ingredients are being introduced into our food systems on an increasing scale. This gives rise to novel challenges in relation to the detection of feed and food fraud and the determination of feed and food authenticity, respectively. In this context, the development and increased implementation of rapid, sensitive and robust molecular methods are key. However, progress in the application of such tools has been hampered by a general lack of well-annotated reference genomes of target species commonly used or newly introduced in feed or food preparations. Our group has been working on the development and implementation of untargeted mass spectrometry-based approaches in regulatory settings to identify, differentiate, and quantify proteic ingredients of animal and plant origin in various food and feed mixes without using any genomic information. Methodology The workflow presented here comprises the enzymatic digestion of samples using trypsin, analysis of extracts using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS), data processing using direct spectra comparison (compareMS2) and proteomics spectra library (SL)-based analyses using tools of the Trans-Proteomics Pipeline (TPP). All data generated and published by our group are being made available on public repositories for MS data such as the Mass Spectrometry interactive Virtual Environment (MassIVE) hosted at the University of California San Diego (UCSD), and also are added to an in-house SL database specifically developed for the species and tissue-specific screening of food and feed samples of marine and terrestrial origin. Results The untargeted proteomics workflow described above has already been used successfully to differentiate processed animal proteins (PAP). It has also been used to identify and authenticate food and feed-grade insect species and to detect whether black soldier fly (BSF) larvae fed on prohibited substrates could be differentiated from conventionally fed insect species. We also found that SL-based untargeted proteomics can be used for the identification of common allergens in food-grade insect samples. Currently, we are investigating whether untargeted MS-based methods can also be used to successfully differentiate and authenticate 30 marine fish species both in pure fillet and mixed samples containing two or more fish species. Discussion The untargeted proteomics SL-based approach implemented in our laboratory was shown to be capable of both species and tissue-specific identification of proteinaceous food and feed ingredients, including processed animal proteins (PAP), insect proteins, and plant, mammalian and fish proteins. Future work is focusing on the differentiation and detection of seafood fraud, which has recently been highlighted as an emerging issue in the global food market. We are also in the process of making all species and tissue-specific MS data collected in the above-listed (and in parts already published) studies available on request to researchers in the field using a dedicated web-based service. The latter is currently being developed in-house and, following proper quality testing, is envisaged to be released publicly to provide research and regulatory laboratories with an easily accessible platform for authenticating and identifying proteinaceous ingredients in feed and food in both pure and mixed samples.
Bioavailability of aflatoxins in cultured fish and animal livers using an in-vitro dialysability approach	M Raquel Domínguez-González	Universidad de Santiago de Compostela (USC)	Innovation in food and feed: keeping safety assessments fit for purpose	DETAILED INFO M Raquel Domínguez-González, Universidad de Santiago de Compostela (USC) Title of work Bioavailability of aflatoxins in cultured fish and animal livers using an in-vitro dialysability approach Co-author(s) Thilini G.D. Madurangika-Jayasinghe, Paloma Herbello-Hermelo, Raquel M. Domínguez-González, Pilar Bermejo-Barrera, Antonio Moreda-Piñeiro E-POSTER DOWNLOAD POSTER ABSTRACT Introduction Aflatoxins (AFs) represent a group of toxic metabolites of mould that significantly affect human and animal health. Animal tissues can retain AF residues, including AF metabolites, giving rise to potential health issues. They can be transferred to the human body from food (including meat). To understand how AFs can transform, sufficient knowledge of the amount of AFs in foodstuffs and the fraction of AFs absorbed by the human body is required. Human bioavailability approaches, mainly encompassing processes such as digestion, absorption, transport, utilisation and elimination, are useful strategies for understanding the fraction of nutrients and pollutants that can theoretically be released into the gastrointestinal tract (GI) and become available for intestinal absorption. Human bioavailability approaches are split into in-vivo and in-vitro assays. The current study is focused on the development of an in-vitro bioaccessibility method, which helps to indicate the maximum fraction of AFs in the muscle and liver that can theoretically be released from foodstuffs into the GI tract (bioaccessible fraction). This then becomes available for entering into the bloodstream. Methodology The AF content in (raw and cooked) muscle and liver samples was extracted with 5 mL of 60:40 acetonitrile/0.1 M aqueous KH2PO4 (pH 7.0) solution and the extract was subjected to a pre-concentration method based on a vortex-assisted liquid-liquid microextraction (VALLME) procedure which consisted of mixing 25 mg of NaCl with 5.0 mL of extract and adding 400 μL of chloroform as an extractant before vortexing at 2000 rpm for 1 min. The chloroform extract was fully dried under an N2 flow and re-dissolved in 100 μL of methanol and injected into the UHPLC-MS-MS instrument. The