Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Systematic review on microbiome-related nutritional interventions interfering with the colonization of foodborne pathogens in broiler gut to prevent contamination of poultry meat.

This systematic review aimed to compile the available body of knowledge about microbiome-related nutritional interventions contributing to improve the chicken health and having an impact on the reduction of colonization by foodborne pathogens in the gut. Original research articles published between 2012 and 2022 were systematically searched in Scopus and PubMed. A total of 1,948 articles were retrieved and 140 fulfilled the inclusion criteria. Overall, 73 papers described 99 interventions against colonization by Escherichia coli and related organisms; 10 papers described 15 interventions against Campylobacter spp.; 36 papers described 54 interventions against Salmonella; 40 papers described 54 interventions against Clostridium perfringens. A total of 197 microbiome-related interventions were identified as effective against one or more of the listed pathogens and included probiotics (n = 80), prebiotics (n = 23), phytobiotics (n = 25), synbiotics (n = 12), organic acids (n = 12), enzymes (n = 4), essential oils (n = 14) and combination of these (n = 27). The identified interventions were mostly administered in the feed (173/197) or through oral gavage (11/197), in the drinking water (7/197), in ovo (2/197), intra amniotic (2/197), in fresh or reused litter (1/197) or both in the feed and water (1/197). The interventions enhanced the beneficial microbial communities in the broiler gut as Lactic acid bacteria, mostly Lactobacillus spp., or modulated multiple microbial populations. The mechanisms promoting the fighting against colonization by foodborne pathogens included competitive exclusion, production of short chain fatty acids, decrease of gut pH, restoration of the microbiome after dysbiosis events, promotion of a more stable microbial ecology, expression of genes improving the integrity of intestinal mucosa, enhancing of mucin production and improvement of host immune response. All the studies extracted from the literature described in vivo trials but performed on a limited number of animals under experimental settings. Moreover, they detailed the effect of the intervention on the chicken gut without details on further impact on poultry meat safety.

Microbiological safety of dry-cured fish from the raw material to the end of processing.

The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption.

Salmonella spp. in poultry production-A review of the role of interventions along the production continuum.

Salmonella is a significant pathogen of human and animal health and poultry are one of the most common sources linked with foodborne illness worldwide. Global production of poultry meat and products has increased significantly over the last decade or more as a result of consumer demand and the changing demographics of the world's population, where poultry meat forms a greater part of the diet. In addition, the relatively fast growth rate of birds which is significantly higher than other meat species also plays a role in how poultry production has intensified. In an effort to meet the greater demand for poultry meat and products, modern poultry production and processing practices have changed and practices to target control and reduction of foodborne pathogens such as Salmonella have been implemented. These strategies are implemented along the continuum from parent and grandparent flocks to breeders, the farm and finished broilers to transport and processing and finally from retail to the consumer. This review focuses on common practices, interventions and strategies that have potential impact for the control of Salmonella along the poultry production continuum from farm to plate.

Persistence of microbiological hazards in food and feed production and processing environments.

Listeria monocytogenes (in the meat, fish and seafood, dairy and fruit and vegetable sectors), Salmonella enterica (in the feed, meat, egg and low moisture food sectors) and Cronobacter sakazakii (in the low moisture food sector) were identified as the bacterial food safety hazards most relevant to public health that are associated with persistence in the food and feed processing environment (FFPE). There is a wide range of subtypes of these hazards involved in persistence in the FFPE. While some specific subtypes are more commonly reported as persistent, it is currently not possible to identify universal markers (i.e. genetic determinants) for this trait. Common risk factors for persistence in the FFPE are inadequate zoning and hygiene barriers; lack of hygienic design of equipment and machines; and inadequate cleaning and disinfection. A well-designed environmental sampling and testing programme is the most effective strategy to identify contamination sources and detect potentially persistent hazards. The establishment of hygienic barriers and measures within the food safety management system, during implementation of hazard analysis and critical control points, is key to prevent and/or control bacterial persistence in the FFPE. Once persistence is suspected in a plant, a 'seek-and-destroy' approach is frequently recommended, including intensified monitoring, the introduction of control measures and the continuation of the intensified monitoring. Successful actions triggered by persistence of L. monocytogenes are described, as well as interventions with direct bactericidal activity. These interventions could be efficient if properly validated, correctly applied and verified under industrial conditions. Perspectives are provided for performing a risk assessment for relevant combinations of hazard and food sector to assess the relative public health risk that can be associated with persistence, based on bottom-up and top-down approaches. Knowledge gaps related to bacterial food safety hazards associated with persistence in the FFPE and priorities for future research are provided.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification 'for production purposes only'; Clostridium tyrobutyricum is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.

