Spent fowl as a source of unintentional egg proteins exposure in Canadian food products.

The occurrence of egg proteins in products containing spent fowl manufactured under current practices was studied to assess the risk these food products may pose to egg-allergic consumers and to determine if Precautionary Allergen Labelling (PAL) was recommended. Spent fowl slaughtering and processing operations in 2 Canadian facilities were observed. Raw hen pieces (n = 134), coming from 2 facilities, and intermediate and processed products containing spent fowl (n = 57), coming from one facility, were analyzed using ELISA. All samples tested positive for egg proteins. Raw pieces were tested using a qualitative method (i.e., swabbing); estimated egg proteins concentrations suggest the presence of highly contaminated samples (>600 mg/kg in 2 hen wing samples). Swabbing was found to be efficient for rapid detection of eggs in raw hen pieces, but not for quantification. A comparison between swab and grind results showed that egg proteins concentration is underestimated by at least a factor 2 for whole carcasses and a factor 10 for breast, wings and drumsticks, when using the swab protocol. For intermediate and processed products, quantitative measurements indicate that egg protein levels were below 16 mg/kg. Additionally, 88 water samples from chiller tanks were analyzed and indicate that this step could be the cause of the global contamination observed with an increase in egg protein concentrations overtime during the production schedule. As egg contamination is not adequately controlled under the current good production practices, the use of PAL would be recommended for raw spent fowl products.

Peanut and hazelnut occurrence as allergens in foodstuffs with precautionary allergen labeling in Canada.

Precautionary allergen labeling (PAL) is widely used by food industries. Occurrence studies revealed that few analyzed products contained the allergen(s) present in the statement, but little is known in Canada. To improve manufacturing practices and better manage allergen cross-contamination, occurrence data is needed to determine the exposure of allergic individuals eating those products. Samples were analyzed for peanuts (n = 871) and hazelnuts (n = 863) using ELISA methods. Within samples analyzed for peanuts, 72% had a PAL (n = 628), 1% had peanuts as a minor ingredient (n = 9) and 27% were claimed "peanut-free" (n = 234). Most hazelnut samples had a PAL for tree nuts/hazelnuts (94%; n = 807) with 6% claimed "nut-free" (n = 56). Peanuts and hazelnuts were found in 4% (0.6-28.1 ppm) and 9% (0.4-2167 ppm) of all samples, respectively. Chocolates were mostly impacted; they should be treated apart from other foods and used in risk assessments scenarios to improve manufacturing practices, reducing unnecessary PAL use.

Probabilistic risk assessment for milk in dark chocolate, cookies and other baked goods with PAL sold in Canada.

The risk of having an allergic reaction in milk-allergic individuals consuming products with precautionary allergen labelling (PAL) for milk has been rarely studied in products such as dark chocolate, cookies, and other baked goods. A probabilistic risk assessment model was developed to estimate potential risks. Milk occurrence and contamination levels were reported in a previous article from our group. Dose-response curves for milk were constructed using values (n = 1078) from published double-blind placebo-controlled food challenges. Canadian consumption data was extracted from a national survey, and a homemade survey involving food-allergic Canadians. Milk eliciting doses (ED) were 0.23 (ED01), 1.34 (ED05), 3.42 (ED10), and 16.3 (ED25) mg of milk protein (Log-Normal distribution). Average exposures, per eating occasion, were 24 mg (dark chocolate), 3.9 mg (baked goods), and 0.20 mg (cookies) of milk proteins. The estimated risk of having a milk-induced allergic reaction by consuming foods with PAL for milk was higher for dark chocolate (16%; 15,881/100,000) than baked goods (3.8%; 3802/100,000) or cookies (0.6%; 646/100,000) in milk-allergic Canadians. Dark chocolate, cookies, and baked goods with PAL for milk, should be avoided by milk-allergic Canadians (consuming or not products with PAL) to prevent allergic reactions.

Occurrence and risk assessment of sesame as an allergen in selected Middle Eastern foods available in Montreal, Canada.

Sesame allergy is a public health problem in many countries around the world. The purpose of this study is to determine the occurrence of sesame allergen in unlabelled or labelled free-sesame Middle Eastern foods with or without Precautionary Allergen Labelling (PAL) 'may contain' and estimate the risk incurred by the Canadian population allergic to sesame with a focus on products purchased in Middle Eastern grocery stores and bakeries in Montreal, Canada. A total of 571 samples were analysed to determine the level of sesame protein. Of the 571 samples analysed, 19% (109/571) contained sesame (results >LOQ) with concentrations of sesame proteins varying between 0.5 and 1,875 mg kg-1 and 35% (199/571) contained traces (a value between LOD and LOQ). Unpackaged products were found to present the highest proportion of sesame containing samples (36%). For packaged products, 16% (27/173) of samples with PAL and 3% (5/173) without PAL were found to contain sesame. A probabilistic approach was used to estimate the risk incurred by the Canadian consumers allergic to sesame. Our evaluation estimated that 33 to 308 allergic reactions may occur out of 10 000 individuals ingesting one type of bakery product contaminated at a level of 0.6-74 mg kg-1 sesame proteins. The incidence and level of sesame cross-contact reported in this study demonstrate that sesame allergic consumers could react if they ignore the precautionary allergen statements on product labels. Attention to sesame as a potential cross-contact agent and as a priority allergen calls for better management, given the growing interest in this ingredient to be included in food formulations. Enhanced risk management efforts must be coupled with targeted risk communication covering both producers and consumers as to the need to adopt and an approach for the application of precautionary allergen labelling based on risk.