in-vitro dialysability procedure was performed with gastric solution (16 % (m/v) pepsin in 0.1 M HCl) and intestinal solution (4.0 % (m/v) pancreatin and 2.5 % (m/v) bile salt solution prepared in 0.1 M sodium hydrogen carbonate, pH 7.4) under the optimum conditions. AF content in dialysable (in PIPES solution) and non-dialysable fractions was evaluated by following the validated VALLME method. A mass balance study was used to assess the accuracy of the bioavailability study and statistically compared (ANOVA test, 95 % confidence interval) with the AF concentration in the standards/foodstuffs prior to the in-vitro bioavailability approach. Results Raw samples showed bioavailability ratios of 41−45 % for aflatoxin B1 (AFB1), 28−38 % for aflatoxin B2 (AFB2), and 42 % for aflatoxin G2 (AFG2). Aflatoxin G1 (AFG1) was not detected. The cooking process (steaming or grilling) was found to change AF bioavailability (higher bioavailability ratios were observed in cooked samples). The cooking process was found to affect the dialysability of AFs in different ways. Steaming does not affect AF bioavailability. Dialysability ratios (42−54 % for AFB1, 29 % for AFB2, and 43 % for AFG2) were quite similar to those found in raw samples. However, grilling increases AF bioavailability, and AFB1 dialysability was found to be within the 54−59 % range, while values of 37 and 56 % were obtained for AFB2 and AFG2, respectively. Statistical dialysability ratios for fried samples were found to be significantly higher (p < 0.05) than those found in raw and steamed samples and in AF aqueous standards. AFB2 was found to be transformed into other compounds during the in-vitro process, and the presence of AFB2 and AFB2 transformation/degradation products was investigated and confirmed by high-resolution mass spectrometry (HRMS). Discussion Cooked samples, predominantly fried samples, produced slightly higher dialysable AF concentrations than raw samples. These findings align with those obtained when assessing the AF content in raw and cooked samples. They are explained by taking into account the moisture content of the samples, which is reduced from 78-83 % in raw samples to 69-76 % in steam samples and 43-59 % in fried samples. A mass balance study was performed by comparing the sum of the AF levels in the dialysable and non-dialysable fractions with AF content in the samples. AFB2 indicates a significant statistical difference between AFB2 in the sample and the sum of AFB2 concentrations in the dialysable and non-dialysable fractions. These results align with those obtained when using AFB2 aqueous standards and should be attributed to AFB2 transformation/degradation into other compounds during the in-vitro assay. Therefore, AFB2 is degraded during the in-vitro dialysability process and several degradation products are present in both dialysable and non-dialysable fractions.
Science-to-policy translation in the One Health EJP: from gap-driven research to dissemination and impact	Ludovico Sepe	German Federal Institute for Risk Assessment (BfR)	Making a difference: bridging EU research and policy	DETAILED INFO Ludovico Sepe, German Federal Institute for Risk Assessment (BfR) Title of work Science-to-policy translation in the One Health EJP: from gap-driven research to dissemination and impact Co-author(s) Pikka Jokelainen, Aura Andreasen, Annemarie Kaesbohrer E-POSTER DOWNLOAD POSTER ABSTRACT Introduction The One Health European Joint Programme (One Health EJP) is a partnership between 43 public health, animal health, and food safety institutes and the Med-Vet-Net Association, that focuses on foodborne zoonoses, antimicrobial resistance, and emerging threats. The One Health EJP aims to improve preparedness, detection, and response to One Health issues by enhancing collaboration, integration of activities and harmonisation of approaches. While conducting gap-driven research is a central activity within the One Health EJP, efficient science to policy translation mechanisms are in place to ensure the produced outcomes are used and have an impact. A dedicated Work Package (WP) of the One Health EJP focuses on science-to-policy translation. Methodology One Health EJP partner institutions have mandates from their national or regional authorities. Activities of the consortium follow a prioritised Strategic Research Agenda that addresses identified needs of both national and international stakeholders, and the scientific work aims at closing prioritised knowledge gaps. Tools of formal interaction with relevant EU and international stakeholders are consolidated and constantly adapted. An important component of the dialogue with the stakeholders is targeted dissemination of outputs of the consortium to national, European, and international decision/policy makers via reports and meetings. The targeted dissemination to stakeholders complements the general dissemination and communication activities of the consortium, responds to efficient identification of stakeholders’ needs, and is tailored in terms of content and language to appeal to the specific audiences. Additional support is provided by an accessible Outcome Inventory and published following the FAIR (findable, accessible, interoperable and reusable) principle. Results The Stakeholders Committee of One Health EJP is one of the main exchange fora. It covers international organisations across the field of One Health (ECDC, EFSA, EEA, EMA, FAO, OIE and WHO-EURO). Dedicated links to point of contacts are established within each organisation, allowing productive dialogue both in regular meetings and at other times. Given particular interest in themes of the meeting, additional representatives of the stakeholders’ organisations are welcome to join. This combination of