Routes of dispersion of antibiotic resistance genes from the poultry farm system.

Poultry farms are hotspots for the development and spread of antibiotic resistance genes (ARGs), due to high stocking densities and extensive use of antibiotics, posing a threat of spread and contagion to workers and the external environment. Here, we applied shotgun metagenome sequencing to characterize the gut microbiome and resistome of poultry, workers and their households - also including microbiomes from the internal and external farm environment - in three different farms in Italy during a complete rearing cycle. Our results highlighted a relevant overlap among the microbiomes of poultry, workers, and their families (gut and skin), with clinically relevant ARGs and associated mobile elements shared in both poultry and human samples. On a finer scale, the reconstruction of species-level genome bins (SGBs) allowed us to delineate the dynamics of microorganism and ARGs dispersion from farm systems. We found the associations with worker microbiomes representing the main route of ARGs dispersion from poultry to human populations. Collectively, our findings clearly demonstrate the urgent need to implement more effective procedures to counteract ARGs dispersion from poultry food systems and the relevance of metagenomics-based metacommunity approaches to monitor the ARGs dispersion process for the safety of the working environment on farms.

Statement on how to interpret the QPS qualification on 'acquired antimicrobial resistance genes'.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications' which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification 'the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials' applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms 'intrinsic' and 'acquired' AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the 'intrinsic'/'acquired' nature of an AMR gene. All AMR genes that confer resistance towards 'critically important', 'highly important' and 'important' antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for 'intrinsic' resistance could be considered as being of no concern in the frame of the EFSA risk assessment. 'Acquired' AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the 'acquired' AMR gene is not leading to phenotypic resistance, further case-by-case assessment is necessary.

Variability in Physicochemical Parameters and Its Impact on Microbiological Quality and Occurrence of Foodborne Pathogens in Artisanal Italian Organic Salami.

Artisanal salami is produced in small-scale production plants, where the lack of full automation might result in higher variability in food intrinsic properties. The aim of the present study was to evaluate the inter- and intra-batch variability in physicochemical parameters and its impact on microbial quality and occurrence of foodborne pathogens on 480 samples collected from six batches of an artisanal Italian production of organic salami. Relatively high total bacterial counts (TBC) were found on the surface of the table in the stuffing room (4.29 ± 0.40 log cfu/cm2). High loads of Enterobacteriaceae in the meat mixture of batch 2 and TBC in batch 5 were associated with a higher occurrence of bacterial pathogens. During ripening, water activity (aw) and pH failed to reach values lower than 0.86 and 5.3, respectively. Six Staphylococcus aureus and four Listeria monocytogenes isolates were collected from the salami meat mixture during ripening and the processing environment. A total of 126 isolates of Enterobacteriaceae were characterized at a species level, with Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Citrobacter freundii isolated from the final products. Results suggest the relevance of first steps of production in terms of the hygiene of raw materials and handling during stuffing procedures, especially when the physicochemical parameters of the final products do not reach values that represent hurdles for foodborne pathogens.

Microbiological hazards associated with the use of water in the post-harvest handling and processing operations of fresh and frozen fruits, vegetables and herbs (ffFVHs). Part 1 (outbreak data analysis, literature review and stakeholder questionnaire).