Occurrence of milk and egg allergens in foodstuffs in Canada.

Food allergies are life-threatening conditions that allergic individuals can avoid by consulting the food labels before consuming. Precautionary allergen labelling (or PAL), to warn against possible allergen cross-contamination, is widely used by food industries, reducing the food choices for allergic individuals. In Canada, there is limited information on the actual occurrence of allergens in products with a PAL related to the given allergen. This study attempted to fill the data gap by evaluating the occurrence of milk and egg allergens in Canadian products with PAL. A total of 1125 samples were analysed for milk and 840 for eggs, with 23% and 7% of these samples showing positive detection of ≥2.5 mg kg-1 for milk and ≥0.245 mg kg-1 for eggs. Chocolate products gave the largest number of positive results. Although the proportion of positive results was low, the levels detected reached 6471 mg kg-1 in a chocolate sample and were indicative of possible health consequences, if PAL was ignored by allergic consumers. The occurrence data generated is destinated to be used in exposure and risk assessments, to support allergen management linked to cross-contamination, with the possible development of allergen action levels that would be used by food industries, thus improving a risk-based approach for the application of PAL.

Summary of the AOAC-Sponsored Workshop Series Related to the Global Understanding of Food Fraud (GUFF): Mobilization of Resources for Food Authenticity Assurance and Food Fraud Prevention and Mitigation.

BACKGROUND: Reports of incidents associated with the misrepresentation of food products as well as the adulteration of their composition leading, at times, to significant public health impacts are being recorded. OBJECTIVE: This paper aims at summarizing the outputs of three workshops dedicated to the theme "Global Understanding of Food Fraud" (GUFF), held in Quebec City in Canada (April 2017), Beijing in the People's Republic of China (October 2017) and Dubai in the United Arab Emirates (October 2018). METHOD: Based on the contributions made at these workshops, the paper reviews current knowledge related to food fraud shared by experts and stakeholders representing the food industry sector, food regulators both domestically and internationally and scientists from Academia. It also discusses approaches available to the industry across the food supply chain to predict, prevent, and possibly mitigate food fraud, inclusive of targeted and non-targeted methods of analysis. RESULTS AND CONCLUSIONS: The paper offers a discussion on areas warranting the mobilization of efforts and resources of the food stakeholder community to reach consistent and accessible guidance on food fraud prevention, validated analytical methods along with an increased emphasis on prevention in food regulatory measures targeting food fraud. Further development is needed to reach consistent and accessible guidance on food fraud prevention, validated analytical methods, along with an emphasis on food fraud prevention. HIGHLIGHTS: Food fraud is receiving increased attention from consumers, regulators, and industry. International food fraud experts were invited to three workshops. Contributions and conclusions from the workshops are reported and discussed.

Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

Optimization of Salmonella detection in garlic, onion, cinnamon, red chili pepper powders and green tea.

Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

A Syst-OMICS Approach to Ensuring Food Safety and Reducing the Economic Burden of Salmonellosis.

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

Novel assay for the detection and monitoring of levamisole resistance in Haemonchus contortus.

A levamisole-sensitive acetylcholine receptor has recently been described in the parasitic nematode Haemonchus contortus. The pentameric receptor is produced from different subunit proteins, one of which is Hco-ACR-8. A truncated transcript, Hco-acr-8b, has been identified in six levamisole-resistant H. contortus isolates and was found to be absent in four levamisole-susceptible isolates, indicating Hco-acr-8b could be a potential marker for levamisole resistance. The Hco-acr-8b transcript contains exons 1 and 2 and terminates with 347bp from within the intron 2. In this work, we investigated genomic DNA sequences of the Hco-acr-8 gene, in a region including exon 2 and exon 3, from a wide range of levamisole-susceptible and resistant H. contortus isolates. Sequences potentially involved in generating the truncated splice variant within the second intron were analysed from individuals and pools of parasites. We found an insertion/deletion (indel) of 63bp located just downstream from the splice acceptor site for the alternative third exon. The sequence of the indel, when present, was similar in the 12 isolates examined. The presence or absence of this indel was statistically (Chi(2) test) correlated with levamisole resistance status. A correlation was also demonstrated between the absence of the indel and the expression of the Hco-acr-8b transcript. We believe this is the first report of a putative DNA marker for levamisole resistance detection. Using this new knowledge, we have developed a novel DNA-based assay for the detection and monitoring of levamisole resistance in parasitic nematodes of animals.

Efficiency of a genetic test to detect benzimidazole resistant Haemonchus contortus nematodes in sheep farms in Quebec, Canada.

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the ß-tubulin isotype 1 encoding gene in Haemonchus contortus.

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the ß-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15 mg/kg), or nine times the recommended dose rate (45 mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)(200) SNP and a decrease in the (TAC)(167) SNP. All worms genotyped were GAA at codon 198.