Multi-laboratory evaluation of a PCR method for detection of ruminant DNA in commercial processed animal proteins

In 2013, the European Union (EU) allowed the inclusion of non-ruminant processed animal proteins (PAP) in fish feeds. Plans also exist to allow non-ruminant PAP in poultry and pig feeds but to keep intraspecies recycling forbidden (e.g. poultry to poultry or pig to pig). However, due to the fear of transmissible spongiform encephalopathy (TSE), PAP from ruminant species remains illegal to use in feeds. To ensure feed safety, nations across Europe need to safeguard that commercial fish feeds do not contain PAP from ruminant species. In this work we report a multi-laboratory study on the detection of ruminant PAP in commercial PAP feed ingredients. A total of 19 non-ruminant PAP, provided by the European Fat Processors and Renderers Association (EFPRA), were analyzed for ruminant content with the European Union Reference Laboratory (EURL)-validated PCR assay by three labs and compared to the result obtained with an immunoassay-based method (MELISA-TEK). Ruminant PAP was detected in five of the 19 non-ruminant PAP samples with both methods, but there were methodological and multi-laboratory differences for the PCR method. Compared to the results with the EU-reference laboratory, 3 false-negatives were found by the two other laboratories. We speculate that the discrepancies seen for some of the samples determined with the PCR method rely on the homogenization and grinding step, which is not described in detail in the EURL Standard Operating Procedure (SOP), shift in the normal distribution of Ct-values near the cut-off of the PCR method, or PCR inhibition. In conclusion, this study demonstrates the presence of ruminant DNA in commercially available non-ruminant PAP by use of two methods for the detection of PAP in commercial feed ingredients, and highlights the importance of thorough homogenization prior to DNA extraction and assessment of normal distribution of Ct-values for successful PCR detection at low ruminant DNA contamination levels.     

Feed for Food: HACCP in the animal feed industry

In this paper, a brief presentation is given a presentation of the quality programme for the Dutch animal feed Industry. The goal of this program is to produce and supply animal feeds which are safe for consumers of animal products, for the animals and for the environment; in a trustworthy way for all stakeholders––the partners in the chain, the consumers and the legislators––which means to do so demonstrably and transparently. An important part of this program is the GMP standard Animal Feed, which was set up in 1992. This GMP standard contains requirements for the quality system (comparable with ISO 9002) and a number of additional generic control measures for production, trade and transport of animal feed materials focussed on additives, undesirable substances and microbiological aspects.The recent feed and food-related safety crises (BSE, dioxin affairs) were particularly important impulses to enhance the quality programme, which has resulted in (i) the integration of HACCP in the GMP standard and (ii) upstream extension of the quality assurance to all suppliers of feed ingredients. By this way the quality system of the food processing industry has been adopted in the animal feed industry. This underlines that the animal feed is part of the food chain, expressed in the slogan ‘Feed for Food’.The main points are:

• •Food safety has a high priority world wide.
• •The animal feed industry, including the ingredient suppliers, is part of the food chain and responsible for the safety of its products.
• •Demonstrable and transparent quality assurance is a ‘licence to produce’.
• •HACCP is a proactive approach which links the feed chain to the food chain.
• •Quality control of raw materials is a mutual concern for suppliers and the animal feed industry.
• •Trade and industry are assuming their responsibility for product safety.

     

A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods

Meat adulteration has posed considerable risks to public health. In this study, we developed a novel real-time quantitative PCR method for the detection of some mammalian and poultry species that are used as meat products or meat adulterants. The method was based on the detection of the single-copy nuclear gene myostatin. The specificity, heterogeneity, and copy number of myostatin were evaluated. Additionally, we determined the sensitivity and precision of the method. The results revealed that myostatin had high specificity and low heterogeneity among different mammalian and poultry species. The limit of detection was 5 pg of animal genomic DNA or 0.001% meat ingredient, and the limit of quantification was 10 pg of animal genomic DNA or 0.01% meat ingredient. The quantification results of 12 blind samples showed that the biases between the measured and true values were <25%. Therefore, the developed quantitative real-time PCR method for mammalian and poultry species is suitable for identification and quantification of different meat ingredients as a reference gene.     