The contamination of water used in post-harvest handling and processing operations of fresh and frozen fruit, vegetables and herbs (ffFVHs) is a global concern. The most relevant microbial hazards associated with this water are: Listeria monocytogenes, Salmonella spp., human pathogenic Escherichia coli and enteric viruses, which have been linked to multiple outbreaks associated with ffFVHs in the European Union (EU). Contamination (i.e. the accumulation of microbiological hazards) of the process water during post-harvest handling and processing operations is affected by several factors including: the type and contamination of the FVHs being processed, duration of the operation and transfer of microorganisms from the product to the water and vice versa, etc. For food business operators (FBOp), it is important to maintain the microbiological quality of the process water to assure the safety of ffFVHs. Good manufacturing practices (GMP) and good hygienic practices (GHP) related to a water management plan and the implementation of a water management system are critical to maintain the microbiological quality of the process water. Identified hygienic practices include technical maintenance of infrastructure, training of staff and cooling of post-harvest process water. Intervention strategies (e.g. use of water disinfection treatments and water replenishment) have been suggested to maintain the microbiological quality of process water. Chlorine-based disinfectants and peroxyacetic acid have been reported as common water disinfection treatments. However, given current practices in the EU, evidence of their efficacy under industrial conditions is only available for chlorine-based disinfectants. The use of water disinfection treatments must be undertaken following an appropriate water management strategy including validation, operational monitoring and verification. During operational monitoring, real-time information on process parameters related to the process and product, as well as the water and water disinfection treatment(s) are necessary. More specific guidance for FBOp on the validation, operational monitoring and verification is needed.

Application of multi-omic features clustering and pathway enrichment to clarify the impact of vitamin B2 supplementation on broiler caeca microbiome.

Background: The results of omic methodologies are often reported as separate datasets. In this study we applied for the first time multi-omic features clustering and pathway enrichment to clarify the biological impact of vitamin B2 supplementation on broiler caeca microbiome. Methods: The caeca contents of broilers fed +50 and +100 mg/kg vitamin B2 were analyzed by shotgun metagenomic and metabolomic. Latent variables extracted from NMR spectra, as well as taxonomic and functional features profiled from metagenomes, were integrated to characterize the effect of vitamin B2 in modulating caeca microbiome. A pathway-based network was obtained by mapping the observed input genes and compounds, highlighting connected strands of metabolic ways through pathway-enrichment analysis. Results: At day 14, the taxonomic, functional and metabolomic features in the caeca of tested broilers showed some degree of separation between control and treated groups, becoming fully clear at 28 days and persisting up to 42 days. In the caeca of birds belonging to the control group Alistipes spp. was the signature species, while the signature species in the caeca of broilers fed +50 and +100 mg/kg vitamin B2 were Bacteroides fragilis and Lactobacillus crispatus, Lactobacillus reuteri, Ruminococcus torques, Subdoligranum spp., respectively. The pathway enrichment analysis highlighted that the specific biochemical pathways enhanced by the supplementations of vitamin B2 were N-Formyl-L-aspartate amidohydrolase, producing Aspartate and Formate; L-Alanine:2-oxoglutarate amino transferase, supporting the conversion of L-Alanine and 2-Oxoglutarate in Pyruvate and L-Glutamate; 1D-myo-inositol 1/4 phosphate phosphohydrolase, converting Inositol 1/4-phosphate and water in myo-Inositol and Orthophosphate. The results of this study demonstrated that the caeca of birds fed +50 and + 100 mg/kg were those characterized by taxonomic groups more beneficial to the host and with a higher concentration of myo-inositol, formic acid, amino acids and pyruvate involved in glycolysis and amino acid biosynthesis. Conclusion: In this study we demonstrated how to perform multi-omic features integration to describe the biochemical mechanisms enhanced by the supplementation of different concentrations of vitamin B2 in the poultry diet. The relationship between vitamin B2 supplementation and myo-inositol production was highlighted in our study for the first time.

Reduction of the microbial load in meat maturation rooms with and without alkaline electrolyzed water fumigation.

