Quantitative determination of tetracyclines in medicated feed for food-producing animals by HPLC-DAD.

Tetracyclines are a group of antibiotic substances largely administered through medicated feed to control diseases in food-producing animals. Fine dosing of antibiotics contained in medicated feed is crucial for the success of the treatment as well as minimising potential threats such as the spread of antimicrobial resistance and the transfer of antibiotic residues in food. A rapid analytical method based on HPLC with diode array detection (HPLC-DAD) was developed to quantify oxytetracycline, chlortetracycline and doxycycline in medicated feed. The reported method underwent in-house validation and was found to be suitable for the quantification of three target tetracyclines within the concentration range of 40-1000 mg kg-1 in official routine analysis. The method was applied to 103 official samples in the framework of the Italian National Plan on animal feed during the years 2021-2023 and nine non-compliant concentrations were identified in swine and fish feed samples.

Mycotoxins and Amyotrophic Lateral Sclerosis: food exposure, nutritional implications and dietary solutions.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder determined by a combination of both genetic and environmental factors. Despite wide investigations, the role of chronic exposure to environmental pollutants is still rather unknown. Among natural toxins, the mycotoxins have received major attention only in the last few years, due to both technical and scientific achievements that allowed to disentangle many important features of the complex fungal biology. Whereas the effects of acute and high-dose mycotoxin exposure are well known, the potential effects of chronic and low-dose exposure on neurodegeneration have not been broadly elucidated. In this review, we have summarized all the studies concerning environmental exposure to unknown substances that caused ALS outbreaks all over the world, reinterpreting in light of the new scientific acquisitions and highlighting the potential and neglected role of mycotoxins. Then, we focused on recent papers about food exposure to mycotoxin, mycobiome and fungal infections in ALS and other neurodegenerative diseases. We analyzed the gaps of current literature that lead to an undervaluation of mycotoxins as detrimental molecules. By listing all the most important mycotoxins and analyzing all the biological pathways that they can affect, we explained the reasons why they need to be considered in the next epidemiological studies on ALS and other neurodegenerative and neuroinflammatory diseases. In conclusion, after suggesting some possible solutions to mitigate mycotoxin exposure risk, we affirm that future collaborations between scientists and policymakers are important to develop sustainable interventions and promote health through dietary diversity.

Optimization and validation of a quick and responsive LC-ESI-MS/MS method to evaluate tetracycline residues in processed animal proteins (PAPs).

Following the bovine spongiform encephalopathy (BSE) in 2001, processed animal proteins (PAPs) reintroduction is envisaged in non-ruminant feed thanks to their high protein content, easy availability and cost-effective characteristics. PAPs must be submitted to rendering practices, providing sterilization of products, under standardized conditions of temperature and pressure, according to Regulation (EC) No 142/2011. However, the chemical risk associated to these raw materials has been never evaluated. The aim of this study was to develop and validate a reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of tetracycline residues in PAPs at µg kg-1 level. The LC-MS/MS method performances were evaluated in terms of specificity, linearity (25-500 µg kg-1), limit of quantitation (LOQ) (25 µg kg-1), accuracy and precision (CV% < 25%), uncertainty, recovery (80-120%) and ruggedness. All the evaluated parameters fulfilled the analytical performance criteria, and the validated LC-MS/MS method fits for purpose as confirmatory method on the occurrence of residues (µg kg-1) of tetracyclines in PAPs. PAPs are a powerful product which could be used both as raw materials in feed and in organic fertilizer production in a circular economy context. Therefore, the lack of regulation and control over antibiotic occurrence should be implemented to avoid a misuse and an increment of antibiotic resistance pressure over the environment and to ensure safety of the feed and food chain.

Preliminary Data on Essential and Non-essential Element Occurrence in Processed Animal Proteins from Insects.

Insects represent a valuable and environmentally friendly protein alternative in food and feed. The Farm to Fork strategy encouraged the reintroduction of animal by-products in feed production to optimise recycling and to valorise under-used resources. In order to grant safe and valuable feed products, this study investigated the black soldier fly (BSF) (Hermetia illucens) chemical risk. Samples collected in different steps of production (8 samples of substrate for culturing, 7 samples of larvae, 15 samples of protein meal, 18 samples of spent substrate) were analysed for microessential elements (chromium, copper, iron, nickel, selenium and zinc) and inorganic contaminants (aluminium, arsenic, cadmium, lead, tin and vanadium) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Microessential elements were found in the following order: Fe > Zn > Cu > Ni > Se > Cr (mg kg-1). Non-essential element concentrations were found lower than the set limits according to the European Union Regulations. The growing demand for alternative protein sources for feed production could be partially compensated by black soldier fly (BSF) (Hermetia illucens) meal, as it appears a good source for high-quality proteins and microessential elements which play a pivotal role in animal growth. In the foreseeable future the current legislation and the official monitoring plans may be implemented and broaden, to focus and assess limits for upcoming matrices, and to ensure feed and food safety.

