Control measures for Shiga toxin-producing Escherichia coli (STEC) associated with meat and dairy products: meeting report

ISSN 1726-5274 Control measures for Shiga toxin-producing Escherichia coli (STEC) associated with meat and dairy products Meeting report 39 MICROBIOLOGICAL RISK ASSESSMENT SERIES

iFood and Agriculture Organization of the United Nations World Health Organization Rome, 2022 39 MICROBIOLOGICAL RISK ASSESSMENT SERIES Control measures for Shiga toxin-producing Escherichia coli (STEC) associated with meat and dairy products Meeting report Required citation: FAO and WHO. 2022. Control measures for Shiga toxin-producing Escherichia coli (STEC) associated with meat and dairy products – Meeting report. Microbiological Risk Assessment Series No. 39. Rome. https://doi.org/10.4060/cc2402en The designations employed and the presentation of material in this information product do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations (FAO) or the World Health Organization (WHO) concerning the legal or development status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. 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Layout: Tomaso Lezzi iii Contents Acknowledgements viii Contributors ix Declaration of interests xi Abbreviations xii Executive summary xiii Background and approach 1 1.1 Scope and objectives 2 1.2 Systematic approach 3 1.3 Beef and dairy 4 1.3.1 Primary production 4 1.3.2 Beef processing and post-processing 5 1.3.3 Dairy processing and post-processing 6 References 7 Primary production control strategies for STEC in beef and dairy 9 2.1 Animal factors 10 2.1.1 Cattle genetics 11 2.1.2 Cattle intestinal microbiome 11 2.1.3 Cattle demography (age, sex, production status) 12 2.2 Environmental factors 13 2.2.1 Biosecurity 13 2.2.2 Animal density 14 2.2.3 Environmental hygiene 14 2.2.4 Manure management issues 15 2.2.5 Seasonal variability and temperature 16 2.3 Water and feed management strategies 16 2.3.1 Drinking water quality and hygiene 16 2.3.2 Drinking water treatment 17 2.3.3 Diet composition, feeding strategies and feed hygiene 17 2.3.4 Feed additives 20 1 2 iv CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 2.4 Vaccines and clinical antimicrobials 22 2.4.1 Vaccines 22 2.4.2 Clinical antimicrobials 23 2.4.3 Beta-agonists / hormones 25 2.5 Dairy production specific interventions 25 2.5.1 Milking environmental hygiene 25 2.5.2 Udder hygiene 26 2.5.3 Milk storage temperature and hygiene 26 2.6 Animal transportation 27 2.6.1 Feed withdrawal prior to slaughter 27 2.6.2 Duration of transportation 28 References 29 Processing control strategies for STEC in beef 41 3.1 Lairage 41 3.1.1 Lairage cleanliness 42 3.1.2 Livestock cleanliness 42 3.1.3 Holding animals in lairage 43 3.2 Hide decontamination 43 3.2.1 Bacteriophage 44 3.2.2 Hide washes with ambient or hot water, organic acids, and other chemicals 44 3.2.3 Hide clipping, coating and chemical dehairing 45 3.3 Slaughter and dressing 45 3.3.1 Speed of processing 46 3.3.2 Hide removal 46 3.3.3 Pre-evisceration and evisceration processes 47 3.3.4 Removal of visible faecal material from carcass 48 3.3.5 Rinsing of Head and cheek meat 49 3.4 Pre-chilling 50 3.4.1 Hot water wash 50 3.4.2 Steam pasteurization 51 3.4.3 Organic acids 51 3.4.4 Oxidizer type antimicrobials 52 3.5 Carcass chilling 53 References 54 3 vOverview of relevant risk assessment approaches 61 4.1 Physical interventions 63 4.1.1 Air-drying heat treatment 63 4.1.2 Condensing steam 63 4.1.3 Hot water 64 4.1.4 Surface trimming 64 4.1.5 Dry chilled ageing 64 4.1.6 High pressure processing (HPP) 65 4.1.7 Irradiation 66 4.2 Chemical interventions 68 4.2.1 Organic acids 68 4.2.2 Other chemical treatments 69 4.2.3 Ozone 70 4.2.4 Lactoferricin B 71 4.2.5 Essential oils 71 4.3 Biological interventions 72 4.3.1 Bacteriophages 72 4.3.2 Lactic acid bacteria (LAB) 72 4.3.3 Colicin 73 References 74 Processing and post-processing control strategies for STEC in raw milk and raw milk cheese 81 5.1 Raw milk processing 81 5.1.1 Bactofugation 82 5.1.2 Microfiltration 82 5.1.3 High pressure processing (HPP) 83 5.1.4 Irradiation (cold pasteurization) 83 5.1.5 Bacteriophage 84 5.2 Raw milk cheese processing 84 5.2.1 Milk fermentation 85 5.2.2 Protective cultures 86 5.2.3 Bacteriophage 86 5.2.4 Acidification, salting and cooking 87 5.2.5 Ripening and ageing 88 5.2.6 Cheese size 88 4 5 vi CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 5.3 Raw milk cheese post-processing 89 5.3.1 Packaging 89 5.3.2 Irradiation 90 5.3.3 Bacteriophage 90 References 91 Primary production and processing control strategies for STEC in other animal species 97 6.1 Small ruminants: primary production 97 6.1.1 Diet composition and feeding strategies 98 6.1.2 Feed additives 98 6.1.3 Vaccination 99 6.1.4 Feed withdrawl prior to slaughter 99 6.2 Small ruminants: processing (meat and dairy) 100 6.2.1 Pre-chill carcass treatments 100 6.2.2 Dairy processing 101 6.3 Other species: primary production and processing 101 6.3.1 Reindeer 101 6.3.2 Yaks 101 6.3.3 Camel 102 6.3.4 Water buffalo 102 6.3.5 Bison 103 6.3.6 Wild game 103 6.3.7 Swine 104 References 105 Laboratory testing 111 7.1 Laboratory testing for STEC during primary processing 111 7.2 Laboratory testing for STEC detection across the beef processing chain 112 7.3 Laboratory testing for STEC detection across the dairy processing chain 115 7.3.1 Raw milk 115 7.3.2 Raw milk cheeses 116 References 119 Conclusions 125 6 7 8 vii ANNEXES Annex 1 Primary production control strategies for STEC in beef and dairy 131 Annex 2 Processing control strategies for STEC in beef 146 Annex 3 Post-processing control strategies for STEC in beef 158 Annex 4 Processing and post-processing control strategies for STEC in raw milk and raw milk cheese 164 References for annexes 1-4 171 viii CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS Acknowledgements The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) would like to express their appreciation to all those who contributed to the preparation of this report through the provision of their time and expertise, data and other relevant information before, during and after the meeting. Special appreciation is extended to all the members of the expert committee for their dedication to this project, in particular, to Todd Callaway for his leadership in chairing of the meeting and his support in preparing the final report. All contributors are listed in the following pages. The preparatory work and expert meeting convened to prepare this report was coordinated by the Secretariat of the Joint FAO/WHO Expert Meetings on Microbiological Risk Assessment (JEMRA). ix Contributors EXPERTS Frederic Auvray, National Veterinary School of Toulouse, France Li Bai, China National Center for Food Safety Risk Assessment (CFSA), China Todd Callaway, University of Georgia, the United States of America Natalia Cernicchiaro, Kansas State University, the United States of America Isabel Chinen, National Infectious Disease Institute, Argentina Paul Cook, Food Standards Agency, the United Kingdom of Great Britain and Northern Ireland Roger Cook, Ministry of Primary Industries, New Zealand Patricia Desmarchelier, Food Safety Specialist / Consultant, Australia Geraldine Duffy, Food Research Centre, Teagasc, Ireland Peter Feng, US Food and Drug Administration (Retired), the United States of America Jorg Hummerjohann, Bacteriological Food Safety, Agroscope, Switzerland Ian Jenson, Meat & Livestock Australia, Australia Musafiri Karama, University of Pretoria, South Africa Tim McAllister, Agriculture and Agri-Food Canada, Canada Sara Monteiro Pires, The National Food Institute, Denmark Camilla Sekse, Norwegian Veterinary Institute, Norway Roberto Vidal, Universidad de Chile, Chile RESOURCE PERSONS Sarah Cahill, Joint FAO/WHO Food Standards Programme, Italy Verna Carolissen-Mackay, Joint FAO/WHO Food Standards Programme, Italy Charmaine Chng, World Organisation for Animal Health (WOAH), France Jose Emilio Esteban, Codex Committee on Food Hygiene, the United States of America x CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS Goro Maruno, Joint FAO/WHO Food Standards Programme, Italy Jenny Scott, Codex Committee on Food Hygiene, the United States of America Hajime Toyofuku, Yamaguchi University, Japan Constanza Vergara, Codex Committee on Food Hygiene, Chile Lingping Zhang, Joint FAO/WHO Food Standards Programme Food, Italy SECRETARIAT Haruka Igarashi, World Health Organization, Switzerland Christine Kopko, Food and Agriculture Organization of the United Nations, Italy Jeffrey LeJeune, Food and Agriculture Organization of the United Nations, Italy Satoko Murakami, World Health Organization, Switzerland Kang Zhou, Food and Agriculture Organization of the United Nations, Italy xi Declaration of interests All participants completed a Declaration of Interests form in advance of the meeting. The Interests declared were not considered by FAO and WHO to present any conflict in light of the objectives of the meeting. All the declarations, together with any updates, were made known and available to all the participants at the beginning of the meeting. All the experts participated in their individual capacities and not as representatives of their countries, governments or organizations. xii CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS Abbreviations and acronyms CAC Codex Alimentarius Commission CCFH Codex Committee on Food Hygiene CFU colony forming units CRISPR clustered regularly interspaced short palindromic repeats DALY disability-adjusted life year DDGS dried distiller's grains with solubles DNA deoxyribonucleic acid eae Escherichia coli attaching and effacing gene EFSA European Food Safety Authority ELISA enzyme-linked immunosorbent assay EPEC enteropathogenic Escherichia coli FAO Food and Agriculture Organization of the United Nations GAP good agricultural practice GHP good hygiene practice GMP good manufacturing practice HACCP hazard analysis and critical control point [system] HUS haemolytic uremic syndrome JEMRA Joint FAO/WHO Expert Meetings on Microbiological Risk Assessment NTS non-type specific PCR polymerase chain reaction PFU plaque forming units SCFA short-chain fatty acids STEC Shiga toxin-producing Escherichia coli stx Shiga toxin gene Stx Shiga toxin TVC total viable bacterial counts US FDA United States of America Food and Drug Administration USDA United States of America Department of Agriculture VTEC vero toxin-producing Escherichia coli WDGS wet distiller's grains with solubles WGS whole genome sequencing WHO World Health Organization WOAH World Organisation for Animal Health xiii Executive summary Shiga toxin-producing Escherichia coli (STEC) are estimated to cause more than 1.2 million illnesses and 128 deaths globally each year. The previous work of FAO and WHO identified beef and other types of meats, dairy products and produce as significant risk factors for STEC infection. As such, at its 42nd Session, the Codex Alimentarius Commission (CAC) endorsed the Codex Committee on Food Hygiene’s (CCFH) recommendation for the development of guidelines for the control of STEC in beef, raw milk and cheese produced from raw milk, leafy greens and sprouts. To facilitate this work, the CCFH requested that FAO and WHO Joint Expert Meeting on Microbiological Risk Assessment (JEMRA) provide scientific advice on the effectiveness and utility of control measures against STEC during primary production, processing and post-processing of raw meat, raw milk and raw milk cheeses. During the meeting, the expert committee reviewed interventions for the control of STEC in cattle, raw beef and raw milk and raw milk cheese manufactured from cows’ milk, and also evaluated available evidence for other small ruminants (goat, sheep), swine and other animals (reindeer, yak, camelids, bison, buffalo and swine). The expert committee was tasked with scoring the degree of support for the effectiveness of interventions for the specific control of STEC as high, medium or low based on the evidence available within the scientific literature. In meat production and processing systems, many approaches to support control of STEC are based on good agricultural practices (GAP) and/or good hygiene practice (GHP) that aim to generally reduce the spread of pathogens and are not specifically focused on STEC. On-farm, these include managing the hygienic conditions of housing, bedding and drinking water hygiene, appropriate animal density and biosecurity measures, effective sanitation of facilities and proper disposal of manure. On-farm, several dietary and herd management strategies with varying levels of impact on STEC populations in beef and dairy animals have been explored. Evidence to support cattle demography (Section 2.1.3), animal density (Section 2.2.2), biosecurity (Section 2.2.1), and environmental hygiene (Section 2.2.3) were rated as having a medium or medium to high degree of support with regards to their ability to impact STEC. Interventions including feeding of forage versus concentrate rations, specific grain types (Section 2.3.3), and the inclusion of citrus products and essential oils in feed (Section 4.2.5) were supported at low to medium or medium degree of support, yet probiotics xiv CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS may be useful with administered to cattle, goats and sheep through feed (Sections 2.3.4.1 and 6.1.1). Some vaccines have been shown to reduce faecal excretion of STEC O157:H7 (Section 2.4.1), but their efficacy is variable depending on the vaccine and the number of doses administered. Long distance transport and the stress of interim unloading/loading have been shown to increase faecal excretion of STEC that can lead to cross-contamination between animals (Section 2.6). Transport distances should be minimized in accordance with best practices for animal welfare, and the evidence related specifically to the control of STEC was supported at a low degree. A summary of primary production control measures for STEC in cattle and their degree of support rating (high, medium, low), based on scientific evidence, is available in Annex 1. Avoiding contamination of the carcass through contact with hides, gut contents or faeces during slaughter is an accepted management practice during meat processing, but evidence supporting the effectiveness and reliability of these measures for the control of STEC was limited. Processing measures where evidence supported a high or medium to high rating for efficacy in STEC reduction included steam vacuuming of visible faecal contamination on carcasses (Section 3.3.4.3), and the use of a hot potable water carcass wash, steam pasteurization followed by 24 h air chilling and combinations of these (Section 3.4). The use of knife trimming to remove carcass tissue contaminated with faecal material is common and is supported by a medium confidence level in the evidence (Section 3.3.4.2). Despite the commercial use of pre-chill carcass decontamination treatments using organic acids and other chemical agents, the confidence in the evidence was low in cattle and other small ruminants due to high variability in results (Section 3.4.3). A summary of processing control measures for STEC in beef and their degree of support (high, medium, low), based on scientific evidence, is available in Annex 2. The efficacy of available control measures for reducing or eliminating STEC on primal cuts, trim, cheek meats, and ground beef was widely varied. Yet, the use chemical antimicrobial dips (Section 4.2) for primals and trims were supported at a low to medium level of confidence, and high-pressure processing (HPP) (Section 4.1.6), gamma irradiation and electron beam sterilization (eBeam) (Section 4.1.7) produced significant reductions of STEC in ground beef and in retail packs. A summary of post-processing control measures, and combinations of these, for STEC in beef and their degree of support (high, medium, low), based on scientific evidence, is available in Annex 3. Pork products and meat from wild game have occasionally been confirmed as vehicles of STEC transmission, but there are no interventions or practices during the processing of these animals that are specific for STEC. Meat from these xv species could be treated post-harvest in a similar fashion as beef to reduce STEC, but reports of the efficacy of these interventions are not available. Contamination of milk with pathogens, including STEC, mainly occurs during milking or via milking equipment, milking personnel, and from the farm environment. Thus, factors affecting the carriage of STEC in live animals and those practices surrounding milking hygiene can reduce, but not assure the absence of contamination of raw milk. The efficacy of the interventions against STEC during the production of raw milk and raw milk cheeses varied greatly depending on the animal origin of the raw milk, manufacturing practices, the scale of production, and the microbial load. Temperature control and hygiene during milking, storage and transportation can significantly affect the microbiological safety of raw milk prior to processing, packaging and sale of milk intended for drinking or for manufacturing of raw milk cheeses. Although these interventions can mitigate the growth of E. coli and other indicator organisms, the degree of support in the evidence for these interventions and the control of STEC ranged from low to medium (Section 2.5). Apart from pasteurization, which is very effective, several technologies have been evaluated to mitigate the presence of STEC in raw milk. Bacteriophages specific to E. coli and STEC have shown some reductions in STEC during refrigeration storage of raw milk (Section 5.1.5). The effect of adding bacteriophage to control E. coli during milk fermentation in the making of cheeses has also been examined with varying results depending on the STEC serovar. The degree of support in the evidence of bacteriophage to specifically control for STEC was evaluated as low (Section 5.2.3). Gamma or eBeam irradiation are very effective at reducing bacterial levels in milk and on cheese surfaces, yet off-flavors are often reported. The degree of support for the evidence was rated as medium (Section 5.3.2). A summary of processing and post-processing control measures for STEC in raw milk and raw milk cheese and their degree of support rating (high, medium, low), based on scientific evidence, is available in Annex 4. The implementation of monitoring plans at the farm level to measure the impact of STEC prevalence is considered impractical, although sampling and testing of beef and raw milk products are a means to verify that food safety program are successful. Because STEC are often present only at low levels in foods, culture enrichment of food samples is a critical step in detecting STEC in meat, dairy and other foods. Since STEC testing is complex, the quantitative detection of non-type specific (NTS) E. coli has been proposed as an alternative hygienic indicator during processing and post-processing stages, although it is not an absolute estimate of STEC levels. xvi CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS The use of molecular techniques, such as PCR, that target STEC virulence genes are highly sensitive and specific for STEC detection but presumptive results must be confirmed by traditional culture-based methods or by immunomagnetic separation (IMS). Methods are needed that enable the efficient and specific isolation of STEC O157:H7 and non-O157 STEC. The expert committee also discussed some of the limitations and gaps regarding the available data. In-plant scientific evaluations of interventions and treatments to control STEC throughout raw beef, raw milk and raw milk cheese production are frequently prohibited due to health risks associated with the potential introduction of pathogens into the food supply and the cost associated with testing large number of samples required for detecting STEC in food matrices. Consequently, surrogate bacteria, such as NTS E. coli, are used as substitutes and the results extrapolated, meaning that evidence of intervention effects specifically for STEC may not be available currently or in the future. Therefore, there is doubt and uncertainty as to whether the detection and reduction levels observed in surrogate studies are truly representative of STEC or of commercial production and processing. Many studies focused on the impact of an individual control measure at a specific stage in the food chain, rather than in the context a total food chain or of the safety of the food available to the consumer. Many food businesses have implemented multiple control measures concurrently or sequentially on farms and in processing facilities, but the overall efficacy of multiple “hurdles” in the total chain remains difficult to quantify It was recognized that with advances in analytical methods, including increasing use of molecular tools, the evaluation of evidence concerning some STEC control measures and interventions may need to be revised in the future. 11 Background and approach Shiga toxin-producing Escherichia coli (STEC) are an important cause of foodborne disease. Infections can result in a wide range of disease symptoms from mild intestinal discomfort and hemorrhagic diarrhea to severe conditions including haemolytic uremic syndrome (HUS), end-stage renal disease and death. In its report on the global burden of foodborne disease, WHO estimated that in 2010 foodborne STEC caused more than 1.2 million illnesses, 128 deaths, and nearly 13 000 Disability Adjusted Life Years (DALYs) (WHO, 2015). The Codex Committee on Food Hygiene (CCFH) has highlighted the importance of STEC in foods since its 32nd Session in 1999, when it prioritized their presence in beef and sprouts as significant public health problems in Member countries (FAO and WHO, 2000). Following a request from the 47th Session in November 2015 (FAO and WHO, 2016), the FAO and WHO published the report Shiga toxin-producing Escherichia coli (STEC) and food: attribution, characterization and monitoring in 2018 (FAO and WHO, 2018). As part of the 50th session of CCFH in November 2018, the FAO/WHO further updated the committee with additional information on STEC that were subsequently published in the report Attributing illness caused by Shiga toxin-producing Escherichia coli (STEC) to specific foods (FAO and WHO, 2019a). The Codex Alimentarius Commission (CAC) at the 42nd Session, July 2019, approved new work on the development of guidelines for the control of STEC in beef, raw milk and cheese produced from raw milk, leafy greens and sprouts (FAO and WHO, 2019b). To support this work, the Joint FAO/WHO Expert Meeting on Shiga toxin-producing Escherichia coli (STEC) associated with Meat and Dairy Products was convened virtually from 1 to 26 June 2020 to review relevant measures for pre- and post-harvest control of STEC in animals and foods of animal origins. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 2 Although STEC can be isolated from a variety of food production animals, they are most commonly associated with ruminants from which we derive meat and milk. Because of the widespread and diverse nature of ruminant-derived food production, coupled with the near ubiquity of STEC worldwide, there is no single definitive solution to STEC risk control that will work alone or in all situations. Instead, the introduction of multiple interventions applied in sequence as a “multiple-hurdle scheme” to reduce STEC at several points throughout the food chain (including processing, transport and handling) will be most effective. It is important to note that complementary approaches to reduce STEC must be selected, however STEC control strategies may impact product quantity, quality or production efficiency. This impact must not unduly impact stable access to high quality and safe dietary protein for the world’s population. When deciding on interventions to implement, it is necessary to consider regulatory status for both local and export markets. It is also essential to consider the required purpose, cost, space availability and infrastructure of the food processing facility, in addition to environmental impact factors, such as waste and effluent disposal (Gagaoua et al., 2022). 1.1. SCOPE AND OBJECTIVES Potential STEC intervention strategies can be applied at various stages throughout the food production-to-consumption continuum. For the purposes of discussion, the expert committee has presented three general categories or stages of food production and processing, whilst recognizing that the diversity and complexity of value chains vary considerably depending upon scale, geography and specific products. Hence certain strategies may be classified by others as being part of a different or of multiple categories. Primary Production control strategies address the stages of animal production until the time of arrival at the slaughter site; and for milk, until milk is stored prior to pasteurization, or for use in raw milk cheese making, or it is further distributed as raw milk. Control strategies during this phase include farm management activities, waste disposal, feed production, milking procedures, and transport prior to slaughter/pasteurization and processing. Processing control strategies include interventions applied at lairage and animal handling facilities at slaughter operations, included as well are slaughter and dressing processes through to carcass chilling and further fabrication. Treatment of fluid drinking milk intended to be consumed without pasteurization and all steps in the production of raw milk cheeses are included as potential processing controls. CHAPTER 1 – BACKGROUND AND APPROACH 3 Post-processing control strategies are focused upon raw meat and raw milk (both fluid milk and products) following initial processing through to consumer handling. This stage includes controls applied following carcass production, chilling of carcasses, and those involved in butchering the meat into primal, subprimal and fabricated cuts, packaging, storage, and transport of meats prior to retail or consumer handling. Post-processing controls for raw milk and raw-milk cheeses include interventions and handling processes applied to products (storage, maturation, packaging transport) prior to distribution to wholesalers, retailers or consumers. During the meeting, the expert committee examined each of these general categories of process steps separately, yet they are clearly interconnected as the production of food is a continuum. Reductions in STEC populations achieved during primary animal production may be nullified by subsequent improper processing and post- processing procedures. Furthermore, there has been inconsistent evidence of a linkage between STEC prevalence in live cattle and contamination of carcasses or finished beef or milk products. 1.2. SYSTEMATIC APPROACH Interventions and strategies to control STEC were considered when there was evidence from either laboratory, pilot plant or commercial scale studies that demonstrated a reduction in the prevalence or concentration of STEC, non-type specific (NTS) E. coli, faecal microorganisms or total microbial load. Moreover, good agricultural practices (GAP) and/or good hygiene practices (GHP) considered to reduce or control the transmission of microorganisms were also included even if there was not specific evidence of their effect on STEC. The quality of evidentiary support varied greatly with study design, scale (laboratory, pilot or full-scale commercial production) and the analytical methods used. The following sections and the tables in Annex 1, 2, 3 and 4 summarize the interventions evaluated for the specific control of STEC at various critical processing points throughout the beef and dairy value chains. They include examples of research and data from relevant publications. Although the outcomes considered included reduction in prevalence or concentrations of NTS E. coli and/or STEC, the scientific evidence was evaluated as to the degree of support based on the interventions ability to specifically control STEC. A scale consisting of the following categories and interpretation was used to qualify the degree of support: • Low: negligible evidence for efficacy of the intervention in reducing STEC presence under field or laboratory conditions and/or documented detrimental effect on animal or derived food product are evident. No obvious pathway or likelihood of immediate adoption. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 4 • Medium: published evidence of efficacy of the intervention in reducing STEC presence through research trials and/or supported by laboratory or field experiments and no detrimental effect on animal or derived food product are evident. However, there are concerns about practicality and implementation in the field. • High: clear and compelling evidence that the intervention, management practice, or procedure reduces the presence of STEC at the application point in the production chain. No detrimental effect on animal or derived food product are evident and widespread application is practical. Many of the practices evalutated at the different stage in the production chain are considered as GAP or GHP and have been scientifically proven to minimizing the potential for microbial contamination, including STEC. Although these well- established best practices have a high degree of support, the purpose of this meeting was to evaluate the scientific evidence and make recommendations specifically regarding their ability to control for STEC. 1.3. BEEF AND DAIRY 1.3.1. Primary production Primary production control measures for STEC were identified using a variety of laboratory, experimental and epidemiological approaches. Although laboratory studies, and to a lesser extent, animal and research-farm/feedlot experiments, provide opportunity for testing hypotheses under controlled conditions, the extent to which results from these studies can be extrapolated and reasonably expected to be repeatable under real life and commercial settings is often limited. Results from epidemiological studies or ongoing monitoring of animals may provide more realistic data on the effectiveness of interventions under certain field conditions, but such studies are more difficult to control and replicate because of biological variability and differences in production practices, environments and species. The reporting of well-designed studies evaluating the impact of individual strategies on farms, as well as those strategies applied in sequence to assess potential synergies, will provide greater confidence on the strength of the associations between the interventions and the outcomes. Moreover, primary production control strategy assessments were frequently based strictly on microbiological criteria, with other important metrics such as animal health, well-being, toxicology, and economic impact seldomly reported. CHAPTER 1 – BACKGROUND AND APPROACH 5 Early microbiological assessments of intervention effectiveness in live animals typically focused on the prevalence of STEC (percentage of animals or farms that are STEC positive) in the study population. Over time some researchers, but not all, included the concentration of STEC present in the specimens as an important outcome variable. Although both prevalence and excretion concentration are important for determining the overall risk of STEC transmission to raw beef or milk, much of the available literature lacks both of these variables, thus limiting the potential for cross study comparisons and understanding the fullest impacts of the interventions and their applicability to the production environment. Thus, both prevalence and concentrations of STEC present are important factors for the determination of intervention efficacy 1.3.2. Beef processing and post-processing Challenge studies using STEC have been performed, but only a few studies investigated the impact of both low and high inoculum and various strains and serogroups were used in these studies. A published meta-analysis on the effectiveness of interventions for the control of E. coli contamination in cattle processing plants found that initial microbial concentrations were significant predictors of intervention effectiveness (Zhilyaev et al., 2017). These results demonstrate that interventions become less effective as E. coli counts decrease, even when publications when results with questionable detection limits were excluded. For post-processing interventions, the experimental approaches were associated with many limitations: • Many studies used a high starting inoculum concentration of one or more STEC strains. Few studies examined the impact of low and high inoculum levels. Low inoculum levels are likely to be more representative of natural contamination cases. • Most studies were conducted using a single or mixed strain of STEC O157:H7. A few studies used non-O157 STEC strains, but most of these studies presumed that STEC O157:H7 and non-O157 behaved similarly in those environments. • Some studies suggested little variation in intervention impacts between different strains, but strain to strain variations have been reported with other interventions, such as bacteriophage treatments. • The log10 reductions in STEC concentrations reported may not include stressed or injured cells which are unable to recover and be detected when selective media are used for enumeration. Nevertheless, injured cells are viable and may recover under favourable circumstances. • The study conditions used in some of the experiments may not reflect actual production practices, for example, temperatures and durations of product storage. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 6 • For interventions where there are many experimental studies (e.g. organic acids), meta-analysis should be used to look at the scale of STEC reduction data in more detail. • Other concerns include the efficiency of methods used in the recovery of STEC cells from different types of beef products, including whether current techniques are capable of recovering all survivors and as such are log10 reductions potentially overestimated, and the recovery media used for different meat product/matrices need to be examined and clarified to understand the impact of viable but not culturable (VBNC) STEC cells in these studies. 1.3.3. Dairy processing and post-processing For this report, rather than being exhaustive, the expert committee’s appraisal of the body of scientific evidence focused on interventions to reduce STEC prevalence and concentration in raw milk and raw milk cheeses, predominantly of bovine and caprine origin. Similarly, most of the interventions were evaluated in challenge studies in the laboratory or implemented in pilot plants rather than under commercial, production-scale conditions. Lastly, it was noted that the efficacy of interventions against STEC in raw milk cheeses varied greatly depending on the animal species of origin of the milk, manufacturing practices, the scale of production, the baseline microbial load and composition of the raw materials, and the STEC serotype. CHAPTER 1 – BACKGROUND AND APPROACH 7 References FAO & WHO. 2000. Report of the thirty-second session of the Codex Committee on Food Hygiene. Codex Alimentarius Commission. Twenty-fourth session, Geneva, Swtizerland, 2–7 July 2001. Cited 31 March 2022. www.fao.org/fao-who- codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace. fao.org%252Fsites%252Fcodex%252FMeetings%252FCX-712-32%252FAl01_13e.pdf FAO & WHO. 2019a. Attributing illness caused by Shiga toxin-producing Escherichia coli (STEC) to specific foods: Report. Microbiological Risk Assessment Series No. 32. Rome, FAO. www.fao.org/3/ca5758en/ca5758en.pdf FAO & WHO. 2019b. Report of the fiftieth session of the Codex Committee on Food Hygiene. Codex Alimentarius. Forty-second session, CICG, Geneva, 7–12 July 2019. Cited 31 March 2022. www.fao.org/fao-who-codexalimentarius/sh-proxy/ en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcod ex%252FMeetings%252FCX-712-50%252FReport%252FREP19_FHe.pdf FAO & WHO. 2018. Shiga toxin-producing Escherichia coli (STEC) and food: attribution, characterization, and monitoring. Microbiological Risk Assessment Series No. 31. Rome, FAO. www.fao.org/3/ca0032en/ca0032en.pdf FAO & WHO. 2016. Report of the forty-seventh session of the Codex Committee on Food Hygiene. Codex Alimentarius. Thirty-ninth session, Rome, Italy, 27 June to 01 July 2016. Cited 31 March 2022. www.fao.org/fao-who-codexalimentarius/sh- proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%2 52Fcodex%252FMeetings%252FCX-712-47%252FReport%252FREP16_FHe.pdf FAO & WHO. 2014. Report of the forty-fifth session of the Codex Committee on Food Hygiene. Codex Alimentarius. Thirty-seventh session, Geveva, Switzerland, 14–18 July 2014. Cited 31 March 2022. https://www.fao.org/fao-who-codexalimentarius/ sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites %252Fcodex%252FMeetings%252FCX-712-45%252FREP14_FHe.pdf Gagaoua, M., Duffy, G., Alvarez, C., Burgess, C. M., Hamill, R., Crofton, E., Botinestean, C., Ferragina, A., Cafferky, J., Mullen, A. M., Troy, D. 2022. Current research and emerging tools to improve fresh red meat quality. Irish Journal of Agricultural and Food Research. doi: 10.15212/ijafr-2020-0141 WHO. 2015. WHO Estimates of the global burden of foodborne diseases. Geneva, WHO. https://apps.who.int/iris/bitstream/handle/10665/199350/9789241565165_eng. pdf?sequence=1 Zhilyaev, S., Cadavez, V., Gonzales-Barron, U., Phetxumphou, K. & Gallagher, D. 2017. Meta analysis on the effect of interventions used in cattle processing plants to reduce Escherichia coli contamination. Food Research International, 93: 16–25. doi: 10.1016/j.foodres.2017.01.005

92 Primary production control strategies for STEC in beef and dairy Primary production controls include strategies that can be used on small and large farm operations for food animal species raised for meat and dairy production. There are many potential control points during the life cycle of food animals where STEC carriage and/or prevalence can be reduced. Because of the diversity of animal production systems world-wide, no single factor can be universally applied to control STEC in every circumstance and condition. Moreover, many control methods discussed herein are broadly impactful, such as the application of GAP, to reduce the spread of many pathogens, including STEC, but are not specifically targeted against STEC. While cattle are not the only ruminant species to carry STEC, beef products or cross-contamination from cattle husbandry (or production) are more frequently associated with human foodborne STEC illnesses. Moreover, in addition to contributions of dairy cattle to human STEC illness via consumption of contaminated milk products, the majority of dairy cattle at the end of their productive lives, also enter the beef production chain; therefore, this report has focused primarily on beef and dairy cattle. Nevertheless, many of the concepts and principles for STEC control may also be applicable for other food animals, especially other ruminants (Section 6). Because of the paucity of literature on the control of STEC in animals other than Bos taurus (and Bos taurus indicus), this section addresses control of STEC in cattle. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 10 Many of the cattle management strategies discussed fall into GAP or GHP, but it is important to note that these practices can have a significant impact on the risk of STEC carriage and transmission to the food supply. Many of the practices reported herein are not necessarily specific to STEC but can also be useful controls for other pathogenic foodborne bacteria such as non-typhoidal Salmonella, Campylobacter, and Listeria. However, the efficacy of any intervention against STEC should not be construed to imply their effectiveness against other foodborne pathogenic bacteria, or vice versa. A summary of primary production control measures for STEC in cattle and their degree of support rating (high, medium, low), based on scientific evidence, is available in Annex 1. 2.1. ANIMAL FACTORS Although STEC are commonly found in ruminants, specific animal factors (e.g. host breed, genetics) can influence host animal carriage and excretion of STEC. Animal-specific differences have not been exploited for the development of intervention strategies per se, yet they represent areas for future investigation (e.g. specific targeting of host epithelial cells via the intimin protein vaccine development) to reduce STEC excretion and prevalence. Super-shedding cattle are defined as those that are excreting more than 4 log10 CFU/g of STEC O157:H7 of faeces (Munns et al., 2014; Munns et al., 2015; Castro et al., 2017). A substantial body of evidence demonstrated that super-shedding events are responsible for the majority of STEC transmission among cattle and is the largest contributor of STEC spread and a threat to food safety. However, the detection of a super-shedding event is dependent on the timing of faecal sampling of an individual animal, possibly a reflection of the natural course of infection in cattle following exposure. To date, efforts to identify distinct genomic markers in the host related to super-shedding has been largely proven unsuccessful. There is evidence that certain strains of STEC O157:H7 are more likely to be associated with super-shedding, but this relationship is not conclusive (Munns et al., 2015). One hypothesis that has been posited is that super-shedding results from STEC O157:H7 biofilms within the lumen of the rectum being sloughed from the intestinal epithelium and entering faecal matter (Munns et al., 2014). This event would account for the high levels of STEC O157:H7 in faeces and the intermittent nature of the phenomena. CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 11 2.1.1. Cattle genetics There is a growing body of evidence indicating that host genetics impact both the phylogenetic diversity and the relative abundance of populations of the intestinal microbiome. Factors such as innate and acquired immunity as well as other host-microbiome communication channels may influence the establishment of STEC within the host and subsequently, STEC excretion (Wang et al., 2016). Suppression of some of the genes involved in immune responses within the epithelium of the recto-anal junction has been observed in cattle that tend to shed more STEC O157:H7 (Jeon et al., 2013). However, currently it is not clear if this reflects a response controlled by host genetics or one that is induced as a result of interactions between STEC O157:H7 and the host epithelium. We are unaware of studies specifically designed to assess the impact of cattle breed on STEC excretion, but it is clear that both beef and dairy cattle can readily become colonized with these pathogens (Jeon et al., 2013; Riley et al., 2003). Production conditions, animal management, diet composition and STEC genomics are likely more important and impactful than cattle host genetics in determining the likelihood of STEC excretion. The degree of support for the use of host animal genetics as an intervention specifically for the control of STEC in cattle herds was low. 2.1.2. Cattle intestinal microbiome The large microbial ecosystem that occupies the gastrointestinal tract of cattle is a vast biochemical/enzymatic/nutrient reservoir that can impact STEC carriage. There is evidence that the phylogenetic composition of this microbial population within the lower gastrointestinal tract varies between super-shedding and non-shedding cattle, with a greater microbial diversity found in non-shedders (Zaheer et al., 2017; Xu et al., 2014). However, distinct members or catabolic niches within the microbial community that confer resistance (or susceptibility) to STEC colonization have not been identified. Furthermore, end products of the gastrointestinal microbial fermentation that enhance or prevent STEC colonization remain unknown. Prebiotics and probiotics may also alter the excretion of STEC through changes in the species composition of the gastrointestinal microbiome. Shifts in the host microbiome may alter nutrient availability for the growth of STEC or metabolic end products from microbial fermentation that inhibit the growth and establishment of STEC within the intestinal tract (VanKessel et al., 2002). However, it is apparent that changes in the microbial population of the gut can provide opportunities for pathogens to colonise the gut, or to be excluded from the complex population. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 12 The degree of support for the modification of the gastrointestinal microbiome as an intervention specifically for the control of STEC in cattle heards was low. 2.1.3. Cattle demography (age, sex, production status) Age of the animal, and the type of cattle have been identified as risk factors for STEC excretion. Younger animals have greater susceptibility to STEC colonization and STEC faecal prevalence is higher in young milk-fed animals as compared to mature adult beef and dairy cows. Differences in colonization are thought to be related to changes in the animal genetic and physiological factors, diet, management and environmental factors that occur as cattle approach physiologic maturity (Ekong, Sanderson and Cernicchiaro, 2015). Grouping cattle into age specific groups is a GAP that minimizes disease spread and prevents exclusion and bullying by dominant animals. Calves shed STEC O157:H7 more frequently than do older cattle, so keeping young cattle in the same groups throughout rearing without introducing new animals is recommended to reduce STEC O157:H7 prevalence (Sanderson et al., 2006; Ellis-Iversen et al., 2008). Other factors such as feeding colostrum from the calf ’s dam instead of from pooled sources, a decrease in serum IgG concentration and a high temperature-humidity index in the pens, were identified as increasing the likelihood for the presence of STEC O157:H7 in pre-weaned calves. These findings supported the idea that immunity and overall health of the calf gastrointestinal tract are important factors that may influence the faecal presence of STEC O157:H7 (Stenkamp-Strahm et al., 2018). To prevent the spread of animal disease and enhance animal health, the housing of young dairy calves and veal calves individually or in small groups is common practice and can contribute to a reduction in the transmission of STEC (Bosilevac et al., 2017). Faecal prevalence of STEC O157:H7 may also be impacted by the production stage of the cattle. However, it should be noted that eventually many dairy animals enter the meat supply at the end of their dairy production stage. The prevalence of STEC serogroups often associated with severe infections in people, such as O157, O26, O111, O103, O121, O45, and O145 was significantly higher in younger animals (veal, young beef and dairy) than in adults (beef and dairy) (Bibbal et al., 2015; Mellor et al., 2016), but the basis for this difference remains to be elucidated. If possible, separation of calves from adult cows and replacement dairy heifers, and lowering the density of cattle in pens, may reduce STEC prevalence by reducing opportunities for animal-to-animal horizontal spread (Ekong, Sanderson and Cernicchiaro, 2015). It has been shown that there was an increased probability of finding a STEC excreting animal where there are larger numbers of finishing cattle, CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 13 or a farm being classified as a dairy unit that also stocked beef animals (Gunn et al., 2007; Synge et al., 2003). The impact of stage of lactation on STEC excretion is inconsistent (Edrington et al., 2004). During the first 30 days (approximately) after calving, dairy cows rapidly increase milk production capacity, which consumes a great deal of dietary energy. During this transition period, many high milk-producing dairy cows cannot consume enough feed to meet their energy demands, resulting in severe negative energy balance, which in turn is linked with many metabolic disorders and is associated with a 30 percent increase in the risk of STEC O157:H7 prevalence and excretion in dairy cows (Venegas-Vargas et al., 2016). There is currently no evidence indicating that there is an effective intervention strategy or control point linked with reducing the effects of the negative energy balance. There are no specific recommendations to address negative energy balance as a hazard-based intervention for the control of STEC in cattle herds, except for following GAP measures to improve animal immunity and overall animal health, which could minimize STEC spread. Grouping of cattle by age and production status is a GAP; however, the degree of support for the use of cattle grouping based on demographic characteristics as an intervention specifically for the control of STEC in cattle herds was medium. 2.2. ENVIRONMENTAL FACTORS 2.2.1. Biosecurity While cattle, sheep and goats are primary reservoirs of STEC, other ruminants as well as monogastric animals can carry STEC. These other animal species can spread STEC between animals on a farm (Bolton, O'Neill and Fanning, 2011), from farm-to-farm, and in the case of birds, over long distances (Cernicchiaro et al., 2012). Mixing of sheep with cattle has been shown to increase the risk of cattle shedding STEC (Stacey et al., 2007), and a positive correlation was found between cattle shedding STEC and sheep density (Strachan, Fenlon and Ogden, 2001). Studies have found that other animals (rodents, insects, birds and boars) can carry STEC at least transiently, which may increase the risk of STEC shedding by cattle (Ahmad, Nagaraja and Zurek, 2007; Branham et al., 2005; Cernicchiaro et al., 2012; Cizek et al., 1999; French et al., 2010; Hancock et al., 1998; Talley et al., 2009; Rice et al., 2003; Sánchez et al., 2010; Wetzel and LeJeune, 2006). The presence of dogs, CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 14 pigs, or wild geese on the farm have also been associated with an increased risk of STEC O157:H7 shedding (Gunn et al., 2007; Synge et al., 2003). These vectors can transfer STEC (and other pathogens) to non-infected groups of cattle within a farm, between farms and even over long distances. Another important consideration is open versus closed herds. Maintaining a stable herd population and not moving cattle between herds is a GAP and contributed to a 48 percent reduction in STEC in a randomised control trial in England and Wales (Ellis-Iversen et al., 2008). On-farm biosecurity is a GAP, and the degree of support for farm biosecurity as an intervention specifically for the control of STEC in cattle herds was medium to high. 2.2.2. Animal density Higher animal density has been linked with an increased risk of carriage of some STEC, including STEC O157:H7, and may also play a role in the horizontal spread of STEC O157:H7 (Frank et al., 2008; Gunn et al., 2007; Vidovic and Korber, 2006), since densely packed animals have a greater chance of contamination via faecal spread in feed and water supplies and on hides. Stocking density of cattle is especially important when super-shedding animals are present as an increase in physical contact between animals results in higher environmental concentrations of STEC. Animal density also plays a role in the recirculation of STEC population within a herd by increasing individual exposures to faeces from other ruminants. The degree of support for reducing animal density on-farm as an intervention specifically for the control of STEC in cattle herds was medium. 2.2.3. Environmental hygiene Sanitary conditions are important for healthy animal production and are regarded as best practices for animal production. Proper sanitation of facilities and disposal of manure are important for interrupting the faecal-oral transmission of STEC and other similarly transmitted bacterial pathogens (Garber et al., 1999). Contaminated materials on pen floor also represent a substantial source of STEC O157:H7 that can drive population dynamics. Contamination spread may be subsequently amplified by flushing alleyways with water intended to remove manure or by rainfall events in open pens. Wet conditions result in muddy pen floors which can impact animal performance, welfare, and can increase STEC survival. CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 15 Pen floor conditions can impact STEC colonization of the hides of feedlot cattle, and muddy pens also increased the chance of STEC excretion (Smith et al., 2001). Minimizing mud and cleaning pen surfaces between groups of cattle are part of GHP that are not specific for STEC but are thought to reduce STEC prevalence in and on cattle. During food animal production, the animals must be restrained to ensure GAP (e.g. vaccination). Often, the use of a restraint system of chutes and squeezes will have physical contact with the animal hide (Mather et al., 2007). As a result, faecal material on the hide of one animal can be transferred horizontally between animals in the same facility and may spread through the entire herd or into receiving pens (Cobbold and Desmarchelier, 2002). Cleaning and disinfection of the common handling area and facilities should reduce risks of horizontal transmission of STEC and other pathogens, though this has not been fully substantiated. Adequate facility, bedding material and pen floor maintenance and hygiene are GAP, and the degree of support for these to be used as an intervention specifically for the control of STEC in cattle herds was medium. 2.2.4. Manure management issues If not properly treated or contained, manure can serve as a source of STEC dissemination on the farm and the broader environment. Manure that is not adequately composted prior to its application to either pasture or cultivated fields as a soil amendment, can contain pathogens that can be transmitted back to the cattle herd via farm-grown forages or silages. STEC in manure can enter either surface or ground water and can pose a risk of contamination of drinking water, wells, ponds or irrigation water supplies (Blaustein et al., 2015). Risks associated with manure application in agriculture can be reduced by secondary manure treatments such as bio-digestion, composting, stock piling or desiccation (Ongeng et al., 2015). As STEC are found to be persistent in cattle faeces, management of manure and slurries on the farm can influence spread of the pathogens (Duffy, 2003; Callaway et al., 2013). Vegetation strips may reduce the flow of STEC with surface water during rainfall events, but if the cattle are allowed to graze on recently manured pastures, the possibility of faecal-oral transmission of STEC is increased. Adequate manure managment is a GAP; however, the degree of support for manure management to be used as an intervention specifically for the control of STEC in cattle herds was low. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 16 2.2.5. Seasonal variability and temperature The seasonality of STEC, especially O157:H7, excretion among cattle (e.g. “summer peak”) and its correlation with the incidence of human illness is well documented (Money et al., 2010; Ekong, Sanderson and Cernicchiaro, 2015). While we still do not know the cause of STEC seasonality, there are multiple potential reasons, several of which are linked to summer months and higher temperatures (e.g. increased growth of E. coli and STEC in water troughs and increased survival in excreted faeces), proliferation of other vectors (e.g. protozoa), longer days, greater animal congregation in shadows and at feeding sites at cooler times of day, and to a lesser extent, drinking behaviours (Bach, Stanford and McAllister, 2005b; Gautam et al., 2011; Besser et al., 2014; Dawson et al., 2018). Heat stress in cattle has shown limited impact on STEC excretion (Brown-Brandl et al., 2009; Edrington et al., 2004). Alleviating heat stress is an animal welfare issue frequently implemented in GAP. Use of sprinklers on animals to alleviate heat stress demonstrated no impact on STEC O157:H7 populations (Morrow et al., 2005). However, sprinklers can increase the presence of mud in pens, which can increase the survival of STEC and can result in increased amounts of STEC contaminated mud and faeces contaminating an animal’s coat (Edrington et al., 2009a). Alleviating heat stress in cattle is a GAP; however, the degree of support for using sprinklers as an intervention specifically for the control of STEC in cattle herds was low. 2.3. WATER AND FEED MANAGEMENT STRATEGIES 2.3.1. Drinking water quality and hygiene Drinking water quality is assumed to be part of GAP in cattle rearing and can have a profound impact on animal production. However, microbiological quality of water itself has not been shown to impact STEC populations or prevalence in cattle. Cleaning of water troughs has ben suggested to reduce the load of NTS E. coli and other bacterial populations in the water troughs, which theoretically would reduce dissemination of STEC in cattle production. However, direct evidence on the efficacy of trough cleaning has not been demonstrated. Keeping water levels high in water troughs was suggested to reduce concentration of STEC O157:H7 via simple dilution, and the ratio of cattle to water volume impacts the concentrations of faecal contamination and the horizontal spread of faecal microbes, including STEC (Beauvais et al., 2018). CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 17 Quality drinking water is a GAP; however, the degree of support for clean drinking water as an intervention specifically for the control of STEC in cattle herds was low. 2.3.2. Drinking water treatment Chlorine is an effective disinfectant that is used extensively as an antimicrobial treatment in potable water and cleaning. It has been broadly assumed by many cattle producers that the addition of chlorine to drinking water could reduce STEC loads in water troughs, however this has not been directly demonstrated. Furthermore, the antimicrobial activity of chlorine is reduced when there are high levels of organic matter in the water and also when exposured to UV from sunlight (LeJeune, Besser and Hancock, 2001). Contamination of water troughs with feed from the oral cavity of cattle during drinking or faecal matter can reduce the effectiveness of chlorination. Chlorine can be used for water trough cleaning and decontamination, but its effectiveness is found to be short term as these surfaces rapidly become re-contaminated shortly after cleaning (Smith et al., 2002). Electrolyzed-oxidized (EO) water administered to drinking water was more effective in reducing STEC populations than chlorinated water but EO is also subject to inactivation by organic matter and UV light (Bosilevac et al., 2005; Stevenson et al., 2004). Drinking water is only one of many possible transmission pathways of STEC in the production environment and it is likely that direct animal-to-animal or faecal-to-animal contact are more significant mechanisms of transmission (LeJeune et al., 2004). The degree of support for the treatment of drinking water as an intervention specifically for the control of STEC in cattle herds was low. 2.3.3. Diet composition, feeding strategies and feed hygiene Using cattle feedstuffs that are free of pathogens is a GAP and subject to regulations by most countries. However, feeds may be contaminated by microbes from a variety of sources, ranging from the time of productions, processing, transportation and storage up until the feed is consumed. It has been suggested that feed troughs are potential reservoirs of STEC contamination; however, there is limited evidentiary support for this or for it to be an effective STEC control point. Additionally, there are numerous logistical hurdles in the production operations that all together, makes consistent feed trough hygiene difficult. As a result, this strategy has limited efficacy. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 18 The type of feed ingredients and the way that ingredients are processed prior to feeding has also been proposed to impact the proliferation or suppression of STEC populations in the ruminant gastrointestinal tract, possibly through modifications of gut microbiome, the presence and proportion of specific metabolites (e.g. volatile or short chain fatty acids) and faecal pH. Forage: Concentrate ratio Escherichia coli populations (both NTS and STEC O157:H7) are present in cattle fed forage-based diets and in those fed rations high in grains. Experimental studies on the duration or magnitude of excretion of faecal STEC O157:H7 populations among calves that were fed high grain or high forage (low quality forage) diets, showed variable results (Callaway et al., 2009, 2013). The degree of support for forage feeding, as compared to rations higher in grain or concentrates, as an intervention specifically for the control of STEC in cattle herds was low to medium. Dietary shifts from grain to forage A rapid shift from a high-grain to a higher fibre ration was initially proposed as a mechanism to control STEC (Diez-Gonzalez et al., 1998). However, subsequent experiments produced inconsistent results (Hancock et al., 2000, Hovde et al., 1999, Keen et al., 1999). Also, dietary shifts to high-forage rations are difficult to implement in feedlots and in arid regions as there are many other logistical challenges. Rapid dietary changes or feeding a high-grain rations results in a shift in the microbial population and creates ecological niches which may be exploited by pathogenic organisms for colonization (Jacob, Callaway and Nagaraja, 2009). However, the mechanism of action of diet change on the concentrations of faecal E. coli is not fully understood. The degree of support for a rapid shift from a grain-based to forage-based rations as an intervention specifically for the control of STEC in cattle herds was low. Grain type For many years, the rumen was thought to be absent of E. coli populations due to the inhibitory effects of short chain fatty acids (SCFA) (Wolin, 1969). However, studies over the past 20 years have found that STEC are frequently isolated from the rumen of cattle that were fed forages and grains. Cereal grains, due to their high starch content, are often included in the diet of ruminant animals to increase their energy density. Most dietary starch is fermented rapidly in the fore-gut (rumen) CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 19 of cattle and results in the production of SCFA and lactic acid; however, not all starch in the rumen is fermented and some passes to the hindgut (cecum and colon) where it undergoes a secondary fermentation. The rate of fermentation of cereal grains varies with grain type and the physical grain processing method used, as cereal grains differ in their composition and starch availability to the microbial population. Starch fermentation results in profound changes in the rumen and the hindgut, including decreases in pH, forage (cellulose and hemicellulose) degradation, as well as decreases in ruminal microbial diversity. The rate of starch fermentation impacts ruminal SCFA production and pH, which in turn, alters the composition of the ruminal microbial ecosystem and the ability for pathogens to survive or colonize ruminants (Berg et al., 2004). Barley is a cereal grain which is more rapidly fermented in the rumen than corn, so little barley starch passes to the hindgut. Feeding barley to cattle increased the incidence of excretion and the concentration of STEC O157:H7 by <0.5 log10 CFU/g in faeces compared to feeding corn. Also, feeding barley to cattle increased the survival time of STEC O157:H7 (above the limit of detection) in faeces by more than 2 fold when compared to corn (Berg et al., 2004; Bach et al., 2005a). These factors could influence the survival of STEC O157:H7 in the lower digestive tract, where STEC appears to colonize primarily. The degree of support for the feeding of diets based on starches with slower fermentation rates as an intervention specifically for the control of STEC in cattle herds was medium. Grain processing Grain used in cattle feed can be processed by various methods to improve digestibility and to change the site of starch fermentation to the rumen or to the hindgut. Dry rolling of corn (cracking), instead of steam flaked corn, increased starch flow to the hindgut, and reduced STEC O157:H7 faecal concentrations (Fox et al., 2007). While, steam flaking of corn increased STEC O157:H7 excretion in heifers as compared to feeding whole corn. Faecal starch concentration and pH were not linked to the faecal excretion of STEC O157:H7, yet post-ruminal starch infusion increased NTS E. coli concentrations in the hindgut. The exact mechanisms that the methods of grain processing exert on STEC populations remains unclear, but steam flaking of corn seems to increase risks of STEC excretion (Depenbusch et al., 2008). The degree of support for avoiding feed with steam flaked corn grains as an intervention specifically for the control of STEC in cattle herds was medium. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 20 Distiller's grains with solubles Industrial ethanol production from the fermentation of corn starch results in a by-product known as distiller’s or brewer’s grains that can be used as animal feed. Distiller’s and brewer’s grains are low in starch, having been removed in the distillation process, and can be fed to cattle as a low-starch protein source. Distiller’s grains (DG) are fed either as wet distiller’s grains with solubles (WDGS) or as dry distiller’s grains with solubles (DDGS), but inclusion of either of these forms of DGS, or the inclusion of brewer’s grain in cattle feed, has been associated with increased faecal prevalence and excretion of STEC O157:H7 (Wells et al., 2011; Berry et al., 2017). The magnitude of effect of DGs on STEC sheding and the survivial of STEC in the faeces of animals fed this feed ingredient may be influenced by the particular characteristics of the DG caused by variability in production conditions. The degree of support for feeding distiller’s or brewer’s grains at levels below 15 percent of ruminant ration as an intervention specifically for the control of STEC in cattle herds was medium. Tannins and essential oils Tannins and essential oils are known to impact the microbial community composition in the gastrointestinal tracts of ruminants, in a unique fashion related to the mode of action of each of these compounds. Inclusion of tannin-containing components or extracts in the ration can reduce STEC O157:H7 and other STEC serotypes in the faeces of cattle, with phlorotannin-containing air-dried brown seaweed being more effective than terrestrial tannin sources (Braden et al., 2004; Zhou et al., 2018). Feeding citrus products and other essential oils also impacted faecal and ruminal STEC O157:H7 concentrations, but these have only been assessed in very small-scale studies (Callaway et al., 2011). The degree of support for the inclusion of tannins and essential oils in ruminant rations as an intervention specifically for the control of STEC in cattle herds was low to medium. 