Dry aging is a process during which meat is stored within maturation chambers at low temperatures and low relative humidity, resulting in improved tenderness and flavor development. The cuts are exposed to the atmosphere by hanging them or setting them on racks in the maturation chamber without any protective packaging. Animals and humans are usually the major sources of bacterial food contamination in the meat industry, but other routes might be involved. Therefore, procedures to reduce or eliminate pathogens from surfaces are crucial for an effective hazard analysis critical control point program in the food industry and other environments. This study aimed to assess the survival of Listeria monocytogenes, Escherichia coli, Salmonella spp., and Staphylococcus aureus on the inner surface of dry aging chambers. Moreover, we tested the efficacy of alkaline electrolyzed water (REW) for its application within a procedure aimed at reducing foodborne pathogens during meat storage. Environmental conditions inside the dry aging cabinet determine a reduction of circa 3 log CFU/cm2 of the considered microorganisms on the inner surface in 24 hours. Additionally, the nebulization of REW with the smoking system increased the count reduction in 24 hours due to environmental conditions for L. monocytogenes (~1 log CFU/cm2) and for S. aureus (~2 log CFU/cm2). In this context, the use of REW can be justified for routine cleaning procedures of the surfaces, with the added value of being safe to handle, not containing environmental pollutants, and making it unnecessary to rinse surfaces due to its instability.

Genomic features of Klebsiella isolates from artisanal ready-to-eat food production facilities.

Increasing reports on K. pneumoniae strains with antimicrobial resistance and virulence traits from food and farm animals are raising concerns about the potential role of Klebsiella spp. as a foodborne pathogen. This study aimed to report and characterize Klebsiella spp. isolates from two artisanal ready-to-eat food (soft cheese and salami) producing facilities, and to track similar genotypes in different ecological niches. Over 1170 samples were collected during the whole production chain of different food batches. The overall Klebsiella prevalence was 6%. Strains were classified into the three Klebsiella species complexes: K. pneumoniae (KpSC, n = 17), K. oxytoca (KoSC, n = 38) and K. planticola (KplaSC, n = 18). Despite high genetic diversity we found in terms of known and new sequence types (STs), core genome phylogeny revealed clonal strains persisting in the same processing setting for over 14 months, isolated from the environment, raw materials and end-products. Strains showed a natural antimicrobial resistance phenotype-genotype. K. pneumoniae strains showed the highest virulence potential, with sequence types ST4242 and ST107 strains carrying yersiniabactin ybt16 and aerobactin iuc3. The latter was detected in all K. pneumoniae from salami and was located on a large conjugative plasmid highly similar (97% identity) to iuc3+ plasmids from human and pig strains circulating in nearby regions of Italy. While identical genotypes may persist along the whole food production process, different genotypes from distinct sources in the same facility shared an iuc3-plasmid. Surveillance in the food chain will be crucial to obtain a more comprehensive picture of the circulation of Klebsiella strains with pathogenic potential.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 18: Suitability of taxonomic units notified to EFSA until March 2023.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 38 microorganisms notified to EFSA between October 2022 and March 2023 (inclusive) (28 as feed additives, 5 as food enzymes, food additives and flavourings, 5 as novel foods), 34 were not evaluated because: 8 were filamentous fungi, 4 were Enterococcus faecium and 2 were Escherichia coli (taxonomic units that are excluded from the QPS evaluation) and 20 were taxonomic units (TUs) that already have a QPS status. Three of the other four TUs notified within this period were evaluated for the first time for a possible QPS status: Anaerobutyricum soehngenii, Stutzerimonas stutzeri (former Pseudomonas stutzeri) and Nannochloropsis oculata. Microorganism strain DSM 11798 has also been notified in 2015 and as its taxonomic unit is notified as a strain not a species, it is not suitable for the QPS approach. A. soehngenii and N. oculata are not recommended for the QPS status due to a limited body of knowledge of its use in the food and feed chains. S. stutzeri is not recommended for inclusion in the QPS list based on safety concerns and limited information about the exposure of animals and humans through the food and feed chains.

Assessment on the efficacy of methods 2 to 5 and method 7 set out in Commission Regulation (EU) No 142/2011 to inactivate relevant pathogens when producing processed animal protein of porcine origin intended to feed poultry and aquaculture animals.