Class 1 integron and Enterococcus spp. abundances in swine farms from the " Suckling piglets" to the "Fatteners" production category.

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

Glyceroltriheptanoate (GTH) occurrence in animal by-products: a monitoring study to minimise safety-related risk of misuse.

Early in this century, the crisis connected to the spread of bovine spongiform encephalopathy caused a great concern related to the use of animal by-products (ABPs). According to the Commission Regulation (EU) No 1069/2009, these materials are classified in three categories according to their related risk. In 2011 Commission Regulation (EU) No 142/2011 established that meat and bone meal (MBM) and fat deriving from ABPs not intended for human consumption (category 1 and 2) are required to be permanently marked with glyceroltriheptanoate (GTH), at a minimum concentration of 250 mg kg-1 of fat, while category 3 processed animal proteins (PAPs) must not contain this compound. PAPs are bio resources, which could be used in a renewable and regenerative way in a circular economy model for a conscious usage of raw materials. The aim of this study was to provide information on GTH occurrence in MBM and, if any, in PAPs. Samples were collected from 2017 to 2021 and analysed by GC-MS. Detected non-compliant samples were exclusively of MBM category 1 and 2, probably due to the addition of an inadequate amount of GTH during the manufacturing processes. These results highlighted the importance of National Monitoring Programs as a useful tool to minimise safety related risk due to the misuse of GTH. Thus, investigating the critical points in feed supply-chain and sharing the information on its occurrence may help to improve animal and human wellness and safety.

Processed animal proteins (PAPs) in animal nutrition: Assessment of the chemical risk of essential and non-essential elements.

BACKGROUND: Processed animal products (PAPs) could be a great alternative to common protein supplements and represent a good example of recycling and valorization of by-products. Due to the reintroduction of certain types of PAPs in feed, a deeper knowledge of these heterogeneous matrices is needed. Thus, the aim of this study is to evaluate the levels of essential elements and inorganic contaminants in 55 PAPs considered as potential alternatives to common protein supplements. METHODS: PAPs samples were analysed for essential (cobalt, nickel, chromium, copper, zinc, iron and manganese) and non-essential elements (arsenic, cadmium, lead and mercury) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS), Graphite Furnace Atomization Atomic Absorption Spectrometer (GF-AAS) and dual cell Direct Mercury Analyzer spectrometer (DMA-80). RESULTS: Essential elements were found with the following decreasing order iron>zinc>copper>manganese>chromium>nickel>cobalt (mg kg-1). Only one sample was found non-compliant to lead concentration according to the European Union Regulation while negligible values of others non-essential elements were found. CONCLUSIONS: This study suggests that PAPs could be a useful supplement for animal diet due to their natural content of essential elements. A careful monitoring of chemical elements should be required and eventually guidelines have to be drafted for a correct use of PAPs to ensure a safe and sustainable feed production.

Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity.

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg-1). Among samples containing more than 25 µg kg-1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg-1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues.

Gravimetric quantitative validation of botanic impurities in feed.

BACKGROUND: Harmful botanical impurities may contaminate feed and feed materials and be a potential danger to animal or human health, or to the environment. The aim of this study was to establish rapid and sensitive methods that can be used in routine official controls to determine botanical impurities such as Datura stramonium, Ricinus communis, Crotaliaria spp., and Ambrosia spp. in animal feed and raw materials. Claviceps sclerotia were also detected in cereals, due to the similarities of the targets and the analytical procedure. Regulation (EU) 625/2017, which replaces Reg. 2004/882/EC, states that EU member states should conduct official controls in assessed and accredited laboratories and that the analytical methods must be validated before use by considering parameters such as specificity, precision, recovery, and measurement uncertainly. RESULTS AND CONCLUSION: The results demonstrate that all of the methods tested are suitable for the official quantitative analyses required by EU official legislation. © 2020 Society of Chemical Industry.

Detection of insect's meal in compound feed by Near Infrared spectral imaging.