SIGMO: A decision support System for Identification of genetically modified food or feed products

Since their introduction in 1994, more and more genetically modified (GM) crops are grown worldwide and introduced in food or feed products. In the European Union (EU), the production, trade and marketing of GM products is strictly regulated, but the situation is becoming more complex due to the increasing number and complexity of GM crops, and asynchronic approval procedures with the major GM crop producing countries. Importers and traders are obliged to assess their respective supply chains for the potential presence of authorised and unauthorised GM organisms (GMOs), where wrong decisions may lead to substantial economic losses. This article presents a decision support system SIGMO aimed at guiding producers and traders with the assessment of the likelihood that their products may comprise authorised or unauthorised GM materials. The assessment is based on traceability data about the product (nature and origin of the raw materials, transportation aspects), as well as analytical results of the presence of GMOs in the final product or its ingredients. The approach uses a combination of data-driven and model-driven decision support. SIGMO is composed of (1) a data base providing data about GMO crop species produced and approved in counties worldwide, (2) a multi-attribute model for the assessment of GMO presence in food/feed products, and (3) an on-line user interface. SIGMO helps producers and traders to better comply to valid EU GMO regulations and to better control their products and supply chains in terms of the unintended presence of (unauthorised) GMOs in a cost-effective way.     

The occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin M1: A review

Mold contamination has challenged the safety of feed production and processing because of its undeniable role in the spoilage and the possible consequent toxicity impact on human health and the economy. Aflatoxin M₁ (AFM₁) is a hepatocarcinogenic derivative of aflatoxin B₁ excreted into the milk after ingestion of feed contaminated by certain molds. Because of the important role of dairy products, especially milk in the human diet, there is a huge concern about the presence of AFM₁ in milk and dairy products. In this article, the occurrence of AFM₁ and the fate of AFM₁ during processing of milk and dairy products, such as yoghurt and cheeses, since 1996 until today, was reviewed. The evaluation of mechanisms by which AFM₁ is affected by each processing step is of major importance to provide useful and accurate information to develop risk assessment studies and risk management strategies.     

Risk factors associated with the presence of aflatoxin M1 in raw bulk milk from Argentina

The objectives of this study were to assess aflatoxin M₁ (AFM1) contamination in bulk tank milk, and to further identify the risk factors associated with the presence of AFM1 in raw milk in Argentina. The presence of AFM1 was investigated in 160 bulk tank milk samples collected from farms located in the most important milk production region in Argentina during one year (four seasons). Samples were analysed using immunoaffinity column (IAC) cleanup and UHPLC-MS/MS method for determining AFM1 at low levels of concentrations (LOQ = 0.003 μg L⁻¹). A survey about the potential factors associated with the presence of AFM1 in milk was performed directly in the field through a questionnaire applied to the farmers. Chi-square and logistic regression were performed with presence of AFM1 in milk as dependent variable, and potential risk factors as independent variables. Incidence of AFM1 in raw milk was 38.8% and, in all samples, AFM1 levels were lower than the Southern Common Market (MERCOSUR) Regulation (maximum level accepted = 0.5 μg L⁻¹). Commercial feed consumption (OR = 4.630, P = 0.001), soybean expeller consumption (>0.95 kgDM/cow) (OR = 3.542, P = 0.019), and cotton seed consumption (>1.5 kgDM/cow) (OR = 2.949, P = 0.089) were associated with the incidence of AFM1 in raw milk. Despite the incidence and the level of AFM1 in milk produced and commercialized in Argentina is not a serious problem for public health. The farm breeding intensification and the supplementation with commercial feed, soybean expeller, and cotton seed seems to be the risk factors that impacts on the AFM1 milk contamination. Therefore, Argentina should improve its monitoring program on mycotoxins in animal feed and milk and improve the management practices in farms.     