An assessment was conducted on the level of inactivation of relevant pathogens that could be present in processed animal protein of porcine origin intended to feed poultry and aquaculture animals when methods 2 to 5 and method 7, as detailed in Regulation (EU) No 142/2011, are applied. Five approved scenarios were selected for method 7. Salmonella Senftenberg, Enterococcus faecalis, spores of Clostridium perfringens and parvoviruses were shortlisted as target indicators. Inactivation parameters for these indicators were extracted from extensive literature search and a recent EFSA scientific opinion. An adapted Bigelow model was fitted to retrieved data to estimate the probability that methods 2 to 5, in coincidental and consecutive modes, and the five scenarios of method 7 are able to achieve a 5 log10 and a 3 log10 reduction of bacterial indicators and parvoviruses, respectively. Spores of C. perfringens were the indicator with the lowest probability of achieving the target reduction by methods 2 to 5, in coincidental and consecutive mode, and by the five considered scenarios of method 7. An expert knowledge elicitation was conducted to estimate the certainty of achieving a 5 log10 reduction of spores of C. perfringens considering the results of the model and additional evidence. A 5 log10 reduction of C. perfringens spores was judged: 99-100% certain for methods 2 and 3 in coincidental mode; 98-100% certain for method 7 scenario 3; 80-99% certain for method 5 in coincidental mode; 66-100% certain for method 4 in coincidental mode and for method 7 scenarios 4 and 5; 25-75% certain for method 7 scenario 2; and 0-5% certain for method 7 scenario 1. Higher certainty is expected for methods 2 to 5 in consecutive mode compared to coincidental mode.

Influence of Fermentation Container Type on Chemical and Microbiological Parameters of Spontaneously Fermented Cow and Goat Milk.

Fermented goat milk is an artisanal beverage with excellent nutritional properties. There are limited data on its physicochemical properties, fatty acids, phenolic acids, and on any insight on microbiota. The aim of this research was to conduct a pilot study to compare these parameters in raw cow and goat milk before and after spontaneous fermentation in a clay pot and glass container at 37 °C for 24 h. Both types of milk and fermentation containers significantly affected the pH, acidity, proximate composition, viscosity, and whiteness index of fermented milks. A total of 17 fatty acids were identified in fermented milks, where palmitic, stearic, and myristic were the main saturated acids, and oleic and linoleic acids were the main unsaturated ones. These profiles were primarily influenced by the type of raw milk used. Three to five phenolic acids were identified in fermented milks, where quinic acid was the major phenolic compound, and salviolinic acid was identified only in raw goat milk. Preliminary metataxonomic sequencing analysis showed that the genera Escherichia spp. and Streptococcus spp. were part of the microbiota of both fermented milks, with the first genus being the most abundant in fermented goat milk, and Streptococcus in cow's milk. Moreover, Escherichia abundance was negatively correlated with the abundance of many genera, including Lactobacillus. Overall, the results of this pilot study showed significant variations between the physicochemical properties, the fatty and phenolic acids, and the microbial communities of goat and cow fermented milk, showing the opportunity to further investigate the tested parameters in fermented goat milk to promote its production.

Monitoring of chronic wasting disease (CWD) (IV).

The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017-28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease-associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high-risk target groups for surveillance should be revised, and 'road kill' removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene (PRNP) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high-risk target groups, sustained over time, using sampling units and a data-driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data-driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double-strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level.

Investigation on the microbiological hazards in an artisanal salami produced in Northern Italy and its production environment in different seasonal periods.

In the present study, the occurrence of Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Escherichia coli VTEC was investigated in two batches of artisanal Italian salami tested in winter and summer. Moreover, enumerations of total bacterial count, lactic acid bacteria and Enterobacteriaceae were performed as well as monitoring of water activity and pH. Samples were taken from raw materials, production process environment, semi-finished product and finished products. The results revealed an overall increase of total bacterial count and lactic acid bacteria during the ripening period, along with a decrease of Enterobacteriaceae, pH and water activity. No significant difference was observed between the two batches. The enterobacterial load appeared to decrease during the maturation period mainly due to a decrease in pH and water activity below the limits that allow the growth of these bacteria. E. coli VTEC, Salmonella spp. or L. monocytogenes were not detected in both winter and summer batches. However, Klebsiella pneumoniae was detected in both summer and winter products. Except for one isolate, no biological hazards were detected in the finished salami, proving the efficacy of the ripening period in controlling the occurrence of microbiological hazard in ripened salami. Further studies are required to assess the virulence potential of the Klebsiella pneumoniae isolates.

Metabolic and microbiota response to arginine supplementation and cyclic heat stress in broiler chickens.