2.3.4. Feed additives Probiotics / Direct fed microbials / Competitive exclusion The prevalence of faecal STEC O157:H7 excretion in cattle can be reduced by using direct-fed microbials (DFM), such as Lactobacillus acidophilus (NP51) and Propionibacterium freudenreichii (NP24) (Wisener et al., 2015). Competitive CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 21 exclusion by probiotic E. coli strains orally administered to calves reduced faecal excretion of STEC O26 and O111 and may also represent a promising control measure for reducing other STEC serotypes in cattle (Zhao et al., 2003). To be effective, the component strains in the product must be consistent and these products must be administered at the recommended CFU/g doses in feed. The impact of DFM against STEC is highly specific, thus a positive response in STEC reduction with one probiotic product cannot be necessarily extrapolated to another product. The degree of support for the use of some DFM as an intervention specifically for the control of STEC in cattle herds was medium. Bacteriophage Bacteriophages have been shown to be very effective at killing STEC on food handling surfaces and on the surface of food, and in some cases reducing STEC O157:H7 to undetectable levels (Liu et al., 2015). In primary production steps, however, the efficacy of bacteriophages has been mixed, with either little reduction in STEC numbers or the establishment of a cyclic effect where STEC numbers decline when phage numbers are high, but as phage numbers decreases, the STEC numbers increases (Sabouri et al., 2017). Phage have been used in a commercial product to control STEC O157:H7 on cattle hides prior to slaughter, but the results were marginal (Arthur et al., 2017). Contact between the phage and the targeted bacteria is essential for the phage to be effective. This is a stoichiometric process that depends on the concentrations of the phage and the host bacterium, as well as the consistency of the matrices that enable adequate mixing of phage and host. Bacteriophages are most effective when large numbers of phage plaque forming units (PFU) can be applied and where contact with the target bacterium can be assured (Wang et al., 2017). Effects of phage may be very broad or may be highly host specific and can be limited to one or a few STEC serotypes. When exposed to phage, STEC can also become resistant to phage either due to the activation of CRISPR/CAS defence mechanisms which prevent phage DNA insertion, or alteration in the host surface receptors which the phage relies on for recognition. Use of phage mixtures has been proposed as means of overcoming phage resistance, but there is also evidence that this approach can select for bacteria that are resistant to multiple phage types. Consistent reductions of STEC by phage treatment are more difficult to achieve in complex matrices and situations such as in the intestinal tract of live animals, where interactions between the phage and the targeted bacterium cannot be assured. The degree of support for the addition of bacteriophage to feed as an intervention specifically for the control of STEC in cattle herds was low. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 22 Colicins Colicins are antimicrobial proteins produced by E. coli and specifically target E. coli, but do not differentiate between NTS E. coli and STEC. Colicins has been shown to kill STEC O157:H7 strains in vitro and in vivo (Callaway et al., 2004; Schulz et al., 2015). E. coli colicin was costly to produce and scarce, but colicin genes have been genetically inserted into plants and fungi to allow for scale up in colicin production. Colicin-producing plants and fungi would be considered genetically modified organisms (GMO) for this purpose. Colicins can be added to cattle rations, however the anti-STEC activity of colicin has been shown to be more promising in ground beef and in cleaning processing facility surfaces than in primary production. Colicins are not currently commercially available. The degree of support for the addition of colicins to feed as an intervention specifically for the control of STEC in cattle herds was low. Sodium chlorate Escherichia coli, including STEC can respire anaerobically using the enzyme nitrate reductase, which reduces sodium chlorate to chlorite, a bacteriocidal agent that accumulates intracellularly. In cattle, experimentally, use of sodium chlorate reduced inoculated STEC O157:H7 populations by 2-3 log10/g digesta throughout the gut (Callaway et al., 2002). Chlorate had no impact on meat quality and radiolabelled residue studies showed that it did not accumulate in the tissue. Use of sodium chlorate has also been studied in other animals, but may not be approved by regulatory agencies and is not currently commercially available as a feed additive. The degree of support for the addition of sodium chlorate to feed as an intervention specifically for the control of STEC in cattle herds was medium. 2.4. VACCINES AND CLINICAL ANTIMICROBIALS 2.4.1. Vaccines Various vaccines have been designed and tested for preventing colonisation and/ or reducing faecal excretion of STEC O157:H7 in cattle. To date, the following vaccine types have been tested: vaccines based on type III secretion proteins (T3SS), vaccines based on siderophores (SRP) and porin proteins, vaccines based on bacterins and bacterial envelopes, vaccines based on flagellin, vaccines based on attenuated Salmonella enterica serovar Dublin expressing intimin, vaccines based on Shiga toxin toxoids and vaccines based on outer membrane vesicles CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 23 (Smith, 2015; Larzábal, Cataldi and Vilte, 2019; Besser et al., 2014; Fingermann et al., 2018). These vaccines have been tested in feedlot cattle, pregnant cattle, calves and mice. However, only a few vaccines have been tested under production conditions and the duration of immunity after vaccination is unknown because the evaluation period in the feedlot studies has been relatively short (Smith, 2015). Furthermore, two vaccines: the T3SS based vaccine (Bioniche Life Sciences Inc., Belville, Ontario, Canada) and the SRP protein-based vaccine (Epitopix, LLC, Wilmar, Minnesota) have been commercialised (Besser et al., 2014). Both have been tested in the United States of America and were shown to be effective in reducing excretion of STEC O157:H7 in cattle faeces (Snedeker, Campbell and Sargeant, 2012; Varela, Dick and Wilson, 2012). The use of a “cocktail” of different vaccine types has been recommended to enhance vaccine efficacy against STEC O157:H7. Adoption of vaccines by producers is hindered if more than one dose is required to achieve an immune response. At present, neither commercialized vaccine is widely used. The degree of support for the use of vaccines as an intervention specifically for the control of STEC in cattle herds was low to high, depending on formulation considered. 2.4.2. Clinical antimicrobials Ruminant animals have a symbiotic relationship with their ruminal and gastrointestinal microbial population, which allows ruminants to thrive on diets that monogastric animals cannot. Unfortunately, this comes at the cost of lower feed efficiency (kg of feed to produce kg of meat/milk). Ionophores, tylosin, chlortetracycline, and oxytetracycline are routinely fed to cattle in some production systems (added to feed and water at subtherapeutic levels) in many countries to improve feed efficiency and improve animal health. The efficacy of clinical antimicrobials against STEC in the gut of cattle has not been demonstrated in vivo or in vitro except for the use of neomycin sulfate (described below). However, it is hypothesized that the use of broad spectrum or bacteriostatic and/or bactericidal agents targeted against Gram-negative bacteria, would most probably reduce STEC colonization and excretion. Key considerations contraindicating the use of antimicrobials would include: 1) Some antimicrobials may induce Stx-encoding bacteriophages that are able to transduce Stx-encoding genes and antimicrobial resistance genes to naive E. coli thereby contributing to the expansion of the STEC pool as shown in in vitro studies (Kimmitt, Harwood and Barer, 2000; Köhler, Karch and Schmidt, 2000); 2) Some antimicrobials may exert selective pressure on intestinal microbiota thereby favouring the survival of antimicrobial resistant STEC (no studies available yet on this issue) and other intestinal microflora; CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 24 and 3) Antimicrobials that inhibit Gram-positive bacteria (which are responsible for rapid starch fermentation, and can include opportunistic pathogens) may favour the dominance of Gram-negative bacteria in the gut, including STEC. The use of antimicrobials to reduce STEC colonization or excretion remains controversial primarily because of concerns with increasing antimicrobial resistance, which may impact public health, globally. No antimicrobial with proven efficacy in reducing STEC in cattle has been demonstrated in scientific studies (Jacob et al., 2008), except neomycin, noted below. Due to valid concerns regarding the use of antimicrobials in animal husbandry, some countries have banned the use of antimicrobials as growth promoters in livestock production. The degree of support for the use of antimicrobials in cattle feed as an intervention specifically for the control of STEC in cattle herds was low to medium. Neomycin sulfate Neomycin sulfate is a commercially available antimicrobial which has been demonstrated to reduce STEC O157:H7 populations in cattle; however, this research study was done on a small scale, so the data is limited and it also raises significant concerns about increasing antimicrobial resistance (Elder et al., 2002). The degree of support for the use of neomycin sulfate as an intervention specifically for the control of STEC in cattle herds was low. The use of neomycin sulfate is not recommended due to potential adverse impacts on public health. Ionophores Ionophores are a class of antimicrobial that inhibit Gram-positive bacteria. Ionophores are not used in human medicine, but have been tested in animals and in beef (and some dairy) cattle feed. They improved feed efficiency by altering the microbial population of the rumen, resulting in a shift in fermentation and end products, including reduced methane production. In theory, inhibition of ruminal Gram-positive bacterial species would benefit Gram-negative species such as STEC. However, this has not been demonstrated in sheep or cattle (Edrington et al., 2003). The percentage of steers excreting enumerable STEC O157:H7 levels was greater in monensin-fed cattle as compared to controls, but feeding ionophores did not change the incidences of STEC excretion in the pens (Hales et al., 2017; McAllister et al., 2006). The degree of support for the use of ionophores as an intervention specifically for the control of STEC in cattle herds was low. CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 25 2.4.3. Beta-agonists / hormones Ractopamine hydrochloride and zilpaterol hydrochloride are beta-agonists that are fed to finishing cattle in North America for a period of 30–40 days prior to slaughter to enhance weight gain. The use of zilpaterol has been largely discontinued, but ractopamine is still widely used in intensive feedlot cattle production. These additives repartition energy from fat towards muscle and improve feed efficiency. It was hypothesized that this shift in metabolism may increase the “metabolic stress” on the animal and result in increased pathogen excretion or that beta-agonists may impact bacterial quorum sensing and increase virulence gene expression in microbial pathogens. A series of studies were undertaken to investigate the impact of these additives on the excretion of STEC O157:H7 (Wells et al., 2017; Paddock et al., 2011; Edrington et al., 2009b). Although there was variation among the studies, none found that the addition of beta-agonists to the cattle diet resulted in an increase in the excretion of STEC O157:H7. One study even found a reduction in STEC O157:H7 faecal excretion as a result of the administration of ractopamine hydrochloride. At this point, the evidence would indicate that beta-agonists do not have an impact on the excretion of STEC O157:H7 in cattle. The expert committee was unable to find other studies that investigated the impact of other hormones such as bovine somatotropin in dairy cattle or estrogenic or androgenic implants/additives in beef cattle. The degree of support for the use B-agonists as an intervention specifically for the control of STEC in cattle herds was low. 2.5. DAIRY PRODUCTION SPECIFIC INTERVENTIONS Contamination of raw milk can occur as a result of infection in the animal prior to milking, from contact with faecal material directly from an animal during milking or via milking equipment, from personnel or from the farm environment. Despite GAP and GHP that are essential to minimize bacterial contamination, several other opportunities for microbial contamination of raw milk occur during processing, thus bacteria cannot be eliminated completely regardless of whether the raw milk is intended for drinking or for use in the production of raw milk cheeses (Jayarao and Henning, 2001). 2.5.1. Milking environmental hygiene Studies have examined risk factors associated with bacterial contamination of raw bulk milk. Verbeke et al. (2014) observed that certain hygiene interventions involving the milking equipment and the milking environment in a Flemish dairy herd were associated with a decrease in bacterial counts including coliforms. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 26 Husbandry and milking procedures do impact bacterial and somatic cell counts in bulk tank milk on dairy farms, but consistent application of a few hygienic practices could significantly improve the microbiological quality of raw milk (Elmoslemany et al., 2010). Adequate milking hygiene is a GHP; however, the degree of support as an intervention specifically for the control of STEC in raw milk was low. 2.5.2. Udder hygiene The epithelium of the udder and teats can be contaminated with STEC (Fremaux et al., 2006). Pre-dipping teats into a disinfectant solution followed by drying is a GHP that has been shown to be an effective teat skin sanitation against many potential mastitis and spoilage organisms (Galton, Petrsson and Merrill, 1986; Gibson et al., 2008). Use of an automated teat scrubber (rotating brushes) for chlorine dioxide disinfection and drying also effectively reduced bacterial loads on teats (Baumberger, Guarín and Ruegg, 2016; Elmoslemany et al., 2010). While none of these basic sanitation processes have been examined against STEC specifically, pre-milking treatment should be used in combination with the control of contamination from the environment, milking equipment and water. Udder hygiene is a GHP and the degree of support as an intervention specifically for the control of STEC in raw milk was medium. 2.5.3. Milk storage temperature and hygiene Temperature control and hygiene in the farming and processing environments as well as during transportation from the farm are critical factors in the commercial milk supply chain and can significantly affect the microbiological quality of raw milk prior to packaging and sale for drinking or for use in the manufacture of raw milk cheeses. Depending on the size of the herd and bulk tank capacity, the bulk tank is a key storage area for milk collected from one or more milkings. Bulk tanks are usually made of stainless steel which aids in cleaning to remove build-up of milk films (and potentially biofilms), but the tanks still have areas that are less accessible to cleaning (e.g. valves, outlet ports and gaskets) (EFSA, 2015). The formation and presence of biofilms in milk bulk tanks is a concern, but should also be considered in all steps between the milking equipment and the tankers hauling milk from the farms (Weber et al., 2019). During processing, temperatures ≥ 6 °C and/or extended storage of raw milk were associated with a significant increase in bacterial counts (ICMSF, 2001 and 2011; CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 27 Vithanage et al., 2017). An increased duration of cold storage from long distance transportation or including water rinses of tanks in between tanker loads had no negative effects on raw milk quality. Although not yet at the level of an accepted standard, a full cleaned-in-place approach where tankers are cleared every 24 h with water rinses along with use of a sanitizer treatment between loads, reduced the presence of surface-associated bacteria in the tankers (Darchuk, Meunier- Goddik and Waite-Cusic, 2015). Temperature change, extended storage time and initial bacterial counts in raw milk during collection, storage and transportation have been associated with increased counts of E. coli in raw milk. Cooling, hygiene practices and reduced storage and transportation times can reduce E. coli and other indicator organism counts, but no specific evidence was found regarding the effect of these practices on STEC. Cleanliness of the bulk tank and temperature control of milk during storage are GHP; however, the degree of support as an intervention specifically for the control of STEC in raw milk was low. 2.6. ANIMAL TRANSPORTATION Pertinent to control of STEC, animal transportation issues that must be addressed include hide contamination (mixing of cattle from different farms, cleanliness of trucks and loading areas), the impact of animal stress from excessive temperature, humidity, loading density and the duration of transport, as all of these can affect the colonization and faecal excretion of STEC. 2.6.1. Feed withdrawal prior to slaughter Cattle should not be fed for a minimum of 8 h to a maximum of 12 h pre-slaughter to avoid very full gastrointestinal tracts, which are more likely to rupture during the evisceration process and increase the potential for spread of STEC onto carcasses. Feed withdrawal reduces faecal output and contamination of the environment and hides, yet because it also results in decreased volatile fatty acid concentrations and increased pH in the gastrointestinal tract, feed withdrawal can lead to an increase in STEC excretion (Pointon, Kiermeier and Fegan, 2012). Most research on the effect of fasting has been conducted on-farm and no studies have investigated the effect of fasting on the reduction of STEC excretion while the cattle are in lairage pens. The degree of support for feed withdrawal prior to slaugher as an intervention specifically for the control of STEC in raw beef was low. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 28 2.6.2. Duration of transportation Bach et al. (2004) evaluated the effects of pre-conditioning (attenuating stress effects) and the duration of animal transport from the pasture on faecal excretion of E. coli and STEC O157:H7 by beef calves. Authors observed an increase in excretion and suggested that the calves’ susceptibility to infection from the environment was likely elevated by the stresses of weaning, transport, and relocation. Faecal excretion increased with lack of pre-conditioning and long periods of transport. Supporting this study, Dewell et al. (2008) observed that lots (groups) of cattle that were transported for long distances (>160 km) had twice the risk of having STEC positive hide samples at slaughter compared with cattle transported a shorter distance. Increased STEC excretion with longer distance of transport is also supported by the study by Arthur et al. (2007). But, other studies did not find an association between transportation stress or general heat stress and temperature with faecal excretion of STEC O157:H7 in cattle (Brown-Brandl et al., 2009; Pfeiffer et al., 2009). Transport density and trailer design, such as multi-level trailers, may also influence the degree that hides are positive with STEC O157:H7 resulting from faecal-coat contamination (e.g. hide tag or dag, or more colloquially “dingleberry”) (Stanford et al., 2011). The degree of support for transportation time as an intervention specifically for the control of STEC in raw beef was low. CHAPTER 2 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 29 References Ahmad, A., Nagaraja, T.G. & Zurek, L. 2007. Transmission of Escherichia coli O157:H7 to cattle by house flies. Preventative Veterinary Medicine, 80: 74–81. doi: 10.1016/j. prevetmed.2007.01.006 Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Guerini, M.N., Kalchayanan, N., Shackleford, S.D., Wheeler, T.L. & Koohmaraie, M. 2007. Transportation and lairage environment effects on prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. 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Comparative transcriptomic analysis of rectal tissue from beef steers revealed reduced host immunity in Escherichia coli O157:H7 super-shedders. PLOS ONE, 11: e0151284. doi: 10.1371/journal.pone.0151284 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 40 Weber, M., Liedtke, J., Plattes, S. & Lipski, A. 2019. Bacterial community composition of biofilms in milking machines of two dairy farms assessed by a combination of culture-dependent and -independent methods. PLOS ONE, 14: e0222238. doi: 10.1371/journal.pone.0222238 Wells, J.E., Shackelford, S.D., Berry, E.D., Kalchayanand, N., Bosilevac, J.M. & Wheeler, T.L. 2011. Impact of reducing the level of wet distillers grains fed to cattle prior to harvest on prevalence and levels of Escherichia coli O157:H7 in feces and on hides. Journal of Food Protection, 74: 1611–1617. doi: 10.4315/0362-028X.JFP-11-160 Wells, J.E., Berry, E.D., Kim, M., Shackelford, S.D. & Hales, K.E. 2017. Evaluation of commercial-agonists, dietary protein, and shade on fecal shedding of Escherichia coli O157:H7 from feedlot cattle. Foodborne Pathogens and Disease, 14: 649–655. doi: 10.1089/fpd.2017.2313 Wetzel, A.N. & LeJeune, J.T. 2006. Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in Northeast Ohio. Applied and Environmental Microbiology, 72: 2621–2626. doi: 10.1128/AEM.72.4.2621-2626 Wisener, L.V., Sargeant, J.M., O'Connor, A.M., Faires, M.C. & Glass-Kaastra, S.K. 2015. The use of direct‐fed microbials to reduce shedding of Escherichia coli O157 in beef cattle: A Systematic Review and Meta‐analysis. Zoonoses and Public Health, 62: 75–89. doi: 10.1111/zph.12112 Wolin, M.J. 1969. Volatile fatty acids and the inhibition of Escherichia coli growth by rumen fluid. Applied Microbiology, 17: 83–87. doi: 10.1128/am.17.1.83-87.1969 Xu, Y., Dugat-Bony, E., Zaheer, R., Selinger, L., Barbieri, R., Munns, K., McAllister, T.A. & Selinger, L.B. 2014. Escherichia coli O157:H7 super-shedder and non- shedder feedlot steers harbour distinct fecal bacterial communities. PLOS ONE, 9: e98115. doi: 10.1371/journal.pone.0098115 Zaheer, R., Dugat-Bony, E., Holman, D., Cousteix, E., Xu, Y., Munns, K., Selinger, L.J., Barbieri, R., Alexander, T., McAllister, T.A. & Selinger, L.B. 2017. Changes in bacterial community composition of Escherichia coli O157:H7 super-shedder cattle occur in the lower intestine. PLOS ONE, 12: e0170050. doi: 10.1371/journal.pone.0170050 Zhao, T., Tkalcic, S., Doyle, M.P., Harmon B.G., Brown, C.A. & Zhao, P.J. 2003. Pathogenicity of enterohemorrhagic Escherichia coli in neonatal calves and evaluation of fecal shedding by treatment with probiotic Escherichia coli. Journal of Food Protection, 66: 924–30. doi: 10.4315/0362-028x-66.6 Zhou, M., Hünerberg, M., Chen, Y., Reuter, T., McAllister, T.A., Evans, F., Critchley, A.T. & Guan, L.L. 2018. Air-dried brown seaweed, Ascophyllum nodosum, alters the rumen microbiome in a manner that changes rumen fermentation profiles and lowers the prevalence of foodborne pathogens. mSphere, 3: e00017-18. doi: 10.1128/mSphere.00017-18 41 Processing control strategies for STEC in beef This section focuses on interventions applied during meat processing to reduce the prevalence and concentration of STEC on beef carcasses and meat products and prevent further cross-contamination of other meat products. Processing stages considered include animal receiving and lairage, slaughter (hide removal, and carcass evisceration, trimming, and dressing) and carcass pre-chilling and chilling. A summary of processing control measures for STEC in beef and their degree of support (high, medium, low), based on scientific evidence, is available in Annex 2. 