Does European Union food policy privilege the internet market? Suggestions for a specialized regulatory framework

The online food and beverages market has grown rapidly over the past decade and a considerable number of consumers conduct their weekly grocery shopping over the internet. However, in the European Union the online distribution channel has been neglected in regard to food safety, and is even privileged in some aspects, as online shops are not necessarily subjected to inspections, while sampling and analysis is often complicated as the demands in regulation (EC) No 882/2004 cannot be completely fulfilled in distance selling circumstances. For example, the food operator's right to apply for a supplementary expert opinion may be infringed. A literature review shows that this lack of control is evidenced by a prevalence for misleading advertising (especially for nutritional supplements), but may also cause consequences for the health of the consumers (e.g. missing labelling of allergens, microbiological risk if the cooling chain is broken during transport, chemical risk if non-approved ingredients are used or contamination occurs). A solution would be a policy framework that specifically targets the internet market. The mandatory food information should be clearly pointed out on the product web-pages before the purchase is concluded. The food safety authorities should be granted the right to sample products by online order, which also needs to be possible anonymously in cases of reasonable suspicion.     

An initial characterization of aflatoxin B1 contamination of maize sold in the principal retail markets of Kigali,Rwanda

Food security considerations have shifted in recent years, with the recognition that available food should also be nutritious and safe. There is a growing evidence base for contamination of maize and other crops by fungal toxins in the tropics and sub-tropics. As an initial snapshot of contamination by one of these toxins in Rwanda, Aflatoxin B1 (AFB1) was analyzed in 684 samples of maize flour collected in seven principal retail markets of Kigali and in 21 samples of animal feed from seven feed vendors. Two rounds of sample collections were carried out, the first in September 2014 and the second in January 2015. A questionnaire given to vendors was used to determine if gender and education level of vendors, origin of maize and awareness of aflatoxins had any significant effect on AFB1 level in collected samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Immuno-affinity fluorimetery were used to analyze samples. Only markets had a significant effect on AFB1 level; for the two collections, differences were inconsistent among markets. In the first round, market means of AFB1 varied between 8.0 ± 5.57 μg/kg and 24.7 ± 23.74 μg/kg and for the second round, between 10.4 ± 8.4 μg/kg and 25.7 ± 25.85 μg/kg. In most animal feed samples AFB1 was >100 μg/kg. None of the vendors interviewed was aware of the risk of mycotoxin contamination in their maize-based flours and feed. Limits set by the United States Food and Drug Administration (20 μg/kg) for total aflatoxins and European Commission (2 μg/kg) for AFB1 for maize flour imports, were varied between 2–35% and 66–100% of samples, respectively. The implications of this study for human and animal health in Rwanda suggest that expanded surveys are needed to understand the scope of contamination, given the influence of environment and other factors on aflatoxin accumulation. Available options to mitigate and monitor aflatoxin contamination can be further deployed to reduce contamination.     

Early detection of microbial contamination in processed tomatoes by electronic nose

Microbial contamination can easily affect processed tomato, thus determining both organoleptic adulterations and potential health risks for customers. Innovative techniques for a rapid and reliable diagnose of spoilage, such as electronic nose technology, are highly requested in order to guarantee food safety and to improve production. In this work canned peeled tomatoes were artificially spoiled with different kinds of microbial flora and then were analyzed by means of an electronic nose based on thin film metal oxide gas sensors. Preliminary analyses by dynamic-headspace gas chromatographic–mass spectrometry showed significant differences in the semi-quantitative volatile compounds profile of spoiled tomato samples just after few hours from contamination, thus suggesting to employ the electronic nose for an early diagnose of microbial presence. The electronic nose was indeed able to reveal contamination, even at early stages depending on the type of contaminant (e.g. for Saccharomyces cerevisiae and Escherichia coli), and to recognize spoiled tomato samples with good classification performances.     