Little attention has been paid to the biological role of arginine and its dietary supplementation in broilers under heat stress (HS) conditions. Therefore, the main aim of this study was to assess the response of broilers to arginine supplementation and cyclic HS, with a focus on liver, pectoral muscle, and blood metabolic profiles and the cecal microbiota. Day-old male Ross 308 broilers (n = 240) were placed in 2 rooms with 12 pens each for a 44-day trial. Pens were assigned to one of two groups (6 pens/group/room): the control group (CON) was given a basal diet in mash form and the treated group (ARG) was fed CON diet supplemented with crystalline L-arginine. The total arginine:lysine ratio of CON diet ranged between 1.02 and 1.07, while that of ARG diet was 1.20. One room was constantly kept at thermoneutral (TN) conditions, while the birds in the other room were kept at TN conditions until D34 and subjected to cyclic HS from D35 onwards (∼34°C; 9:00 A.M.-6:00 P.M.). Blood, liver, Pectoralis major muscle, and cecal content were taken from 2 birds per pen (12 birds/group/room) for metabolomics and microbiota analysis. Growth performance data were also collected on a pen basis. Arginine supplementation failed to reduce the adverse effects of HS on growth performance. Supplemented birds showed increased levels of arginine and creatine in plasma, liver, and P. major and methionine in liver, and reduced levels of glutamine in plasma, liver, and P. major. HS altered bioenergetic processes (increased levels of AMP and reduced levels of fumarate, succinate, and UDP), protein metabolism (increased protein breakdown to supply the liver with amino acids for energy production), and promoted the accumulation of antioxidant and protective molecules (histidine-containing dipeptides, beta-alanine, and choline), especially in P. major. Arginine supplementation may have partially counterbalanced the effects of HS on energy homeostasis by increasing creatine levels and attenuating the increase in AMP levels, particularly in P. major. It also significantly reduced cecal observed diversity, while HS increased alpha diversity indices and affected beta diversity. Results of taxonomic analysis at the phylum and family level are also provided.

Feeding broiler chickens with arginine above recommended levels: effects on growth performance, metabolism, and intestinal microbiota.

BACKGROUND: Arginine is an essential amino acid for chickens and feeding diets with arginine beyond the recommended levels has been shown to influence the growth performance of broiler chickens in a positive way. Nonetheless, further research is required to understand how arginine supplementation above the widely adopted dosages affects metabolism and intestinal health of broilers. Therefore, this study was designed to assess the effects of arginine supplementation (i.e., total arginine to total lysine ratio of 1.20 instead of 1.06-1.08 recommended by the breeding company) on growth performance of broiler chickens and to explore its impacts on the hepatic and blood metabolic profiles, as well as on the intestinal microbiota. For this purpose, 630 one-day-old male Ross 308 broiler chicks were assigned to 2 treatments (7 replicates each) fed a control diet or a crystalline L-arginine-supplemented diet for 49 d. RESULTS: Compared to control birds, those supplemented with arginine performed significantly better exhibiting greater final body weight at D49 (3778 vs. 3937 g; P < 0.001), higher growth rate (76.15 vs. 79.46 g of body weight gained daily; P < 0.001), and lower cumulative feed conversion ratio (1.808 vs. 1.732; P < 0.05). Plasma concentrations of arginine, betaine, histidine, and creatine were greater in supplemented birds than in their control counterparts, as were those of creatine, leucine and other essential amino acids at the hepatic level. In contrast, leucine concentration was lower in the caecal content of supplemented birds. Reduced alpha diversity and relative abundance of Firmicutes and Proteobacteria (specifically Escherichia coli), as well as increased abundance of Bacteroidetes and Lactobacillus salivarius were found in the caecal content of supplemented birds. CONCLUSIONS: The improvement in growth performance corroborates the advantages of supplementing arginine in broiler nutrition. It can be hypothesized that the performance enhancement found in this study is associated with the increased availability of arginine, betaine, histidine, and creatine in plasma and the liver, as well as to the ability of extra dietary arginine to potentially ameliorate intestinal conditions and microbiota of supplemented birds. However, the latter promising property, along with other research questions raised by this study, deserve further investigations.