Insects have recently emerged as a new protein source for both food and feed. Some studies have already demonstrated that insects' meal can be successfully added to animal feed without threaten animals' growth indices. However, effective and validated tests to individuate insects' meal in feed are strongly needed to meet traceability and safety concerns and to support the European legislation under development. Spectroscopic techniques represent valuable rapid and non-destructive methods that can be applied for in-situ analysis in feed production plants or in farms. In this work a Fourier Transform Near Infrared spectroscopy imaging (FT NIR) as a potential screening method for the detection and quantification of insects' meal in feed is presented. Discriminant analysis was used for the automatic recognition of insects' meal fragments into the feed matrix. Moreover, the possibility to quantify insect's meal in feed sample was successfully tested. The proposed method is a rapid and green strategy for feed contamination screening analysis.

Updated quantitative risk assessment (QRA) of the BSE risk posed by processed animal protein (PAP).

EFSA was requested: to assess the impact of a proposed quantitative real-time polymerase chain reaction (qPCR) 'technical zero' on the limit of detection of official controls for constituents of ruminant origin in feed, to review and update the 2011 QRA, and to estimate the cattle bovine spongiform encephalopathy (BSE) risk posed by the contamination of feed with BSE-infected bovine-derived processed animal protein (PAP), should pig PAP be re-authorised in poultry feed and vice versa, using both light microscopy and ruminant qPCR methods, and action limits of 100, 150, 200, 250 and 300 DNA copies. The current qPCR cannot discriminate between legitimately added bovine material and unauthorised contamination, or determine if any detected ruminant material is associated with BSE infectivity. The sensitivity of the surveillance for the detection of material of ruminant origin in feed is currently limited due to the heterogeneous distribution of the material, practicalities of sampling and test performance. A 'technical zero' will further reduce it. The updated model estimated a total BSE infectivity four times lower than that estimated in 2011, with less than one new case of BSE expected to arise each year. In the hypothetical scenario of a whole carcass of an infected cow entering the feed chain without any removal of specified risk material (SRM) or reduction of BSE infectivity via rendering, up to four new cases of BSE could be expected at the upper 95th percentile. A second model estimated that at least half of the feed containing material of ruminant origin will not be detected or removed from the feed chain, if an interpretation cut-off point of 100 DNA copies or more is applied. If the probability of a contaminated feed sample increased to 5%, with an interpretation cut-off point of 300 DNA copies, there would be a fourfold increase in the proportion of all produced feed that is contaminated but not detected.

LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

Gravimetric quantitative determination of packaging residues in feed from former food.

Valorisation of former foodstuff products (FFP) in feed is part of a long-term strategy for sustainability. An approach to valorise FFP outside the waste value chain is their use as an alternative source of feed materials, with a subsequent optimisation of the environmental impact of products. In the current practice of food production, food packaging is provided to ensure the maintenance of food quality and safety during transport and storage. One of the problems of reusing FFP is how to deal with packaging materials or remains that can become residues in the feed. The aim of this study is to propose a fast and sensitive gravimetric method, fit for routine official controls, for the determination of packaging residues in feed. The developed method can briefly be summarised as: (1) visual selection of the undesired ingredients which can be identified as remnants of packaging materials; (2) weighing of the selected materials; (3) defatting; (4) dehydration; (5) final weighing; and (6) reporting of weight and percentage. Moreover, the method has been validated through the determination of some of the parameters listed in Council Regulation 2004/882/EC (i.e., specificity, limit of quantification (LOQ), recovery, repeatability, within-laboratory reproducibility and measurement uncertainty).

Species-specific detection of processed animal proteins in feed by Raman spectroscopy.

The existing European Regulation (EC n° 51/2013) prohibits the use of animals meals in feedstuffs in order to prevent Bovine Spongiform Encephalopathy infection and diffusion, however the legislation is rapidly moving towards a partial lifting of the "feed ban" and the competent control organisms are urged to develop suitable analytical methods able to avoid food safety incidents related to animal origin products. The limitations of the official methods (i.e. light microscopy and Polymerase Chain Reaction) suggest exploring new analytic ways to get reliable results in a short time. The combination of spectroscopic techniques with optical microscopy allows the development of an individual particle method able to meet both selectivity and sensitivity requirements (0.1%w/w). A spectroscopic method based on Fourier Transform micro-Raman spectroscopy coupled with Discriminant Analysis is here presented. This approach could be very useful for in-situ applications, such as customs inspections, since it drastically reduces time and costs of analysis.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

Light microscopy with differential staining techniques for the characterisation and discrimination of insects versus marine arthropods processed animal proteins.