Distribution of Salmonella clonal groups in four Brazilian feed mills

Feed is one of the major vehicles for Salmonella transmission to pigs, and its spread during the milling process may play an important role in feed contamination. Therefore, a cross-sectional study was carried out in four feed mills in order to evaluate the frequency of Salmonella isolation at various stages of feed production and to track the spread of genotypically related isolates grouped by pulsed-field gel electrophoresis (PFGE). From a total of 1269 analyzed samples, 63 (4.96%) showed the presence of Salmonella. Evaluated feed mills (A, B, C and D) presented 3.5% (n = 11/317), 1.7% (n = 5/289), 7.5% (n = 23/308) and 7.0% (n = 25/355) positive samples, respectively. Twenty-three serovars were identified, with the most frequently detected being Montevideo (n = 14, 22.2%), Anatum (n = 8, 12.7%) and Senftenberg (n = 8, 12.7%). The isolation of Salmonella was significantly higher (p = 0.002) in samples with the presence of total coliforms (36/489; 7.36%) than in the coliform-negative samples (27/780; 3.46%). Conveyors (OR = 4.43, 95% CI: 2.43–8.09) were the most likely sites of Salmonella isolation, followed by dust settled on the feed mill's floor (OR = 2.88, 95% CI: 1.41–5.33). Isolates indistinguishable on PFGE or belonging to pulsotypes with a high similarity (>95%) were identified in serovars Agona, Infantis, Montevideo, Orion, Senftenberg and Worthington. In particular, clonal groups of serovars Montevideo and Senftenberg were found to be disseminated among different sample types (ingredients, dust collected from the premise's floor and complete feed) or to be endemic in the feed mills. The dissemination of Salmonella clonal groups demonstrates the importance of control measures to avoid dust and debris accumulation on equipment surfaces.     

Occurrence and legislation of mycotoxins in food and feed from Morocco

Mycotoxins are natural food and feed contaminants, mainly produced by moulds of genera Aspergillus, Penicillium and Fusarium. The number of mycotoxins known to exert toxic effect on human and animal health is constantly increasing as well as the legislative provisions taken to control their presence in food and feed. Morocco, a North African country, surrounded by the Mediterranean Sea and Atlantic Ocean, has a climate characterized by high humidity and high temperature which favor growth of moulds. This paper gives an overview about the contamination levels and the occurrence of some mycotoxins (e.g. aflatoxins, ochratoxin A, and Fusarium toxins) in cereals, bread, milk, spices, wine, olives, poultry feeds, dried fruits and nuts; the average of contaminated samples was often above 50%. A section on mycotoxin regulations by Moroccan authorities is discussed with a comparison with international and European limits. Recent data about the contamination of foods and feed from Morocco by mycotoxins are considered in this review. Finally, the paper gives a last part with conclusions and principal prospectives and recommendations that should be undertaken by authorities and scientists during monitoring of mycotoxins in food and feed produced and/or commercialized in Morocco.     

Prevalence of food-borne pathogens in ready-to-eat meat products in seven different Chinese regions

Microbiological surveillance of ready-to-eat (RTE) meat products provides empirical data to inform scientific guidance for improving food safety. In this study we conducted the first nation-wide survey of food-borne pathogens in RTE meat products in China in 2013. We analyzed the prevalence of food-borne pathogen in different product categories, market distribution, packaged forms, seasonal variation and regions. In total, 4047 samples were collected from all seven regions in China representing distinct geographical areas: Northern, Northeastern, Eastern, Central, Southern, Southwestern, and Northwestern Regions. Samples were collected from catering, retail, and wholesale sources in different seasons throughout the course of the year. The presence of Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and diarrheagenic Escherichia coli in all the samples was examined by China's national food safety standard method. All the surveillance data were analyzed according to meat product category, market distribution, packaged form, seasonal variation and regions, respectively. Microbial prevalence of L. monocytogenes, Salmonella spp., S. aureus and diarrheagenic E. coli were detected in 1.43% (57/3974), 0.64% (26/4035), 0.79% (32/4047) and 1.06% (40/3774), respectively. The prevalence of heat-treated category RTE meat products was high, probably due to cross contamination. Prevalence data for Salmonella spp. and S. aureus were higher in the third quarter of the year. We found packaging method played a consistent role in the prevalence of all four food-borne pathogens in packaged ready-to-eat meat products. Bulk ready-to-eat meat products were significantly more likely to contain contamination than portion packaged products. In all, this study implied a potential public health problem. It is necessary to improve control measures of RTE meat products.     