flexibility together with having dedicated contacts, ensures that the dialogue and dissemination activities reach the right persons in the stakeholders’ organisations. Dialogue with the stakeholders and identification of their needs guides targeted dissemination, including thematic reports and dissemination workshops. Impact is also achieved by providing ad hoc support. Discussion The One Health EJP contributes to the prevention, detection, and response to One Health issues including foodborne zoonotic threats at the national and European level by supporting 1) evidence-based policy decisions by contributing to risk assessments, and 2) practical and sustainable implementation of the consortium’s outcomes. Engagement with stakeholders is crucial to gain perceived and measurable impact. The One Health EJP also gathers policy intelligence, which will shape post-One Health EJP initiatives in the field of One Health. In parallel, the solutions developed and the lessons learned from the international, multidisciplinary One Health EJP will support stakeholders’ future initiatives in light of the increasingly important One Health paradigm. Science-to-policy is at best a dialogue.
Evaluation of an adverse outcome pathway network for thyroid hormone system disruption across taxonomic groups	Lucia Vergauwen	University of Antwerp	Endocrine disruptors: exploring present challenges and future developments (in memory of Alfonso Lostia)	DETAILED INFO Lucia Vergauwen, University of Antwerp Title of work Evaluation of an adverse outcome pathway network for thyroid hormone system disruption across taxonomic groups Co-author(s) Jason M. O’Brien, Carlie A. LaLone, Daniel L. Villeneuve, Imke Van Dingenen, Lisa Baumann, Thomas Braunbeck, Henrik Holbech, Dries Knapen E-POSTER DOWNLOAD POSTER ABSTRACT Introduction Thyroid hormone system disrupting chemicals (THSDCs) are widely regarded as potential threats to human and environmental health. Thus, efforts are under way within the human health and ecotoxicology communities to develop screening assays capable of identifying THSDCs and to describe adverse outcome pathways (AOPs) that link thyroid hormone system disruption (THSD) to adverse outcomes. The Horizon 2020 project EndocRine Guideline Optimisation (ERGO) aims to break down the wall between human and environmental health assessment of chemicals using an AOP network (AOPN) approach and promoting the use of non-mammalian assays for predicting effects in mammals, including humans, and vice versa. Methodology In recent years, a fish-specific AOPN, which consists of 5 AOPs linking the inhibition of enzymes which are important for the synthesis and activation of THs to impaired swim bladder inflation, has been developed. This network is currently being expanded to include AOPs leading to altered visual function. When further expanding the network to include all AOPs for THSD in different species (either endorsed or under development), a cross-species AOPN for THSD emerges. This broader AOPN, which includes AOPs currently applicable to fish, amphibians or mammals, provides a scientifically plausible and evidence-based foundation for the measurement of endpoints using fish and amphibian assays to predict outcomes in humans and vice versa. In the present work, we evaluated which AOPs have already been defined for more than one taxon, which AOPs are taxon-specific and thus irrelevant to other taxa, and which novel pathways linking molecular initiating events to adverse outcomes in a particular taxon are potential targets for dedicated AOP development. Based on this evaluation, we identified assays in fish and amphibians that are likely to predict effects in humans and vice versa. Results By filtering the AOPN based on the currently described taxonomic domain of applicability of the AOPs (i.e. fish, amphibians or mammals), it was found that most AOPs have been developed with a focus on one specific taxonomic group and information on the taxonomic domain of applicability is often missing. The AOPN currently includes 14 molecular initiating events (MIEs) leading to THSD and the coverage of MIEs is highest in the AOPs applicable to amphibians. Furthermore, while mammalian AOPs largely focus on developmental toxicity (DNT) as the adverse outcome, fish and amphibian AOPs thus far mostly lead to impaired swim bladder inflation and altered amphibian metamorphosis – both taxon-specific adverse outcomes. Other adverse outcomes include thyroid carcinoma, kidney toxicity, altered visual function, hearing loss and reduced fertility. Discussion Based on this evaluation, we identified data gaps and prioritised AOP development efforts. AOPs leading to altered visual function are currently being developed as a first explicit case of cross-species AOP development. Based on the evaluation of the AOP network, priority endpoints to be added to fish and/or amphibian tests include thyroid hormone levels, swim bladder inflation, impaired eye development, thyroid histopathology and expression of thyroid-related genes. Since developmental neurotoxicity (DNT) is often considered the most important outcome of THSD in humans, and no AOPs for DNT in fish and amphibians exist, DNT was highlighted as an additional priority for cross-species AOP development and a potential route to expedite the use of fish and amphibian assays for predicting effects in humans. Other than DNT, there are adverse health effects with the potential for cross-species extrapolation, including reduced fertility, hearing loss or neurosensory development in general, and kidney toxicity. Lastly, another significant omission is the lack of AOPs describing THSD in birds and reptiles. The content of this abstract neither constitutes US EPA policy nor necessarily reflects such policy.