3.1. LAIRAGE It is well recognized that the hide of ruminants presented for slaughter is the most important source of microbial contamination for carcasses and the processing environment (Cernicchiaro et al., 2020). Most of the microorganisms found on the hide are of faecal origin with some originating from the farm environment. Pathogens such as STEC may be present in the faecal material, hence on the hides of the cattle, posing a risk for cross-contamination of other animals in the lairage. Upon arrival at the processing plant, cattle are unloaded and directed through common alleys to lairage pens to be held until slaughtered. Upon exit from the lairage pens, cattle are directed through more common alleys before reaching the area for stunning and shackling. These common use alleys can spread contamination between the animals. Proper lairage pen and working facility hygiene is a GHP that will not reduce STEC carriage in an animal, but may reduce contact transfer of STEC on the hides of cattle. 3 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 42 3.1.1. Lairage cleanliness Animals presented for slaughter are a source of microbial contamination for lairage areas and pens (Small et al., 2003). A study by Small et al. (2007) demonstrated that the use of pressure washing with water and quaternary ammonium sanitizers and/ or steam under pressure reduced E. coli and Enterobacteriaceae levels in lairage surfaces, ranging from 0.9-5.8 log10 CFU/cm2. If the lairage pens and alleys that have previously housed other lots of animals are not cleaned or sanitized in between animal lots, hides contaminated with STEC during transport can transfer STEC within and between animal lots. The cleanliness of the animal intake, lairage and animal handling environments (washing trailers, cattle handling facilities, holding pens in between use) are important to ensure that hides contain a minimum amount of faecal material to reduce STEC cross- contamination. Similarly, Dewell et al. (2008) reported that cattle lots held in STEC O157:H7-positive lairage pens were eight times more likely to have hides that test positive at slaughter than cattle held in lairage pens that tested negative for STEC O157:H7. Maintaining hygienic environmental conditions (e.g. cleaning, disinfecting and dry conditions), and limiting the amount of time spent in lairage are common animal health and animal welfare management practices. Implementing these steps can reduce animal stress, hide contamination, faecal output, potential gastrointestinal content spillage, and carcass contamination during processing. The efficacy of interventions (primarily in the form of GHP) applied during animal intake and lairage is unclear and likely dependent on the amount of time the animals spent in lairage, the level of stress experienced by the cattle and the animal density of the lairage pens. Lairage cleanliness is a GHP; however, the degree of support as an intervention specifically for the control of STEC in raw beef was low. 3.1.2. Livestock cleanliness Recognizing the role of dirty animals in introducing microbial contamination into the slaughter plant, many countries have introduced policies around cleanliness of livestock (Gagaoua et al., 2022). The management of animals classified as dirty can be used to reduce the risk of microbial contamination. Meat processors can implement a range of interventions and GHP’s from holding cattle for a period of time on straw in lairage to changing logistics in slaughter (e.g. slaughtering the dirtiest animals at the end of the day to reduce cross contamination; clipping hide after kill and before hide removal; and slowing the slaughter line speed to allow for more care with hide removal). Limited studies that examined animal CHAPTER 3 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 43 dirtiness have generally showed a direct correlation between visual cleanliness of cattle hide and lower microbiological counts for aerobic organisms (aerobic plate counts [APC], Enterobacteriaceae and E. coli) on derived carcasses (Blagojevic et al., 2012). Studies conducted in commercial processing plants in the United States of America, found a significant association between hide cleanliness scores with STEC O157:H7 prevalence after controlling for season of sampling (Cernicchiaro et al., 2020), and similar associations with hide cleanliness were observed with STEC O145 (Schneider et al., 2018; Antic et al., 2010b; Nastasijevic, Mitrovic and Buncic, 2008; Smith et al., 2005; Van Donkersgoed et al., 1997). Livestock cleanliness is a GHP; however, the degree of support as an intervention specifically for the control of STEC in raw beef was low. 3.1.3. Holding animals in lairage To comply with animal welfare regulations, the amount of time that animals are held in lairage should be minimized. Researchers have investigated changes in faecal and hide-on prevalence and concentration of STEC from feedlot to lairage pens and then subsequently, during later processing stages, however, studies specifically evaluating the effect of the time spent in lairage pens on controlling STEC excretion have not been reported. It is recognized however, that spending less time in holding pens can reduce faecal contamination among pen-mates by decreasing exposure to other animals defecating and/or to contaminated facilities. Yet different hide decontamination and disinfection procedures remain useful and a key issue to reduce faecal contamination and keep animals dry and mud-free. The degree of support for miminizing time spent in liarage as an intervention specifically for the control of STEC in raw beef was low. 3.2. HIDE DECONTAMINATION Most hide decontamination interventions applied before stunning consist of live animal hide washes. Washing cattle with water, ozonated or electrolyzed water or water with the addition of chemicals can reduce visible hide contamination and the level of generic organisms (Bosilevac et al., 2005); however, most of these studies were laboratory-based and the results were inconsistent in terms of specifically reducing potential spread of STEC from hides to carcass surfaces. Washes and hair removal before or after stunning can reduce visible hide contamination and can lower levels of generic microbes. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 44 3.2.1. Bacteriophage Bacteriophage cocktails specific for STEC O157:H7, may be applied as a spray or a mist, to the hides of live cattle in the holding pens, up to 4 h before slaughter and hide removal. Such treatments are predominantly used in warm summer months. A laboratory-based study showed a 1.50 log10 CFU reduction in STEC O157:H7 on hides treated with bacteriophage (Coffey et al., 2011); however, when phage treatment was done under commercial beef plant conditions, STEC O157:H7 prevalence on hides was only reduced from 57.6–51.8 percent, indicating no significant impact (Arthur et al., 2017). The efficacy of bacteriophage treatment on hides remains unclear and further in-plant studies are needed. Different regulatory positions in the different countries may also affect implementation. A further limitation of this intervention is that at present, most of the phages developed for use only targets STEC O157:H7. The degree of support for the application of bacteriophage to hides as an intervention specifically for the control of STEC in raw beef was low. 3.2.2. Hide washes with ambient or hot water, organic acids, and other chemicals A range of hide washes may be applied, most commonly after stunning but before hide removal. Washing cattle hides using pressure hoses for 3 min removed faecal contamination and decreased STEC O157:H7 prevalence on inoculated hides (Byrne et al., 2000). The use of ozonated and electrolyzed oxidizing water reduced the concentration of generic organisms (Bosilevac et al., 2005). The use of chlorinated water (Arthur et al., 2007; Carlson et al., 2008), sodium hydroxide wash with a chlorinated (1 ppm) water rinse (Bosilevac et al., 2006) or washes with 1.0 percent cetylpyridinium chloride (CPC) (Bosilevac et al., 2004) reduced the prevalence of STEC O157:H7 on hide-on surfaces. However, another study reported an increase in the concentration of generic organisms after the application of water with chemicals (Mies et al., 2004). Electrolyzed-oxidized (EO) water applied to the hide is subject to inactivation as a result of interaction with organic matter and UV light, as is chlorine (Stevenson et al., 2004). Using inoculated hides, EO water reduced STEC O157:H7 hide concentrations by up to 4.3 log10 CFU/100 cm2, and reduced hide prevalence from 82 percent to 35 percent. The primary application for EO water currently is within processing and post-processing environments (Bosilevac et al., 2005). Addition of 220 ppm hypobromous acid to wash water reduced STEC O157:H7 hide prevalence from 25.3 to 10.1 percent (Schmidt et al., 2012). Meta-analysis of the literature found that the use of sodium hydroxide or lactic acid was effective CHAPTER 3 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 45 for hide decontamination, but the incorporation of water washes along with the antimicrobial washes were largely ineffective, as water diluted and removed antimicrobials (Zhilyaev et al., 2017). Other factors to consider include, as antimicrobial washes drain down the carcass, they may redistribute microbial contamination to other parts of the carcass. Acid washes can potentially select for acid-resistant microorganisms that may accelerate spoilage and the appearance of undesirable products. The need for specialized equipment/infrastructure to implement and the possibility for increases in equipment corrosion. Exposure to chemicals also raises environmental and employees' health and safety concerns. The degree of support for hide washes using ambient or hot water, organic acids and other chemicals as an intervention specifically for the control of STEC in raw beef was low. 3.2.3. Hide clipping, coating and chemical dehairing Hair removal may be beneficial in reducing overall contamination of carcasses, however, its efficacy at reducing STEC is controversial. Concerns about environmental and worker health and safety limits the utility of hair removal. Coating hides with shellac, which immobilizes bacteria, could also reduce pathogen transmission to the carcass. The use of food-grade resin in ethanol (shellac) to coat inoculated hides was reported to successfully reduce STEC O157:H7 prevalence on hides (Antic et al., 2010a) and when applied under commercial conditions, also reduced the level of generic organisms (Antic, Blagojevic and Buncic, 2011). Even if proven to be efficacious, the environmental issues associated with waste disposal, lack of large-scale studies, need for specific infrastructure to implement, personnel health concerns associated with exposure to the chemicals used, and the scarce evidence supporting their use, the practicality of these interventions is considered limited. The degree of support for hair removal as an intervention specifically for the control of STEC in raw beef was low. 3.3. SLAUGHTER AND DRESSING Pre-evisceration measures to prevent contamination of carcasses with fecal material or remove visible faecal material from carcasses are hygienic practices that in principle, reduce general microbial contaminants, including pathogen prevalence and concentration. Important factors for consideration in their use are worker skill, equipment operational maintenance, potential for re-distribution of the carcass contamination or cross-contamination and destruction or loss of the carcass surface and meat. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 46 3.3.1. Speed of processing The speed at which animals are moved along the processing line has been reported to have an effect on microbial levels on carcasses following dressing (Sheridan, 1998). But, the evidence is conflicting in studies from different countries and data comparisons are complicated by the variable conditions within and among the plants (e.g. efficiency of management systems, worker skill and working conditions, time allowed) and the use of decontamination systems that could mask the effects of rapid line speeds. No consistent evidence was found on the impact of processing line speed on the microbial load or STEC contamination on dressed carcasses, but this factor varies with worker and processing practices in an establishment. However, plants may decide to alter line speeds based on the amount of mud coat on the hide to reduce microbial cross contamination. The degree of support for reducing processing speed as an intervention specifically for the control of STEC in raw beef was low. 3.3.2. Hide removal Contamination from hide to carcass may occur each time the hide is incised through its surface (Huynh et al., 2016). Contamination can also occur from the operator’s hands, utensils and equipment used to create opening cuts and to pull the hide. Slaughter facilities should rely on good dressing procedures during de-hiding to prevent or minimize any contact with the carcass (Gagaoua et al., 2022). Workers should sanitize the knives in a hot water bath between carcasses to minimize cross- contamination. Studies showed that holding knives and steels in water at 82 °C for at least 30 s or an equivalent combination of conditions, resulted in a 2 log10 CFU reduction of E. coli on the knives (Eustace et al., 2007; McEvoy et al., 2001). To minimize contamination more efficiently, the use of a two-knife system is recommended, as one knife can be held in hot water while the other knife is being used (EFSA, 2013). The use of hide pullers is the final step in hide removal and allows for a clean pull of the hide from the carcass without damaging the hide. Downward hide pullers are the most common type but there are some limited users of upward pullers. As the hide is removed, microorganisms on the hide are released into the air as part of droplets and particulate matter that may settle on the carcasses. Kang et al. (2019) reported that a downward pulling system resulted in lower bacterial loads on the carcasses while Kennedy et al. (2014) found no significant differences. However, the designs of these two studies were not directly comparable. CHAPTER 3 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 47 Evidence regarding the choice of up- or downward hide pulling system to minimize microbial contamination was evaluated as low. Knife and steel hygiene procedures are considered GHP and widely implemented to minimize transfer of microbial contamination from hides to carcasses and between carcasses. Good dressing procedures are GHP; however, the degree of support for hide removal practices as an intervention specifically for the control of STEC in raw beef was low. 3.3.3. Pre-evisceration and evisceration processes Pre-evisceration and evisceration of the beef carcass include the removal of the organs, respiratory tract, rumen and other parts of the gastrointestinal tract. In this process, the abdominal cavity is opened, and the contents are removed by cutting away the fat, membrane, and connective tissue attaching the abdominal contents to the carcass. Good dressing practices are particularly important during evisceration to ensure that the rumen and intestinal tract are not punctured, which could result in gross carcass contamination with digesta. Good dressing procedures should also ensure that each end of the gastrointestinal tract of the animal is sealed off before evisceration, to prevent spillage of gastrointestinal content, to prevent carcass contamination with faecal microorganisms. “Bunging” and “weasanding” are the two practices that are used to seal off the rectum and the esophagus, respectively and ensure that the connective tissue attaching both the esophagus and rectum are separated from the carcass. These two practicesmay be associated with lower STEC contamination of carcasses (Stopforth et al., 2006; Sheridan, 1998). Yet, in a meta-analysis on E. coli interventions, Greig et al. (2012) found that while these pre-evisceration practices are commonly used, there were few studies that reported on their effectiveness to prevent contamination, and this precluded their inclusion in the meta-analysis of available research evidence. Practices to prevent the leakage of contents from the gastrointestinal tract prior to evisceration are commonly used and recommended as GHPs. When appropriately applied, they generally reduced contamination of carcass with gastrointestinal microorganisms that may include pathogens such as STEC. Good dressing procedures are GHP, and the degree of support for eviceration practices that prevent leakage of gastrointestinal tract contents as an intervention specifically for the control of STEC in raw beef was medium. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 48 3.3.4. Removal of visible faecal material from carcass A variety of practices may be carried out to remove visible faecal material, and the associated faecal microorganisms from the carcass, though most of these are not STEC-specific. Water washing, targeted trimming, and steam vacuuming have all been used to reduce visible faecal material contamination on carcasses, though most of these methodologies generally reduce bacterial levels on the carcass. Water washing of carcass As a GHP, pre-evisceration washing of carcasses may be carried out just after hide removal. Cold or warm water, or organic acid washes have been used to remove visible carcass contamination (Antic, 2018). In a meta-analysis by Greig et al. (2012) on the interventions applied to beef carcasses to control E. coli, hot water (74 °C applied with a nozzle pressure of 700 lb/in2 for 5.5 s) and a 2 percent lactic acid wash warmed in an online spray cabinet, reduced the prevalence of STEC O157:H7 by 81 percent and 35 percent respectively; but sequential treatment of these interventions had no additional benefit (Bosilevac et al., 2006). Depending on the water volume and pressure used, this intervention may actually redistribute contamination on the carcass surface. Removal of visible faecal material from carcasses is a GHP; however, the degree of support for the use of water washing as an intervention specifically for the control of STEC in raw beef was low. Trimming of carcass After carcass splitting and spinal cord removal, knife trimming may be carried out to remove visible faecal contamination. This is a GHP commonly adopted by commercial beef processing plants and has been shown to reduce APC of spoilage organisms from 3.0–4.3 log10 CFU/cm2 (Castillo et al., 1998a; Prasai et al., 1995). Horchner et al. (2020) reported that at commercial plants, trimming reduced total viable bacterial counts (TVC) by 0.44 log10 CFU/cm2 and the prevalence of NTS E. coli by 29.1 percent. In a study with inoculated carcasses, trimming reduced STEC O157:H7 by 3 log10 CFU/100 cm2; however, spread of contamination was observed to occur which required further tissue removal (Castillo et al., 1998a). When trimming was combined with another intervention like hot water, lactic acid, or steam vacuum, TVCs was reduced by 0.61 log10 CFU/cm2 and the prevalence of E. coli by 36.8 percent (Castillo et al., 1998b). Trimming was considered a GHP, but it was noted that there are conflicting results on its impact in reducing TVCs, E. coli and inoculated STEC O157:H7. CHAPTER 3 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 49 The efficacy of this intervention is very dependent on the workers’ skill level and operational maintenance of the equipment. Trimming may also contribute to possible redistribution of contamination on the carcass or cross-contamination of other carcasses from knives and personnel hands/gloves. Trimming also resulted in losses of carcass meat, including surface fat and tissue that can lead to drying, degradation of meat cuts, and may impact product aesthetics for the consumers, thus it has low evidence for use as an STEC reduction strategy. Removal of visible faecal material from carcasses is a GHP and the degree of support for trimming as an intervention specifically for the control of STEC in raw beef was medium. Steam vacuuming Steam vacuuming utilizes a hand-held device comprised of a vacuum wand with a hot spray nozzle, which delivers water at 82-95 °C to the carcass surface under pressure, while simultaneously vacuuming the area to remove faecal material. This treatment has been reported to reduce STEC O157:H7 by 5 log10 CFU/cm2 on experimentally inoculated beef (Dorsa, Cutter and Siragusa, 1996). Commercial steam vacuum systems have been reported to reduce E. coli by 2.8-5.5 log10 CFU/ cm2 (Castillo et al., 1998b; Moxley and Acuff, 2014). On naturally contaminated carcasses under commercial processing conditions, steam vacuuming after carcass trimming was reported to reduce mean TVCs by 0.4-0.9 log10 CFU/cm2 (Hochreutener et al., 2017). In a meta-analysis, the average reduction of E. coli on beef carcasses was 3.1 log10 CFU/cm2 (Zhilyaev et al., 2017). The effectiveness of steam vacuuming depends on worker diligence and skill, operational maintenance of the equipment, exposure time and application temperature. It has been reported that a non-permanent, discolouration of the carcass surface can occur. Based on a meta-analysis of literature and its applicability under commercial conditions (in-plant studies), steam vacuuming remains a valuable tool to reduce surface faecal contamination from carcasses. Removal of visible faecal material from carcasses is a GHP, and the degree of support for the use steam vacuuming as an intervention specifically for the control of STEC in raw beef was medium to high. 3.3.5. Rinsing of head and cheek meat Head