Growth capacity of Listeria monocytogenes in ingredients of ready-to-eat salads

Raw minimally processed vegetables and fruits as well as boiled pasta and potato are the main ingredients of ready-to-eat (RTE) salads. These ingredients are diverse in their ability to support growth of Listeria monocytogenes. The objective of this study was to determine to what extent different ingredients can contribute to growth of L. monocytogenes in RTE salads. This was done by assessing the growth capacity of L. monocytogenes in ingredients of RTE salads by means of challenge tests and determination of the effect of product characteristics and presence of competitive flora. The majority of the tested products did not support or hardly supported growth of L. monocytogenes. The calculated maximum increase of 3.4 log CFU/g was exceeded in Galia melon, potato and pasta products. In RTE dinner salads, growth of L. monocytogenes was supported by the carbohydrate component. Galia melon, which is often used as ingredient of fruit salads, can contribute to growth of L. monocytogenes in these products. Growth inhibition, or differences in relative increase, could not always be explained by a_w, pH or presence of competitive flora. The effect of competitive flora, especially lactic acid bacteria, on growth inhibition was observed in non-pasteurized potato, white cabbage and mango.     

Quantitation of ochratoxin a in cereals and feedstuff using sequential injection analysis with luminescence detection

Ochratoxin A is one of the most important mycotoxins due to its global incidence and toxicity. Therefore, many countries have adopted regulations or guidelines to limit exposure and protect human and animal health. In this paper, we have developed two different flow-through optosensors using sequential injection analysis, and compared them in terms of analytical parameters. One of them used fluorescence detection, whereas the other one used terbium-sensitized luminescence detection. In both cases, an appropriate solid support was placed inside the flow-cell to retain the analyte and increase the selectivity and sensitivity of the system.The extraction of ochratoxin A from cereals and animal feedstuff was performed with a modified QuEChERS method. The obtained detection limit made it possible to fulfill the Commission of the European Communities Recommendation guidance values for this contaminant in feed. Recovery experiments were carried out in cereals and several feed samples at different fortification levels. Recoveries in the 86–112% range were obtained, therefore demonstrating the applicability of the proposed analytical method for quality control analyses.     

Aflatoxins occurrence through the food chain in Costa Rica: Applying the One Health approach to mycotoxin surveillance

Aflatoxins (AFs) are toxic metabolites produced by Aspergillus spp. and commonly found in crops, grains, feedstuff, and forages. Exposure to AFs has been associated with increased risk of liver cancer and growth retardation in humans, liver damage, immunosuppression, embryotoxicity in both animals and humans, and decreased milk, egg and meat production in animals. For the first time, the Costa Rican national mycotoxin surveillance programs for animal feed and food are considered as a whole, applying the One Health approach to the mycotoxin epidemiological research. Therefore, the aim of this study was to determine the occurrence of AFs in cereals, nuts, grains intended for animal and human consumption in Costa Rica.In animal feed and feed ingredients, 970 samples were analyzed for AFs from 2010 to 2016 with an overall prevalence of positive samples of 24.0% (ranging from 0.01 to 290 μg kg⁻¹). Only 2.5% of the samples failed to comply the regulation for total AFs (20 μg kg⁻¹ feed). From 5493 samples of agricultural commodities intended for human consumption analyzed from 2003 to 2015, there was an overall prevalence of AF positive samples of 10.8% (ranging from 0.48 to 500 μg kg⁻¹), and 2.8% did not comply the regulation for AFs (20 μg kg⁻¹). In both feed and food, the highest AF prevalence corresponded to corn ingredients (27.8%) and white corn (38.6%), respectively. Among the commodities intended for human consumption, red beans had the highest aflatoxin concentrations (500 μg kg⁻¹).     

Enteropathogens in cocoa pre-processing

The European community lists cocoa among the products associated with major salmonellosis outbreaks in humans. Though cocoa products are not the only ingredients that may introduce Salmonella into chocolate, they have been implicated as the most prominent potential source of some outbreaks. The objective of this study was to investigate the presence of Salmonella, Escherichia coli and the level of total coliforms throughout the four different steps of cocoa pre-processing. The presence of Salmonella was detected in only one of the 119 samples analyzed – a sample of stored beans. Contamination by total coliforms and E. coli was highest during drying and storage, with percentages of up to 100% and 89% of positive samples. The environment, including the presence of vectors, intense handling and storage conditions appear to be the main critical points during pre-processing of cocoa contributing to contamination by these enteropathogens.     