Characterisation of different Black Soldier Fly (Hermetia illucens) protein extracts and potential application as an emulsifier in oil-in-water emulsions	Lucas Sales Queiroz	Technical University of Denmark	Innovation in food and feed: keeping safety assessments fit for purpose	DETAILED INFO Lucas Sales Queiroz, Technical University of Denmark Title of work Characterisation of different Black Soldier Fly (Hermetia illucens) protein extracts and potential application as an emulsifier in oil-in-water emulsions Co-author(s) Federico Casanova, Betul Yesiltas, Fatemeh Ajalloueian, Aberham Feyissa, Antonio Carvalho, Italo Perrone, Charlotte Jacobsen, Mohammad Mohammadifar E-POSTER DOWNLOAD POSTER ABSTRACT Introduction According to the UN Food and Agriculture Organization (FAO) the global population is expected to reach 9 billion in 2050. However, current agricultural systems are not prepared, in terms of sustainability, to address such a huge demand on the food supply –something that was made more evident during the COVID-19 pandemic, when there was reduced meat supply on the market. Hence, there is an urgent need to explore alternative protein sources, which must provide good nutritional value and functionality and must be sustainable. Edible insects have drawn interest from the FAO and the scientific community, being reported as a promising protein source with potential technological applications. Recently, the European Union has authorised the placing on the market of the first insect as a novel food (2015/2283). Black soldier fly larvae (BSFL) has largely been utilised for animal feed. Due to its interesting composition, BSFL shows great potential for being further implemented in the human diet. In addition, sustainable methods can be applied to optimise protein extraction from insects and improve their techno-functional properties, such as the emulsifying properties. Methodology In this study, defatted BSFL powder was treated by ohmic heating (BSFL-OH), ohmic heating and ultrasound (BSFL-UOH) and compared to sodium caseinate (CAS) for their emulsifying properties. To have wide knowledge on the protein structure and profile, Fourier-transformed infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and the interfacial properties for oil-in-water emulsion were evaluated. Emulsions were produced with cod liver oil-in-water using a microfluidizer system and analysed during 10 days of storage. The emulsion stability was evaluated by Turbiscan, considering the TSI value. In addition, droplet size and z-size were also evaluated. The oxidative stability of each emulsion was analysed by the peroxide value (PV) method for primary oxidative stability, tocopherol content and secondary oxidation products by dynamic headspace GC-MS. Results A clear difference in the secondary structure was observed between BSFL and the treated samples, considering mainly the amide I region and the qualitative analysis of the presence of b-sheets, a-helices, random coils and b-turns. The protein extraction was conducted by the alkaline method for all the samples and the yield of protein content was 62 %, 67 %, and 66 % for BSFL, BSFL-OH and BSFL-UOH. A decrease in the denaturation temperature for samples treated with ohmic heating and then ultrasound was observed, when compared to untreated BSFL samples. The best stability was reported by BSFL-OH emulsion according to TSI value. CAS showed the smallest droplet size followed by BSFL-OH, BSFL and BSFL-UOH. z-potential had values higher than -30 mV and was similar for all emulsions including CAS. Among the insect samples, BSFL-OH was shown to reduce the interfacial tension more efficiently compared to the other samples. CAS showed the lowest oxidation after 10 days, followed by BSFL, BSFL-OH and BSFL-UOH. The secondary volatile compounds were higher for the emulsions prepared with insect protein as emulsifier, with BSFL-OH and BSFL-UOH showing the highest values over 10 days of storage. Discussion The secondary structure of BSFL-OH showed a unique pattern and the b-turns region was shown to be more affected by ultrasound treatment. For DSC, an increase in enthalpy was observed for the BSF-UOH sample, probably due to protein aggregation and bond formation after treatment. An increase in enthalpy and decrease in temperature is observed for BSFL-UOH, which might be due to the long exposure time of samples to ultrasound treatment causing protein aggregation. Considering emulsion stability by TSI, the more uniform heating and higher protein content provided by ohmic heating treatment was shown to be more interesting for emulsifying stability. The increase in particle size noted more evidently for BSFL-UOH can directly impact the emulsion stability, due to the presence of aggregates. The lowest particle size observed in BSFL-OH could also contribute to a more interesting decrease in the interfacial tension of the oil-in-water interface. All insect emulsions showed a high PV and volatile value after 10 days of storage. Therefore, as this was a pioneering study using BSFL for oxidative stability tests, further treatments might bring promising results in the future.