and cheek meats are excised in a separate step from the carcass dressing. Head and cheek meat have been found to have high levels of microbial contamination that occur either naturally, due to contaminants being washed down on inverted and vertically railed carcass during washing, or due to poor CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 50 GHP during processing and chilling. Washing animal heads using water or water treated with chemicals has been proposed as a treatment to reduce STEC O157:H7 contamination of the associated head meat. There is some evidence of STEC O157:H7 reduction specifically on masseter muscles (cheek meat) following head washing (Kalchayanand et al., 2008). Most of these results were obtained from challenge studies or simulations and their practicality requires further evaluation with studies in a slaughter plant. Rinsing of head and cheek meat is a GHP; however, the degree of support for the use of this practice as an intervention specifically for the control of STEC in raw beef was low. 3.4. PRE-CHILLING Interventions may be applied to the carcass post-dressing and pre-chill to reduce microbial contamination on the carcass surface (Gagaoua et al., 2022). These interventions may be physical, chemical or biological. Based on a meta-analysis of literature, there is good (high quality of evidence) evidence that the use of hot potable water carcass wash, steam pasteurization and 24 h air chilling and combination of these, are effective in reducing NTS E. coli and potentially pathogen contaminants on beef. 3.4.1. Hot water wash Water at varying temperatures may be used to wash the dressed carcass at the pre-chill stage. The temperature achieved on the carcass surface will have the most impact in terms of microbial reductions and it is affected by the temperature, the pressure and the volume of water applied, and the distance between the spray nozzles and the carcass (Gagaoua et al., 2022). The application of hot water to a carcass may result in a bleached discolouration of the meat, but this generally disappears after chilling. The use of very high-pressure water may also drive bacteria into the carcass tissue rather than removing them. A commercial hot water wash cabinet set at 74 °C for 5.5 s reduced TVC and Enterobacteriaceae counts by 2.7 log10 CFU/cm2 and reduced the prevalence of carcasses that were positive for STEC O157:H7 by 81 percent (Bosilevac et al., 2006). A meta-analysis of interventions for E. coli on beef carcasses (Zhilyaev et al., 2017) estimated that the efficacy of wash water against E. coli was increased by 0.014 log10 CFU/cm2 per °C increase in temperature. Using a cocktail of STEC serogroups inoculated onto beef flank, hot water (85 °C) in a spray cabinet at 1.05 kg/cm2 (60 cycles) pressure reduced STEC levels by 3.3 and 4.2 log10 CFU/cm2 CHAPTER 3 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 51 (Kalchayanand et al., 2012). Similarly, stx gene prevalence was reduced on carcass following hot water spray (82 °C) (Signorini et al., 2018). The degree of support for the use of hot water carcass wash at pre-chill as an intervention specifically for the control of STEC in raw beef was high. 3.4.2. Steam pasteurization Steam (100 °C) has a higher heat capacity than water at the same temperature and therefore, should better penetrate the carcass meat surface to target microorganisms present. At the carcass surface, a temperature of ≥ 82.2 °C for 6 s to 11 s is reached using steam. Steam may result in carcass discolouration, but acceptable colour is restored after 24 h of chilling. Steam pasteurization has been demonstrated to effect significant reductions in the concentration of TVC and coliforms (below detectable levels) on pre-chill carcasses. A commercial trial showed a reduction in E coli levels (0.5 log10 CFU/cm2) at rump sites only, along with reductions in Enterobacteriaceae (0.8 log10 CFU/cm2) levels at all carcass sites examined (Minihan et al., 2003). When used on inoculated pre-rigor beef, STEC O157:H7 was reduced by 3.5 log10 CFU/cm2 (Phebus et al., 1997). The degree of support for the use of hot steam pasteurization at pre-chill as an intervention specifically for the control of STEC in raw beef was high. 3.4.3. Organic acids Organic acids (e.g. lactic, formic, propionic, citric, fumaric, L-ascorbic, acetic and mixtures) may be applied to carcasses after trimming and inspection but before chilling. Internationally, solutions of lactic or acetic acids (1 to 3 percent) are commonly used chemical interventions in commercial plants for beef dressing (Gagaoua et al., 2022) and can effect reductions of 0.02 to 3 log10 CFU/cm2 for APC, Enterobacteriaceae, coliforms and NTS E. coli (Dormedy et al., 2000; Bosilevac et al., 2006; Signorini, 2018). Lactic acid produces reductions of 2–3 log10 CFU/cm2 of STEC O157:H7 on beef carcasses (Ransom et al., 2003). Reductions in E. coli levels from treatment with 2 percent solutions of lactic, acetic and citric acids applied manually or automatically, ranged from 0.08–0.83 log10 CFU/cm2 depending on acid type, temperature and the mode of application. In most cases, automatic application had greater impact than manual. Organic acids have been shown to be most effective when applied as a warm rinse (50 °C to 55 °C) (Acuff, 2005). A 3 percent solution of lactic acid at 55 °C and applied automatically, gave a 1.03 log10 CFU/cm2 reduction in microbial levels and also a significant reduction (29.3 percent) in stx gene prevalence on carcasses (Signorini et al., 2018). CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 52 There is a lot of variability in the literature in terms of the cited reductions in STEC that can be achieved through the use of organic acids. This is mainly due to differences in the concentrations and types of acids used by different researchers, the method of application, the types of samples tested, and the initial microbial load of samples. Carcass surfaces treated with organic acids often display some discolouration of tissue or fat surfaces (Meat Industry Services, 2006). However, discolouration becomes less evident after chilling and may be less apparent if preceded by a hot water (~90 °C) carcass wash. The degree of support for the use of organic acids on carcasses at pre-chill as an intervention specifically for the control of STEC in raw beef was low. 3.4.4. Oxidizer type antimicrobials Other agents which act as oxidative biocides can be used as a carcass wash or spray. Ozone is a water-soluble gas and a strong oxidizing agent which must be generated at the point of use. Application of 0.5 percent ozonated water on beef tissue reduced total bacterial counts by 2.5 log10 CFU/cm2 (Gorman et al., 1995, 2007). However, a study of the effectiveness of an ozone treatment in reducing STEC O157:H7 and Salmonella Typhimurium contamination on hot carcass surfaces show ozone treatment had no significant improvement over a water wash in reducing pathogens on beef carcass surfaces (Castillo et al., 2003). Potential exposure of ozone to workers also poses safety concerns. Electrolysed water is generated by passing electric current through a dilute saline solution. At present, there is limited evidence of its efficacy to reduce microorganisms on beef carcass. Peracetic/peroxyaectic acid can be applied to carcasses generally at levels of around 200 ppm. Reported reductions of STEC O157:H7 levels on meat carcasses have varied from 0.7 log10 CFU/cm2 (King et al., 2005); 1 to 1.4 log10 CFU/cm2 (Ransom et al., 2003) to 2.2 log10 CFU/cm2 (Penney et al., 2007). Peracetic and peroxyacetic acids treatments are commonly used in beef processing either alone or in combination with other agents. Acidified sodium chlorite (ASC) can be used at concentrations between 500 and 1200 ppm as a wash/spray on beef carcasses. There are conflicting reports on its effectiveness ranging from limited reduction in APC and E. coli levels (Gill and Badoni, 2004) to 0.6–2.3 log10 CFU/cm2 reduction of different STEC serogroups (Kalchayanand et al., 2012). CHAPTER 3 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 53 The degree of support for the use of ozonated and electolyzed water and other chemicals on carcasses at pre-chill as an intervention specifically for the control of STEC in raw beef was low. 3.5. CARCASS CHILLING The biochemical processes and structural changes that occur in beef during the first 24 h post-mortem are critical in determining product quality and palatability (Reid et al., 2017). The objective of chilling carcasses is to cool the meat quickly enough to prevent bacterial growth, but not so quickly to cause cold shortening (toughening) of the meat. The higher the carcass surface temperatures, greater the likelihood of bacterial growth, including spoilage bacteria (Gagaoua et al., 2022), which may result in higher bacterial counts and shorter product shelf-life. Chilling, obtained by setting a critical limit of ≤ 4 °C surface temperature within 24 h, is considered a critical control point in the Hazard Analysis and Critical Control Point (HACCP) plans of many processing plants. Conventional air chilling has been reported to reduce levels of APC and indicator microorganisms by 0.5 to 2 log10 CFU/cm2 on carcasses, but there is some evidence to suggest that this reduction may be an artefact, as the bacteria are only stressed and given appropriate conditions, the stressed bacterial cells may recover (Mellefont, Kocharunchitt and Ross, 2015). Spray chilling of carcasses uses water micro droplets with a chilling regime for approximately 14 h with intermittent spraying cycles. Spray is commonly used for beef and is designed to reduce carcass weight loss. There is no evidence that spray chilling has a substantial effect on microbial populations, including STEC. The use of spray chilling in combination with oxidizer type of antimicrobials has also been reported. In a laboratory study, aqueous ozone applied as a spray chill on inoculated beef showed significant reductions in STEC O157:H7 and APC levels as compared to spray chilling with water alone (Kalchayanand, Worlie and Wheeler, 2019). Simulated spray chilling with chlorine dioxide (> 20 ppm) and peroxyacetic acid (> 200 ppm) on inoculated beef striploins reduced E. coli by ≥ 4 log10 CFU/cm2 (Kocharunchitt et al., 2020). The use of such combination approaches show potential, but more evidence is needed on their performance under commercial processing conditions. Spray chilling and incorporating antimicrobial oxidizers is a GHP; however, the degree of support for the use of this practice specifically for the control of STEC in raw beef was low. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 54 References Acuff, G. R. 2005. 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Journal of Food Protection, 69: 1452– 1455. doi: 10.4315/0362-028x-69.6.1452 Van Donkersgoed, J., Jericho, K.W.F., Grogan, H. & Thorlakson, B. 1997. Preslaughter hide status of cattle and the microbiology of carcasses. Journal of Food Protection, 60: 1502–1508. doi: 10.4315/0362-028x-60.12.1502 Zhilyaev, S., Cadavez, V., Gonzales-Barron, U., Phetxumphou, K. & Gallagher, D. 2017. Meta-analysis on the effect of interventions used in catt le processing plants to reduce Escherichia coli contamination. Food Research International, 93: 16–25. doi: 10.1016/j.foodres.2017.01.005 61 Post-processing control strategies for STEC in beef The intended use of raw beef is an important factor to consider in the selection and implementation of methods for STEC control. If the product is not intended to remain intact, STEC present on the exterior of meat may be internalized during the non-intact production process, such as grinding and mechanical tenderization. In such cases, cooking to a rare or medium-rare internal temperature may not be sufficient to destroy STEC throughout the product. It is critical therefore, that primal, sub-primal and other cuts intended to be non-intact products should be treated by interventions to reduce or eliminate STEC. During carcass fabrication, the carcass is broken down into consumer portions, which includes additional product preparation and handling. As all these steps increase surface area of the product, the likelihood of contamination spread is great, therefore the application of inventions to reduce STEC at fabrication can be impactful. During mechanical tenderization of meats, the needles or blades used in the process of tenderization can physically transfer foodborne pathogens from the surface into the interior of the beef cuts. This has prompted the development of interventions that can reduce internalization of surface STEC (Currie et al., 2019). Some nations have required registered plants to affix a label (Mechanically Tenderized Beef [MTB]) to products and to include safe cooking instructions for the consumers, stating “Cook to a minimum internal temperature of 63 °C” (Health Canada, 2014). Raw ground beef and ground beef-based products (e.g. hamburger patties), pose a higher risk to human health than intact beef because of its greater contact surface and the higher degree of handling and processing involved with production. 4 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 62 During the mincing/grinding process, microbial transfer from the external surfaces into the mass of the ground beef is likely to occur; therefore, it is important to implement GHP, GMP and HACCP principles as well as intervention measures throughout the ground beef production chain to minimize STEC exposure and contamination. In several nations, all beef used in grinding is required to be tested for contamination by specific STEC serotypes (USDA, 2016, 2017). Despite all the control measures applied at the previous stages of production, contamination of STEC in ground beef can still be detected, albeit mostly at low concentration. This remains a critical issue however, because of the low infectious dose of STEC, hence interventions still need to be applied at all stages of ground beef production, product manufacturing, packaging and distribution. Since ground beef is perishable, it is important to apply control measures properly during transport and storage of the carcasses/beef cuts before grinding. Maintaining temperature (< 7 °C) is an important parameter that should be controlled throughout the ground beef production chain to reduce growth of STEC through distribution, retail sale, and until the product reaches the consumer (Duffy et al., 2005). Packaging processes, including interventions, for ground/ minced products are also critical for ensuring STEC control. Product labels should contain sufficient information about interventions applied, while also guiding the purchaser with safe handling and preparation guidelines (e.g. use-by dates and the need for thorough cooking on the label). Although the implementation of the interventions in the post-processing phase are mostly to improve microbial safety of fresh ground beef, other essential parameters must also be considered, such as extension of product shelf-life and consumer acceptance (e.g. maintenance of sensory qualities without altering organoleptic characteristics; inclusion of package labelling regarding the treatment, guidance for safe handling). The antimicrobial interventions implemented throughout the beef production chain can vary depending on the country’s regulation and the volume of production as well as destination of the product (e.g. local consumption vs export market). Intervention strategies used in post-processing should be safe and suitable to be broadly approved by the regulations of different nations. A summary of post-processing control measures, and combinations of these, for STEC in beef and their degree of support (high, medium, low), based on scientific evidence, is available in Annex 3. CHAPTER 4 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 63 4.1. PHYSICAL INTERVENTIONS 4.1.1. Air-drying heat treatment Air-drying heat treatment consists of a dry air decontamination apparatus, which produces repeatable and known heating time–temperature cycles onto food surfaces (McCann et al., 2006). The use of air-drying has been proposed for the decontamination of surfaces of (smaller) meat pieces but has only been examined on a laboratory scale. Beef sample surfaces inoculated with STEC O157:H7, heated at 60 °C, 75 °C, 90 °C and 100 °C using fast and slow heating rates and subsequently held at these temperatures for up to 600 s were found to have reductions of STEC O157:H7 by 4.18–6.06 log10 CFU/cm2 at the higher temperatures (90 °C and 100 °C) (McCann et al., 2006). However, the significant number of microorganisms that survived posed potential concerns. Osmotic and thermal protective traits of the resistant bacterial population must be investigated before this method can be proposed as a decontamination process. Air-drying heat treatment also changed meat appearance and colour, which limited the utility of this intervention to products used for catering and institutional preparations rather than for retail sales. The degree of support for the use of air-drying heat treatment as a post- processing intervention specifically for the control of STEC in raw beef was low to medium. 4.1.2. Condensing steam Steam is an important intervention for pathogen reduction used during processing but has also been developed for use on finished meat and have been tested at a laboratory scale. Logue et al. (2005) used steam treatment temperatures of 55 °C, 65 °C and 75 °C on food surfaces for 10 min, 18 s, and 10 s, and found populations of inoculated STEC O157:H7 (~6 log10 CFU/cm2) were reduced the most at higher temperatures (75 °C for 10 s at 38.6 KPa, 5.59 - 3.48 log10 CFU/cm2). However, post-process storage conditions were also important to ensure that no re-growth of the pathogen occursed and this was best achieved through storage under vacuum at 0 °C. This study indicated that sub-atmospheric steam could have significant application in the decontamination of post-fabrication meat primals immediately prior to packaging (Logue et al., 2005). The degree of support for the use of condensed steam as a post-processing intervention specifically for the control of STEC in raw beef was low. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 64 4.1.3. Hot water Hot water treatment was examined as an intervention to minimize the risk of internalizing inoculated STEC O157:H7 on the surfaces of sub-primal cuts undergoing blade tenderization or moisture enhancement (Heller et al., 2007). Evaluated under laboratory conditions, round pieces cut from sub-primals were inoculated with a STEC O157:H7 cocktail at 4.2 log10 CFU/100 cm2. Application of hot water (82 °C for 20 s) sprayed onto the surface, resulted in 1.0 log10 CFU/100 cm2 reductions in STEC O157:H7 levels. However, another study showed that use of hot water treatments (82 °C, aerobically or anaerobically [559 mm/Hg vacuum] for 3 min in a tumbler) of beef trimmings before grinding did not reduce any microorganism populations (Stivarius et al., 2002). The degree of support for the use of hot water treatment as a post- processing intervention specifically for the control of STEC in raw beef (sub-primals) was low. 4.1.4. Surface trimming Trimming of beef carcasses by slaughterhouse operators removes visible surface contamination and is effective at reducing STEC O157:H7 on sub-primals under lab conditions (Heller et al., 2007). When an STEC O157:H7 cocktail (4.2 log10 CFU/100 cm2) was inoculated onto round pieces cut from sub-primal meats, by trimming away the external surface with a sterile knife resulted in 1.1 log10 CFU/100 cm2 reduction in inoculated STEC O157:H7 levels (Heller et al., 2007). Another trial conducted under laboratory conditions also found that full-surface trimming (removal of 5 mm of the dorsal and ventral surfaces) and partial-surface trimming (removal of 5 mm from the dorsal surface only) of sub-primals, significantly decreased STEC O157:H7 levels by more than 2 log10 CFU/cm2 (Lemmons et al., 2011). The need to use sterile equipment for trimming poses some limitations to implementation at many locations. The degree of support for the use of surface trimming as a post-processing intervention specifically for the control of STEC in raw beef (primals) was low to medium. 4.1.5. Dry chilled ageing Dry ageing of carcasses at refrigeration temperatures was the most common intervention used for the reduction of STEC O157:H7 in small processing plants (Tittor et al., 2011). A survey found dry chilled and aged meat samples, which were suspended in refrigerators/chillers (3 °C) with an air velocity of 0.25 m/s and a relative humidity of 80 percent, resulted in a reduction of 4 log10 CFU/cm2 CHAPTER 4 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 65 on day 28 of storage (Tittor et al., 2011). Many practices commonly used by large facilities to control pathogens and reduce faecal contamination are difficult to implement for small and very small plants. Dry chilled ageing is a critical control point and could be used as a potential intervention for small processing plants (Tittor et al., 2011). The degree of support for dry ageing of carcasses at refrigeration temperatures as a post-processing intervention specifically for the control of STEC in raw beef was low to medium. 4.1.6. High pressure processing (HPP) High pressure processing (HPP) is a safe and effective non-thermal processing method that improves the microbial safety of fresh ground beef (Zhou, Karwe and Matthews, 2016). HPP could be applied to packaged commodities, thus eliminating the potential for microbial survival and post-packaging contamination. The effect of single- and multiple-cycle HPP treatments on the survival of STEC O157:H7 in ground beef was investigated (Morales et al., 2008). When HPP was applied to ground beef at 450 MPa for 15 min at refrigeration temperature (4 °C to 7 °C), more than 5 log10 CFU/g reduction in populations of a cocktail of O26:H11, O45:H2, O103:H2, O111:NM, O121:H19, O145 and O157:H7 serotypes was observed (Hsu et al., 2015). Sensory qualities of HPP-treated products remained unchanged so there is greater consumer acceptance than irradiated foods (Doona and Feeherry, 2007). Some research suggested that ground beef patties subjected to HPP were drier and less flavourful compared to untreated patties (Hayes et al., 2014). HPP treatment retains many of the fresh qualities of the commodity. It also denatures enzymes, extends shelf-life, and reduces the need for preservatives, without significantly altering organoleptic qualities. Combining vacuum-packaged and HPP treatment reduced STEC O157:H7 levels in ground beef by 3 log10 CFU/g and produced substantial sublethal injury in the surviving STEC population that resulted in further reductions in levels during frozen storage (Black et al., 2010). Vacuum-packaged and HPP treatments (four, 60 s cycles, 400 MPa, 17 °C) produced > 2.0 log10 CFU/g reductions of E. coli DH5α and the major seven (O103, O111, O26, O145, O121, O45, O157:H7) STEC serogroups (Jiang et al., 2015). The colour and texture of ground beef patties exhibited significant changes when more severe HPP and vacuum treatments were applied. Refrigerated or frozen storage of HPP-processed ground beef served as an additional intervention to limit the survival and recovery of STEC O157:H7 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 66 (Black et al., 2010). Combining HPP with 24 h storage of ground beef at 4 °C or at -20 °C achieved a 5 log10 CFU/g reduction in STEC O157:H7 populations. HPP treatment with cold storage regardless of whether the pressure applied was cyclic or static, had no significant effect on the colour of ground beef (Zhou et al., 2016). The degree of support for the use of HPP treatment alone or in combination with other interventions such as vacuum packaging, refrigerated or frozen storage as a post-processing intervention specifically for the control of STEC in raw beef was medium. 