Dioxins and polychlorinated biphenyls contamination in poultry liver related to food safety – A review

The present article reviews the most important scientific literature on dioxins and PCBs found in poultry liver and their relation with food safety and consumers' health. Dioxins and polychlorinated biphenyls (PCBs) are persistent organic pollutants with high chemical stability; they are lipophilic compounds and they are not destroyed by microbial, photochemical, chemical or thermal degradation. Dioxins and PCBs are ubiquitous environmental contaminants, which are present in all marine plant and animals, birds, mammals and humans and bioaccumulate through the food chain. In the last years, there has been concern regarding food contamination with different chemical substances and their effect on food safety. More particularly, at the end of the 20th and the beginning of the 21st century, there were a series of incidents related to dioxin and PCBs, which directly affected human or contaminated the soil and accumulated in feed and then in food products, such as poultry liver. It was shown in case of dioxin incident that tetra and penta-chlorinated congeners (the most toxic ones) accumulates selectively in poultry livers. Maximal concentrations have been fixed in the European legislation for dioxins and PCBs in food from animal origin, in order to protect the consumer. Data about background poultry liver contamination are scarce and the few available show levels below the legal limit for dioxins, but data are still lacking for DL-PCBs.     

Phenotypic and molecular typing of Listeria monocytogenes isolated from the processing environment and products of a sandwich-producing plant

Listeria monocytogenes as a food-borne pathogen has significant public health and economic impacts. The present study investigated the diversity of L. monocytogenes in a Swiss sandwich-producing plant over a 12-months period. L. monocytogenes were detected by culture after enrichment in 70 (3.5%) of 2028 environmental swabs and 16 (7.4%) of 217 samples from ingredients and sandwiches. Of the 86 L. monocytogenes strains, 93% belonged to serotype 1/2a and genetic lineage II. Rep PCR and PFGE analysis yielded each six profiles. Sixty-seven (77.9%) strains belonged to only one genotype, which was repeatedly found on/in slicers, conveyor belts, tables, a bread-feeding machine, spattles, air blow-guns, salmon, and egg sandwiches. Strains of this genotype persisted for more than 9 months in the processing environment, in particular on slicers and conveyor belts, which probably contributed to the contamination of sandwiches. After revision of the cleaning and disinfection procedures, L. monocytogenes were no longer found on slicers, conveyor belts, or in products. Besides, these results emphasize the importance of environmental monitoring schemes to identify potential contamination sources and as an early warning system for food business operators.     

Investigation into the risk of exposure to antibiotic residues contaminating meat and egg in Ghana

In Ghana and many developing countries, little is known about food safety in relation to antibiotic residues. The objectives of the study were to (i) determine the prevalence of antibiotic residues in animal source food and estimate the risk to consumers (ii) identify factors predisposing animal source food to contamination with antibiotic drug residues. A total of 634 samples of various animal source foods, including beef, chevon, mutton, pork and egg were screened for drug residues. Additionally, epidemiological data related to antibiotic usage on animals and consumption patterns of animal source food was collected from animal farmers and consumers of animal source food respectively. Overall, the prevalence of drug residues in animal source food was 21.1%, which translates to an average risk of exposure every fifth time animal source food is consumed. The prevalence rates of drug residues in the various animal source foods were; 30.8% (beef); 29.3% (chevon); 28.6% (pork); 24% (mutton); and 6.8% (egg). The trends of consumption of the animal source foods were similar, and majority of the consumers consumed the food every week. Considering the relatively high contamination rates of animal source food as well as their high rates of consumption, it is likely that consumers experience a high risk of exposure to drug residues, especially through beef. Specific causal factors associated with non-adherence of withdrawal requirements of drugs, and therefore contaminations of food were mainly knowledge related factors. This underscores the importance of health education of farmers in dealing with the problem of drug residues contaminating animal source food.