The aim of this study was to evaluate the use of light microscopy with differential staining techniques for the discrimination of insect material from marine arthropods - classified as fishmeal. Specifically, three samples of single-species insect material, Hermetia illucens (HI), Bombyx mori (BM) and Tenebrio molitor (TM), and two samples of marine arthropods, shrimp material and krill, were analysed and compared after staining by two reagents to enhance fragment identification. Alizarin Red (AR) and Chlorazol Black (CB), which react respectively with calcium salts and chitin, were tested for their potential efficacy in distinguishing between insect and marine materials. Results indicated that AR failed to stain HI, BM and TM materials. By contrast, the three insect species materials tested were stained by CB. When shrimp fragments and krill were considered, AR and CB stained marine materials reddish-pink and light blue to black, respectively. By combining these results, it can be suggested that CB staining may efficiently be used to mark insect materials; AR does stain shrimp fragments but does not stain the tested insect material, indicating a possible approach for discriminating between insects and marine arthropods. However, since the present study was performed on pure materials and a small set of samples, possible implementation of this technique still needs to be confirmed in complex matrices such as compound feed.

Rapid and sensitive detection of melamine in milk with gold nanoparticles by Surface Enhanced Raman Scattering.

A rapid and sensitive method to detect melamine in liquid milk based on Surface Enhanced Raman Scattering (SERS) spectroscopy is presented, exploiting the selective binding of gold nanoparticles (AuNPs) with this analyte. This interaction promotes the aggregation of the AuNPs inducing a huge enhancement of the melamine signals in the Raman spectrum due to the formation of SERS "hot spots". An external standard calibration method was employed for quantitative analysis and the method was validated for linearity, sensitivity, repeatability and recovery. A good linearity (R(2)=0.99) was found in the concentration range of 0.31-5.0 mg l(-1) in milk with a limit of detection of 0.17 mg l(-1). This method does not require a long extraction procedure (total analysis time can be lower than 30 min) and can be reliably used for melamine detection in milk matrix in accordance with the European law limits.

European interlaboratory trial regarding the official microscopic method for the detection of the presence of animal constituents in feedstuffs.

The bovine spongiform encephalopathy epidemic is thought to have occurred as a consequence of feeding prion-infected material to cattle. To avoid the risk of bovine spongiform encephalopathy diffusion, the European Commission (Directive 2003/126/EC) established an official method to detect the presence of animal-derived constituents in feedstuffs, using microscopic examination. This method allows easy identification of bone fragments among other animal constituents. The analysis is based on morphological conformation of the fragments and their characterization (mainly of the shape of lacunae) to discriminate among mammalian, poultry, and fish tissues. The aim of this study was to assess the performances of nine European laboratories through a ring trial of the official microscopic method, and to calculate accuracy and reproducibility of the method. In general the reproducibility of the microscopic method was very good (kappa overall = 0.83), with a high sensitivity for all laboratories. Concerning the analysis on the different animal-derived constituents, the results show values of sensitivity with large variability between fish and poultry or mammal. It was generally more difficult to discriminate between mammalian and poultry tissues than fish tissue.

Validation of a dissociation enhanced lanthanide fluorescence immunoassay for the screening of 17beta-estradiol in bovine serum according to European Union decision 2002/657/EC.

A validation study was carried out in order to evaluate the performances of a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) for rapid screening of 17 beta-estradiol in bovine serum. This validation was performed according to European Union (EU) Decision 2002/657/EC, which establishes criteria and procedures for determination of detection capability (CCbeta), selectivity/specificity, and applicability/ruggedness/stability for qualitative screening tests. To determine these performance characteristics, 20 blank serum samples of cattle were collected and spiked with 17 beta-estradiol at 40 pg/mL, corresponding to the maximum residue limit permitted by Italian legislation. According to the EU Decision CCbeta criterion, spiked samples must have <5% probability to be classified as a false negative. 17 beta-Estradiol was detected in each spiked sample, and the CCbeta results were <40 pg/mL. There was also no observed interference effect due to chemically related substances or from the matrix. Moreover, slight variations of some critical factors in the DELFIA procedure, deliberately introduced for ruggedness evaluation, did not result in any negative effect on the 17beta-estradiol detection. The proposed method is suitable for qualitative screening analysis of 17 beta-estradiol according to EU performance requirements.