4.1.7. Irradiation The primary purpose of food irradiation is to eliminate microbial pathogens and improve food safety. Irradiation is accomplished using carefully controlled doses of ionizing radiation for a short time. One of the most common energy sources used in food irradiation is the electron beam (eBeam), which consists of highly energetic electrons, generated from commercial electricity rather than a radioactive source and it does not penetrate deeply into the product. Another common energy source is gamma radiation produced by radioactive Cobalt60 or Cesium137 which penetrate deeply into the product. Both eBeam and gamma irradiation have been approved by many different countries for non-thermal processing of foods. The potential of eBeam to control STEC O157:H7 populations was examined using meat samples inoculated with STEC O157:H7 at levels ranging from 3–6 log10 CFU/cm2. A low 1-kGy dose of eBeam radiation reduced STEC O157:H7 by at least 4 log10 CFU/cm2. The impact of eBeam on organoleptic quality was assessed using flank steak and ground beef and showed that the sensory qualities of the products were not affected by the 1 KGy dose (Arthur et al., 2005). Similarly, a 1 KGy dose effected a 4 log10 CFU/cm2 reduction of STEC O157:H7 and a 3.9–4.5 log10 CFU/cm2 reduction was observed using a cocktail of other STEC strains (Kundu et al., 2014). Several studies examined the effects of low dose gamma irradiation to control STEC O157:H7 and other STEC. One study found that a 2.5 KGy dose reduced STEC O157:H7 populations seeded onto meat trim by 5 log10 CFU/g and there were no sensory changes on the product until the dosage was increased to 5 KGy (de la Paz Xavier et al., 2014). Cap et al. 2020, used gamma irradiation on five STEC strains (O26, O103, O111, O145 and O157) seeded at 7 log10 CFU/g on beef trim and found that both a low (0.5 KGy) and a high (2.0 KGy) dose reduced STEC levels by 1.5 and > 5 log10 CFU/g, respectively. The effect of gamma irradiation on 40 STEC strains seeded onto lean ground beef was examined and results showed that the D10 value, defined as the dose needed to reduce levels by 1 log10 CHAPTER 4 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 67 (90 percent), ranged from 0.16–0.48 kGy, with a mean of 0.31 kGy for the 40 STEC isolates (Sommers et al., 2015). These studies illustrated that both eBeam and gamma irradiation are very effective at reducing levels of STEC and STEC O157:H7. These interventions may be applicable to meats prior to the mechanical tenderization to minimize internalization of pathogens into meat or for treatment of trim prior to grinding. However, installing an irradiation system in a plant is costly, brings up security and safety concerns, and consumer perception and reluctance to buy irradiated foods may also affect the marketability of the product. The degree of support for the use of eBeam and gamma irradiation as a post-processing intervention specifically for the control of STEC in raw beef was medium. Irradiation and organic acids The potential of using high (HDI-2 KGy) and low (LDI-0.5 KGy) dose gamma irradiation, along with lactic acid (LA-5 percent), caprylic acid (CA-0.04 percent) or combinations of these, were evaluated as intervention measures to control five STEC strains inoculated at 7 log10 CFU/g on beef trim. Low dose gamma irradiation alone or with CA caused a 1.4 log10 reduction. Low dose gamma irradiation with LA showed a 1.7 log10 reduction. But the most effective treatment was HDI, which gave a > 5 log10 reduction. Minimal changes in meat quality parameters and sensory factors were noted with all treatments, except for LDI + LA (Cap et al., 2020). Li et al. (2015) examined samples that were treated with 5 percent LA at 55 °C, were aerobically or vacuum packed, and kept at 4 °C. Irradiation with 1 KGy reduced STEC by 4.5 log10 and the addition of LA did not further reduce STEC levels. Studies showed no additional benefits of combining LA and CA with irradiation, regardless of irradiation dose in fresh beef, yet there was some benefit in frozen product. The degree of support for the use of eBeam and gamma irradiation in combinaion with organic acids as a post-processing intervention specifically for the control of STEC in raw beef was medium. Irradiation and packaging The effectiveness of controlling STEC O157:H7 in ground beef by combining eBeam and vacuum or modified atmosphere packaging (MAP) was examined using an inoculated mix of 5 STEC O157:H7 strains (5 log10 CFU/g) in ground beef patties packaged in 99.6 percent CO2 and 0.4 percent CO or in vacuum. Patty packages that were irradiated with eBeam at 0.5, 1.0 or 1.5 KGy, showed log10 CFU CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 68 reductions of 0.5 to 0.7, 1.0 to 2.2 and 3.0 to 3.3, respectively. The D10-values for STEC O157:H7 was similar in vacuum (0.47 ± 0.02 kGy) or in MAP (0.50 ± 0.02 kGy) and irradiated packages stored at 4 °C for 6 weeks showed no bacterial growth. However, storage at 25 °C showed growth in the vacuum packaged samples but not in MAP, suggesting that MAP was more effective than vacuum in controlling microbial growth post irradiation (Kudra et al., 2011). The effect of combining gamma irradiation and MAP was examined with raw meat ball samples seeded with 6 log10 CFU/g of STEC O157:H7 and packed in MAP (3 percent O2 and 50 percent CO2 and 47 percent N2) or packaged aerobically and irradiated at 0.75, 1.5, and 3 kGy before storage at 4 °C for 21 days. The D10 value for STEC O157:H7 was 0.24 KGy and it was totally inactivated by 1.5 KGy. In the aerobic packages, irradiation caused significant loss of product colour and sensory quality, but not in the MAP packages, suggesting that MAP better inhibited irradiation‐ induced quality degradations during the 21-day storage (Gunes et al., 2011). The degree of support for the use of eBeam and gamma irradiation in combinaion with MAP as a post-processing intervention specifically for the control of STEC in raw beef was medium. 4.2. CHEMICAL INTERVENTIONS 4.2.1. Organic acids Organic acids, particularly lactic acid, have a long history of use as food preservatives as well as decontamination treatments for foods, including meat. Lactic acid is probably the most widely used organic acid for meat decontamination and is already approved or in use in a number of countries. Several studies have examined the efficacy of lactic acid, although relatively few have specifically considered its efficacy on STEC O157:H7 or non-O157 STEC. EFSA (2011) reviewed studies looking at the efficacy of lactic acid treatments at a variety of concentrations for decontamination of beef carcasses, beef cuts and trimmings. For the studies which examined STEC on beef cuts and trimmings, there were reductions of 0.1 to 1.4 log10 CFU/g for beef cuts (Echeverry et al., 2009) and 1.1 to 2.3 log10 CFU/g for trimmings (Harris et al., 2006) as compared to untreated controls. Wolf et al. (2012) compared the effectiveness of 4.4 percent lactic acid dip or spray application on beef trim and in ground beef and found that dip application was more effective than spray, and decreased the levels of STEC O157:H7 by 0.91 to 1.41 log10 CFU/g, and non-O157 STEC were decreased by 0.48–0.82 log10 CFU/g. In challenge studies that examined the use of lactic acid to decontaminate sub-primals, trimmings and cheek meat, reductions ranged from 0.2 to 2.8 log10 CFU for STEC O157:H7 and 0.2-3.4 log10 CFU for NTS E. coli (Antic, 2018). CHAPTER 4 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 69 As previously noted in Section 3.4.3, the efficacy of lactic acid treatment varied widely and was dependent on lactic acid concentration, how it was applied, the length of application, temperature and the microbial load on the meat surfaces. Other factors such as the inoculum level, STEC strain(s) used, and the recovery methods used in the studies were also impactful. In practice, lactic acid may be used in combination with other chemical or physical treatments, such as hot water or vacuum/modified atmosphere storage, which add a further level of complexity to understanding the efficacy of each treatment applied individually versus in combination. The degree of support for the use of organic acids on sub-primals, trim, and cheek meat as a post-processing intervention specifically for the control of STEC in raw beef was low to medium. 4.2.2. Other chemical treatments A wide range of chemical treatments have been explored for the decontamination of beef, with some chemicals used individually or in various combinations. Kalchayanand et al. (2015) examined the efficacy of hypobromous acid, neutral acidified sodium chlorite and two citric acid-based antimicrobial compounds against strains of seven STEC serogroups (i.e. O26, O45, O103, O111, O121, O145 and O157). The chemicals when applied as spray treatments on the surface of STEC-inoculated pre-rigor beef flank at 4 °C, resulted in reductions in STEC populations of 0.7–2.0 log10 CFU/cm2 after treatment and 1.2 to 2.3 log10 CFU/ cm2 after 48 h at 4 °C. No differences were observed in the efficacy of the four antimicrobial compounds between the strains of STEC O157:H7 and the six non-O157 STEC serovars. However, when the seven STEC strains were inoculated at low concentrations, none of the four antimicrobial treatments resulted in the complete elimination of STEC. Muriana et al. (2019) examined the effectiveness of 14 different commercially- available chemical treatments with a wide range of pH values (0.8–13.1). The chemicals were sprayed at commercially recommended concentrations onto lean beef wafers (cut from cores of beef sub-primals) inoculated with a four- strain cocktail of STEC O157:H7. Reductions achieved ranged from 0.1–1.18 log10 CFU/cm2 after 1 h, 0.44–2.07 log10 CFU/cm2 after 1 day, and 0.37–3.61 log10 CFU/cm2 after 7 days of storage. In a separate experiment, inoculated beef cores from sub-primals were subjected to antimicrobial organic acid treatments prior to blade tenderization. None of the antimicrobial treatments eliminated STEC O157:H7 post-tenderization, but there was a significant reduction in the number of positive samples when the antimicrobial treatments were used before tenderization. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 70 Scott-Bullard et al. (2017) investigated the efficacy of a sulfuric acid-sodium sulfate mix against a mixture of five strains of STEC O157:H7 and 12 strains of non-O157 STEC on pre-rigor beef tissue. Treatments lowered STEC populations by 0.6–1.5 log10 CFU/cm2, depending on the inoculum type and the recovery culture medium used. Similar results were obtained with samples seeded with NTS E. coli, supporting its suitability as a surrogate organism for STEC in efficacy validation studies of sulfuric acid-sodium sulfate in beef plants. The degree of support for the use of other chemical treatments on sub- primals, trim, and cheek meat as a post-processing intervention specifically for the control of STEC in raw beef was low to medium. 4.2.3. Ozone Ozone is a powerful oxidising agent that exhibits antimicrobial activity against a wide range of microorganisms by damaging their cell walls and membranes leading to lysis. Ozone has been considered for use on a wide range of foods of both animal and non-animal origin with varying degrees of success, but it is most effective as an antimicrobial treatment for low pH foods (such as fruits) due to the lower decomposition of ozone in those conditions (Kumar and Sabikhi, 2019). Ozone treatments can also impact organoleptic properties of the meat, such as colour and aroma. Coll Cárdenas et al. (2011) found that treatment of beef primals with 72 ppm of gaseous ozone at 0 °C and 4 °C for 3 h reduced E. coli counts by 0.6–1.0 log10 CFU/g with the reduction being slightly higher at 0 °C than at 4 °C. Compared to the 3 h exposure, a longer exposure time (24 h) to ozone resulted in a greater reduction in E. coli counts (0.7–2.0 log10 CFU/g) but had a significant effect on surface colour of the beef due to lipid oxidation. Novak and Yuan (2003) examined the effect of 3 ppm aqueous ozone treatment on a cocktail of three STEC O157:H7 strains which were applied to irradiated, sterilised beef. Ozone treatment resulted in a reduction of 0.85 log10 CFU/g and produced some ultrastructural changes but did not alter the visual appearance of the beef (Novac and Yuan, 2003). McMillin and Michel (2000) examined high concentrations of ozone (500, 3500 and 5000 ppm) to treat E. coli inoculated into minced beef and obtained up to 2 log10 CFU/g reductions in a dose-dependent manner; and no organoleptic changes were reported (McMillin and Michel, 2000). The degree of support for the use of ozon on primals and ground beef as a post-processing intervention specifically for the control of STEC in raw beef was low. CHAPTER 4 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 71 4.2.4. Lactoferricin B Lactoferricin B is an antimicrobial peptide derived from acid-pepsin digestion of bovine lactoferrin and it is bactericidal to a wide range of bacteria. The antimicrobial properties of lactoferricin B are reduced at acid pH or completely inhibited by the addition of 5 percent cow’s milk (Jones et al., 1994). Venkitanarayanan, Zhao and Doyle (1999) investigated the effect of Lactoferricin B on coarsely ground top round beef steak which were inoculated with a five-strain mixture of STEC O157:H7. The ground beef samples were treated with lactoferricin B (100 µg/g), mixed and stored at 4 °C and 10 °C for 3 days. Lactoferricin B significantly reduced STEC O157:H7 counts by approximately 0.8 log10 CFU/g at both storage temperatures. However, there was no significant impact of lactoferricin B on total aerobic plate counts (Venkitanarayanan, Zhao and Doyle, 1999). Reductions in STEC O157:H7 achieved in ground beef were much lower than the 5 log10-cycle reductions in 1 percent peptone water at 37 °C that was reported by Shin et al. (1998). The study by Venkitanarayanan, Zhao and Doyle (1999) used a 10-fold lower concentration of lactoferricin B and the storage temperatures used were also much lower to simulate conditions at retail and in the home. There was no sensory analysis of the treated ground beef, so the effects of lactoferricin B on product quality remain unknown. The degree of support for the use of lactoferricin B in ground beef as a post-processing intervention specifically for the control of STEC in raw beef was low. 4.2.5. Essential oils A wide range of plant essential oils have a long history of antimicrobial activity particularly in in-vitro studies (Quinto et al., 2019; Valdivieso-Ugarte et al., 2019). Antimicrobial activity of essential oils has been examined in foods including meat and meat products, but the concentrations of essential oils required to be effective can result in organoleptic changes. Solomakos et al. (2008) examined the antimicrobial effect of thyme essential oil, nisin (a bacteriocin) or their combination against two strains of STEC O157:H7 in minced beef during refrigerated storage. Thyme oil (0.6 percent) was added to samples of minced meat inoculated with STEC O157:H7, mixed and stored at 4 °C or 10 °C for up to 12 days. There was no significant reduction in STEC O157:H7 counts with storage at 4 °C but, approximately a 1 log10 CFU reduction in counts were obtained at 10 °C after 2 days. The addition of 500 or 1000 IU/g of nisin along with 0.6 percent thyme oil reduced STEC O157:H7 counts by 1.0 log10 CFU/g at 4 °C, compared with either thyme oil or nisin alone. Sensory evaluation revealed that the organoleptic properties of 0.6 percent thyme oil treated minced beef was acceptable, however this may not be the case with other essential oils and therefore, each will have to be examined individually. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 72 The degree of support for the use of essential oil on muscle tissue and ground beef as a post-processing intervention specifically for the control of STEC in raw beef was low. 4.3. BIOLOGICAL INTERVENTIONS 4.3.1. Bacteriophages The potential of using bacteriophages to control STEC at post-processing of meats has been explored. A bacteriophage specific to STEC O157:H7 and tested to lyse STEC O157:H7 strains, reduced STEC O157:H7 levels by 2.7 log10 CFU on meat incubated at 37 °C (Hudson et al., 2013). Phage treatment at 10 log10 PFU on enteropathogenic E. coli (EPEC) and STEC, reduced STEC by ~0.77 logs in 3 h, and 1.15 log10 after 6 h at 24 °C (Tomat et al., 2013). Bacteriophage insensitive mutants (BIM) emerged at low frequency, although the use of a phage cocktail could potentially limit this development. A cocktail of six phages specific to E. coli, EPEC and STEC reduced STEC populations by 3–3.8 log10 at 37 °C, with effectiveness being time- and temperature-dependent (Tomat et al., 2018). A different phage cocktail tested reduced STEC levels by 0.48 and 1.97 log10 at 4 °C and 24 °C, respectively, over 24 h (Hong, Pan and Ebner, 2014). Bacteriophage do not impart sensory changes to beef. Most of the studies have been performed on small pieces of meat or packages, so it is uncertain if and how this technology can be scaled up for use in the production plant. Other concerns include the lengthy incubation time and the temperatures of 24 °C to 37 °C needed for optimal phage lytic activity, which are not well suited for conditions in the post- processing of meats. The degree of support for the use of Bacteriophage treatment in ground beef as a post-processing intervention specifically for the control of STEC in raw beef was low. 4.3.2. Lactic acid bacteria (LAB) Lactic acid bacteria (LAB) are antagonistic to the growth of other bacteria, including STEC. The effectiveness of LAB treatment on STEC and STEC O157:H7 was evaluated. After refrigerated vacuum ageing of beef strips for 14–28 days, LAB treatment reduced the level of a STEC cocktail by 0.4 log10 CFU/cm2 (Kirsch et al., 2017). After 3 days of storage, LAB-treated ground beef showed STEC O157:H7 population had reduced by 2 log10 at 5 °C and LAB caused no sensory changes on the meat (Smith et al., 2005). Treatment with LAB reduced STEC and STEC O157:H7 populations on beef, however the lengthy time required for LAB to be CHAPTER 4 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 73 effective limits its use mostly to the ageing and storage phases of the final product. Impacts of LAB on shelf-life, stability and organoleptic properties of the product have not been clearly elucidated to support the use of LAB to reduce STEC in beef. The degree of support for the use of LAB in ground beef as a post-processing intervention specifically for the control of STEC in raw beef was low. 4.3.3. Colicin Colicins are antimicrobial proteins produced by E. coli that kill other E. coli, including STEC O26, O45, O103, O111, O121, O145, O157 and O104:H4. Pieces of pork steak drip-inoculated with STEC and a solution of colicin M (3 mg/kg) and colicin E7 (1 mg/kg) showed that STEC levels were reduced by 2.3 and 2.7 log10 CFU/cm2 after 1 h and 24 h, respectively (Schulz et al., 2015). E. coli colicins are costly to produce and available only in small amounts. However, recombinant colicin can now be made in yeast, tobacco, spinach and bean plant tissues in quantities that are feasible for commercial application. Colicins do not appear to effect sensory changes in the product, but more extensive studies are needed to better evaluate the use of colicins to control STEC in meats. The degree of support for the use of colicin in sub-primals as a post- processing intervention specifically for the control of STEC in raw beef was low. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 74 References Antic, D. 2018. A critical literature review to assess the significance of intervention methods to reduce the microbiological load on beef through primary production. University of Liverpool. Food Standards Agency Project FS301044. www.food.gov. uk/print/pdf/node/4506 Arthur, T. M., Wheeler, T. L., Shackelford, S. D., Bosilevac, J. M., Nou, X. & Koomaraie, M. 2005. Effects of low-dose, low-penetration electron beam irradiation of chilled beef carcass surface cuts on Escherichia coli O157:H7 and meat quality. Journal of Food Protection, 68: 666–672. doi: 10.4315/0362-028x-68.4.666 Black, E. P., Hirneisen, K. A., Hoover, D. G. & Kniel, K. E. 2010. 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81 Processing and post-processing control strategies for STEC in raw milk and raw milk cheeses This section will discuss interventions to reduce STEC prevalence and concentration in raw fluid milk and raw milk cheeses from processing to post-processing, including packaging. Interventions applied to raw milk and raw milk cheeses produced from all milk-producing domestic species (e.g. dairy cattle, sheep, goat, buffalo, yak, camel, small ruminants) were briefly discussed, however, only those that applied to products of bovine and caprine origin were included in this section of the report. A summary of processing and post-processing control measures for STEC in raw milk and raw milk cheese and their degree of support rating (high, medium, low), based on scientific evidence, is available in Annex 4. 