The carriage of antibiotic resistance genes in the environment: the case of Campylobacter in surface water and wild birds in Luxembourg	Louise Hock	Luxembourg Institute of Science and Technology (LIST)	Tackling antimicrobial resistance in food producing environments	DETAILED INFO Louise Hock, Luxembourg Institute of Science and Technology (LIST) Title of work The carriage of antibiotic resistance genes in the environment: the case of Campylobacter in surface water and wild birds in Luxembourg Co-author(s) Cécile Walczak, Juliette Mosser, Maureen Feucherolle, Christian Penny, Catherine Ragimbeau, Henry-Michel Cauchie. E-POSTER VIDEO POSTER DOWNLOAD POSTER ABSTRACT Introduction Each new technology brings its own advantages and risks. Even if antibiotics allow us to fight severe bacterial diseases, their unreasonable use increases the selection of resistant bacteria that currently have a negative impact on Public Health. The transmission and widespread prevalence of bacteria carrying antimicrobial resistance (AMR) genes in wildlife and the environment could represent an additional challenge by turning the environment into an AMR hotspot of putative critical epidemiology Campylobacteriosis is recognised as the principal bacterial gastroenteritis in industrialised countries. Luxembourg presents one of the highest incidence rates of Campylobacter infections in the EU: 103.8 cases per 100,000 inhabitants in 2018 (EFSA and ECDC, 2019). Campylobacter jejuni is implicated in more than 80 % of human cases whereas Campylobacter coli accounts for the majority of the rest. Although campylobacteriosis is usually self-limiting, antimicrobial treatment is necessary in the case of severe infections. Our aim is to assess the prevalence of AMR in C. jejuni and C. coli strains isolated from surface water and wild birds to obtain a picture of the AMR genes carriage in Luxembourg environment. Methodology An environmental collection (set under the FNR Core Campylomic project 11684203) of 93 C. coli and 80 C. jejuni isolated from water surface (N=99) and wild bird faeces (N=74) were characterised according their antimicrobial resistances using disk diffusion methods following EUCAST recommendations (EUCAST, 2021). These phenotypic resistance results were compared to “in silico” antibiograms generated from NGS data (Next Generation Sequencing, Illumina technologies) submitted to the ResFinder database. Results A total of 6.5 % of the C. coli and 21 % of the C. jejuni isolates presented at least one antibiotic resistance whereas half of the Campylobacter isolates possessed known AMR genes. The most prevalent resistance observed was to ampicillin and quinolone antibiotics (8 and 5 % of isolates, respectively): nalidixic acid and ciprofloxacin. The single mutation of the DNA gyrase A (C257T) conferring the quinolone resistance was encountered in 4 % of the collection. No florfenicol (phenol), amoxicillin (beta-lactam) and gentamicin (aminoglycoside) phenotypic resistances were observed whereas aminoglycoside resistance genes were detected in 14 % of the Campylobacter isolates. Resistant strains were found in 17.5 and 10 % of the wild bird and surface water samples, respectively. Two C. jejuni isolates from water samples were multiresistant (Beta-lactam, Quinolone and Tetracyclin). Discussion In Luxembourg, resistance to at least one antibiotic was demonstrated by one in eight Campylobacter isolates from wild birds and surface water. Isolates are initially exposed to antibiotics both in animal production systems and in human medicine through drug treatments. Resistant strains emerging from this selection pressure could then reach the environment via faeces and wastewater. Moreover, some wild birds, which are well adapted to anthropogenic environments, come into close contact with livestock, domestic animals and humans. Therefore, they become reservoirs of antimicrobial resistance and contribute to the multi-host spread of AMR. Consequently, the surveillance of resistant bacteria in wilds animals and the environment is necessary to guide public health policies to control foodborne illnesses in humans.