5.1. RAW MILK PROCESSING For the pupose of this report and in accordance with the definitions included within Codex General Standards for the Use of Dairy Terms (CXS 206-1999) (FAO and WHO, 1999) and the Code of Hygienic Practices for Milk and Milk Products (CAC/RCP 57-2004) (FAO and WHO, 2009), raw milk is describe as follows: Raw milk: Milk (defined as the normal mammary secretion of milking animals obtained from one or more milking) which has not been heated beyond 40 °C or undergone any treatment that has an equivalent effect. This definition excludes milk that has been proceeded using methods where heat treatment above 40 °C have been applied. 5 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 82 Some farms produce raw drinking milk for sale at the farm gate, by a vending machine, or for wider sales distribution, including via the internet. However, there is a significant body of evidence from outbreaks and from testing that depending on the animal species of origin, raw milk can be a potential source of microbiological hazards including Campylobacter, Salmonella, STEC, Brucella melitensis, Mycobacterium bovis and tick-borne encephalitis virus (EFSA, 2015). In the EFSA Scientific Opinion (2015) on the public health risks related to the consumption of raw drinking milk, STEC was recognized as a potential hazard in raw drinking milk derived from cows, sheep, goats, horses and donkeys but not from camels. While pateurization is a very effective at killing off harmful pathogens in raw milk, the processing techniques described in this section have been evaluated as alternative treatments to mitigate the presence of STEC in raw milk. 5.1.1. Bactofugation Bactofugation separates microorganisms and spores from milk by their differences in density. It is used in the dairy industry to remove bacterial spores, but its efficiency strongly depends on viscosity, so milk must be heated to 55–60 °C to reduce viscosity prior to treatment. Bactofugation removes 90–99 percent of the bacterial spores from milk, but its efficiency in removing vegetative cells is less consistent (Euster and Jakob, 2019). Faccia et al. (2013) showed that bactofugation reduced Enterobacteriaceae counts by 72 percent and Kosikowski et al. (1968), used a double bactofugation procedure at 54.4 °C to remove 95 percent of the NTS E. coli population in raw milk. By heating the milk to the required 55–60 °C prior to bactofugation, it no longer meets the definition of raw milk. In addition, bactifugation is also limited by added equipment costs and the limited volume of milk that can be quickly and efficiently processed. The degree of support for the use of bactofugation as a processing intervention specifically for the control of STEC in raw milk was low. 5.1.2. Microfiltration Microfiltration typically uses ceramic membranes of 1.4 µm pore size, which provide a bacterial retention rate of 99.93–99.99 percent (Trouvé et al., 1991) in raw milk. Microfiltration needs raw milk to be heated to 50–60 °C to reduce viscosity before treatment, also excluding it from the definition of raw milk. Furthermore, it only works with skimmed milk, so the cream needs to be separated, as a result, its use in treating milk intended for making cheese is uncommon. Elwell and Barbano (2006) demonstrated that microfiltration removed up to 2 log10 CFU/mL of aerobic bacteria from raw milk. The need to heat the milk to 50 °C prior to microfiltration CHAPTER 5 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESES 83 is a limitation, however, studies have also shown that cold microfiltration (6°C) can remove 3–5 log10 CFU/mL of aerobic bacteria down to non-detectable levels (Fritsch and Moraru, 2008; Griep, Cheng and Moraru, 2018). The effectiveness of microfiltration to remove NTS E. coli or STEC has not been examined. The investment in equipment and operating costs and the volume of milk that can be quickly and efficiently processed are limitations to the implementation of microfiltration. The degree of support for the use of microfiltration as a processing intervention specifically for the control of STEC in raw milk was low. 5.1.3. High pressure processing (HPP) Experimental work using high pressure to kill bacteria and extend product shelf-life was explored at the end of 19th century by Hite (1899) who found that high pressure treatment at 600 MPa for 1 h at room temperature extended the shelf-life of raw milk by 4 days. Use of a lower pressure treatment (200 MPa) delayed spoilage by about 24 h. Researchers have explored the effects of high pressure on a wide range of microorganisms under different conditions, as well as the underlying mechanisms of inactivation of bacteria and spores (Farkas and Hoover, 2000; Balasubramaniam, Martínez-Monteagudo and Gupta, 2015; Georget et al., 2015). Few studies have specifically examined the effect of HPP on the survival of STEC O157:H7 (Patterson and Kilpatrick, 1998; Kalchayanand et al., 1998), and most studies have used heat processed, rather than raw milk. Patterson et al. (1995) found that STEC O157:H7 strains were more resistant to HPP in ultra high temperature (UHT) milk than treatment on poultry meat and there was a significant difference in pressure sensitivity of different STEC strains. Patterson and Kilpatrick (1998) found that a 15 min treatment with 400 MPa at 50 °C, above the minimum temperature for raw milk, resulted in approximately a 5.0 log10 CFU/mL reduction of STEC O157:H7 in UHT milk. Studies have also investigated the use of HPP as a treatment option for human milk intended for milk banks. Viazis et al. (2008) observed that when human milk was treated with 400 MPa at 21–31 °C, NTS E. coli in peptone solution was inactivated more quickly than in human milk. The degree of support for the use of HPP as a processing intervention specifically for the control of STEC in raw milk was low to medium. 5.1.4. Irradiation (cold pasteurization) Food irradiation is non-thermal and has the potential to reduce pathogen load in raw milk to increase product safety. Most studies on milk have investigated the effect of either gamma or eBeam irradiation. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 84 The use of eBeam on raw milk was evaluated using different bacteria, including STEC O157:H7. Raw milk irradiated with a 2.0 kGy dose reduced aerobic plate count from 4 log10 CFU/mL to below detectable limits and the D10 value for STEC O157:H7 was 0.062 kGy (Ward et al., 2020). Irradiation eBeam doses of 1 to 2 kGy did not alter the organoleptic and nutritional quality of raw milk, and although the level of vitamin B2 was decreased by 31.6 percent, it was within USDA nutritional guidelines, and no lipid oxidation was detected. However, after 7 days of refrigerated storage, there was 58 percent lipid oxidation but the oxidation did not result in the development of off-odors (Ward, Kerth and Pillai, 2017). Installing an irradiation system in a plant is costly and consumer perception and reluctance to purchase irradiated foods may also affect the marketability of the product. The degree of support for the use of irradiation or cold pasteurization as a processing intervention specifically for the control of STEC in raw milk was medium. 5.1.5. Bacteriophage A cocktail of six phages, specific to E. coli, EPEC and STEC, including STEC O157:H7, reduced E. coli and STEC O157:H7 counts in raw milk from 2 log10 CFU/mL to below the detection limit after 1 day at 4 °C. However, counts of EPEC and other STEC required 7–13 days of incubation to become non-detectable, demonstrating strain to strain variation in specificity and resistance to the phage. At 24 °C, reductions by phage treatment averaged 4 log10 CFU/mL for STEC (Tomat et al., 2018). The temperature and long incubation times required for optimal phage lytic activity are not well suited for raw milk processing conditions. The degree of support for the use of bacteriophage as a processing intervention specifically for the control of STEC in raw milk was low. 5.2. RAW MILK CHEESE PROCESSING For the pupose of this report and in accordance with the definitions included within Codex General Standards for the Use of Dairy Terms (CXS 206-1999) (FAO and WHO, 1999) and the Code of Hygienic Practices for Milk and Milk Products (CAC/RCP 57-2004) (FAO and WHO, 2009), raw milk cheese is describe as follows: Raw milk cheeses: Cheeses made from raw milk. For technical purposes, cheese curd might be “cooked” (i.e. processed by application of heat). CHAPTER 5 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESES 85 Raw milk cheeses are a potential source of STEC human infections. The ability of STEC to survive raw milk cheese production processes depends primarily on their stress-response mechanisms, which include: general, acidic, osmotic, and heat shock stress-responses in E. coli (Peng et al., 2011). Different NTS E. coli, and STEC serogroups or even strains within a serogroup can exhibit high variability in response to physiological properties. For example, STEC O157:H7 clones were not significantly more acid resistant than the NTS E. coli, and overall, the STEC O157:H7 clonal group was not exceptionally acid resistant (Large, Walk and Whittam, 2005). However, there are strain to strain variations in acid resistance among STEC. The cheese manufacturing process varies widely depending on the starter cultures used, the mode and extent of the acidification and salting and the conditions and duration of ripening/ageing. All of these factors, which contribute toward the ultimate composition, body, taste and texture of the cheese, profoundly impact the fate of STEC or indicator organisms within a finished cheese product. Due to the large variety of raw milk cheese types and the different technologies used in their production, lactic cheeses will be split into soft, semi-hard or hard cheese varieties where applicable; as each of these categories have different curd warming (cooking) temperatures (not to be confused with heat treatment of milk), acidification conditions and ripening periods (Annex 4). The diverse technologies used in making raw milk cheeses will have variable effects on strains of STEC and E. coli in cheeses. These impacts have been studied using artificial challenge studies in laboratory scale pilot plants, which poses a clear limitation to the supporting evidence for actual production situations. Furthermore, the STEC strains were often inoculated at concentrations higher than those found in naturally contaminated milk, thereby, raising further concerns about applicability of these data. STEC populations can grow exponentially under certain conditions during the first hours of the cheese manufacturing process. As a consequence, the quality of raw milk used is essential to ensure the safety of raw milk cheeses. Two physicochemical factors can be used to inhibit the growth of STEC during the first hours of cheese manufacture: rapid acidification and high temperature, both of which can reduce bacterial cell numbers observed during cheese ripening. 5.2.1. Milk fermentation The microbial ecosystems of raw milk can have a protective effect or inhibit the growth of some pathogenic and spoilage microorganisms (Quigley et al., 2013). Lactic acid bacteria (LAB) can be used as biocontrol agents against foodborne CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 86 pathogens. LAB produce organic acids which reduce pH and also antimicrobial substances including H2O2, diacetyl and bacteriocins (Dal Bello et al., 2010), but the evidentiary support for the use of LAB to reduce STEC in raw milk was low. The degree of support for the use of LAB as starter cultures as a processing intervention specifically for the control of STEC in raw milk cheese was low. 5.2.2. Protective cultures Protective bacterial cultures may be used to reduce or eliminate pathogens in cheeses. However, very few protective cultures are currently marketed for cheese production, underlining the difficulty of developing effective protective cultures for the cheese industry. Additionally, the non-specific nature of their antimicrobial activity indicate that the use of protective cultures can affect the activity of desired cheese flora, starter cultures, ripening bacteria, yeasts and molds (Gensler et al., 2020), which thereby, impacts the sensory quality of the product. A cocktail of Hafnia alvei, Lactobacillus plantarum and Lactococcus lactis reduced STEC O26:H11 and STEC O157:H7 populations by up to 2 log10 CFU/g in pasteurized and raw milk cheeses when inoculated into milk at 102 CFU/mL (Callon, Arliguie and Montel, 2016). For cheeses made from different raw milk batches inoculated with STEC O26:H11 at very low concentrations (0.5 and 0.05 CFU/mL) that closely simulated natural contamination levels, the protective culture cocktail reduced STEC levels (average of 2.8 log10 CFU/g) in all cheeses. Differences in the growth and inhibition of E. coli in the cheeses depended on the natural microbial composition of the raw milk batches. Further research utilizing metagenomics and transcriptomic approaches should improve our understanding of the interactions between the endogenous milk microbiota and STEC contamination (Fretin et al., 2020). The efficacy of protective culture appears to be strain-dependent and further experimental challenge studies using raw milk cheeses along with actual production-relevant studies are needed to evaluate the potential of using live cultures as an STEC intervention. The degree of support for the use of protective cultures as a processing intervention specifically for the control of STEC in raw milk cheese was low. 5.2.3. Bacteriophage The effect of adding bacteriophage during fermentation in the making of cheeses has been examined. Milk samples were inoculated with E. coli and STEC (including STEC O157:H7) and treated with an E. coli-specific phage cocktail that CHAPTER 5 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESES 87 did not inhibit starter cultures. The phage cocktail completely inactivated E. coli and STEC O157:H7 strains after 8 h, but only reduced other STEC strains by < 1 log10 CFU/mL (Tomat et al., 2013). No sensory changes were associated with the use of bacteriophage; however, the time and temperature needed for optimal phage lytic activity is not well suited to the cheese making process. There is also STEC inter-strain variation in response to phage treatment, as well as concerns about the emergence of phage-resistant strains or bacteriophage insensitive mutants (BIMs). The degree of support for the use of bacteriophage as a processing intervention specifically for the control of STEC in raw milk cheese was low. 5.2.4. Acidification, salting and cooking Rapid acidification and high temperature inhibit the growth of STEC during the early stages of cheese manufacturing. A long acidic coagulation step (pH <4.5 for 24 h) involving lactic acid production prevented growth of STEC O26, O103, O145 and O157 and reduced their concentrations to below enumeration limits. However, acidification resulting from enzymatic coagulation was not sufficient to prevent the growth of STEC when the pH did not fall below 5 (Miszczycha et al., 2013). For salting, sodium chloride stress caused differences in transcriptional induction, which were associated with the survival of phenotypes of E. coli strains in cheese. The E. coli strain that lacked significant induction in the three salt-stress response genes investigated survived poorly in cheese compared to the other E. coli strains (Peng et al., 2014). For making hard and extra-hard cheeses, curd cooking temperatures of 53 °C or higher applied to remove water from the curd grains, also rapidly reduced STEC and E. coli populations (~2–4.5 log10 CFU/g of thermotolerant E. coli), even when very high bacterial levels were present before cooking (Ercolini et al., 2005; Miszczycha et al., 2013; Peng et al., 2013b). An E. coli strain carrying the “locus of heat resistance” is more thermotolerant and survived curd cooking well as compared to a strain without this gene cluster. However, a thermotolerant E. coli strain carrying the “locus of heat resistance” was decreased by 4 log10 CFU/g to < LOD in 24-h-cheese, indicating that the combination of several other stresses inhibited this strain as well (Peng et al., 2013b). The degree of support for the use of acidification, salting and cooking as a processing intervention specifically for the control of STEC in raw milk cheese was low to medium. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 88 5.2.5. Ripening and ageing The ripening step of soft cheeses can reduce STEC populations, and reductions were higher for STEC O157:H7 than for strains of STEC O26, O103 and O145 (Perrin et al., 2015; Miszczycha et al., 2016). However, STEC was not eliminated completely by ripening, and different STEC serogroups displayed variations in acid-resistance and survived the ripening process of soft cheeses (Montet et al., 2009). For uncooked, pressed cheeses, the duration of ripening impacted STEC survival. A short ripening period (40 days) did not promote a significant reduction in water activity (aw), or a reduction of STEC populations. In contrast, when aged for 240 days, the aw decreased under the minimum value of 0.95 resulting in reduction, but not total elimination of several STEC serotypes (Miszczycha et al., 2013). Similarly, STEC reduction and survival was proportional to the duration of ripening in “bleu” type cheeses, where after 240 days, STEC O26, O103 and O157 were either not detected or below the enumeration limit (Miszczycha et al., 2013). A combination of factors during the ripening step (e.g. temperature, low aw of 0.898, acidic pH and the presence of antagonistic microorganisms) likely helped to reduce STEC populations in the end products. Semi-hard cheeses are comprised of a large variety of types, so risk assessment- based challenge studies are required to examine the effects of various specific manufacturing processes on STEC. Approximate reductions of ≥ 1 log10 CFU/g of STEC or E. coli per month was observed during ripening of semi-hard cheeses (Miszczycha et al., 2013; Peng et al., 2013a, 2013b). Bacterial populations decreased more rapidly in the cheese core which had higher maturation temperatures as compared with the rind. It has been speculated that differences in CO2 partial pressure in the cheese core also contributed to faster decrease of STEC or E. coli populations (Peng et al., 2013b). The degree of support for the role of ripening and ageing during cheese production as a processing intervention specifically for the control of STEC in raw milk cheese was low to medium. 5.2.6. Cheese size STEC or E. coli are rarely detected in hard cheeses after cooking at ≥ 53 °C for 30 min. But, STEC could still be detected in the rind zone (although only after culture enrichment), as this area cools down most rapidly compared to the core zone. The size of a cheese block, therefore, contributes to differences in temperature profile and to the survival of pathogens. During ripening, a thermotolerant E. coli strain was detected in a number of samples (Peng et al., 2013b), including at the CHAPTER 5 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESES 89 end of the ripening period. However, the inoculation level, size (8 kg instead of 35 kg for commercial cheese) and the conditions used (53 °C instead of up to 57 °C cooking temperature) in this model cheese production study were more favorable for the survival of E. coli. In commercial production, the potential of E. coli surviving until the end of ripening in cooked hard raw milk cheese is expected to be low (Peng et al., 2013b). Ercolini et al. (2005) developed a curd-cooling model where 55 °C cooking temperature was used for cooked hard cheese made from raw milk. Variation in temperature across the young cheese which was combined with a challenge test using different pathogens including STEC O157:H7, led to conclusions that pathogens in the cheese core are eliminated by cooking at 55 °C, but may survive in the more favorable thermal conditions on the crust (absent other pathogen reduction factors), as the rind cools more rapidly than does the core. Therefore, the size of a cheese block impacts the temperature profile and the survival of pathogens (e.g. the larger the cheese, the lower the ability of survival in the core of cooked cheeses). However, other factors such as acidification, long ripening period and high NaCl concentration of the crust contributed to the reduction of pathogens in this part of the cheese. Semi-hard cheeses with cooking temperatures of 40 °C and 46 °C (which allows growth of E. coli) had a significantly higher decrease in E. coli populations in the core than in the rind (Peng et al., 2013a). This has clear implications on the outcome of STEC challenge studies, hence studies should simulate as closely as possible the size of the actual product made by the industry. If the cheese size used in the challenge test is too small, the results would reflect more the conditions that STEC would experience in the cheese rind, but not those in the core of an actual cheese product. The degree of support for the size fo the cheese block as a processing intervention specifically for the control of STEC in raw milk cheese was low. 5.3. RAW MILK CHEESE POST-PROCESSING 5.3.1. Packaging Active packaging is used in the food industry primarily to extend shelf-life, but it can also prevent pathogen growth (Yildirim et al., 2018). Although not as effective against STEC O157:H7, the use of technology such as modified atmosphere packaging can retard the growth of other pathogenic bacteria (e.g. L. monocytogenes and S. aureus) in hard cheeses made with raw sheep’s milk (Solomakos et al., 2019). CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 90 However, the application of active packaging in cheese is still limited and further studies are needed to explore the potential and efficacy of these technologies (Speranza et al., 2020; Al-Moghazy, Mahmoud and Nada, 2020). The degree of support for the use fo active packaging as a post-processing intervention specifically for the control of STEC in raw milk cheese was low. 5.3.2. Irradiation The efficacy of eBeam to control pathogens on the surface of raw milk cheeses was evaluated using L. monocytogenes inoculated onto the surface of Camembert and Brie. Samples packaged under vacuum and irradiated with eBeam doses of 1.27 and 2.59 kGy controlled the growth of L. monocytogenes. No significant differences were noted in the sensory attributes of the Camembert samples treated with doses up to 2.59 kGy (Velasco et al., 2015). For cheese slices, other technologies such as X-ray, UV-C irradiation, and pulsed-light can be applied to reduce pathogens, including STEC O157:H7 (Park and Ha, 2019; Proulx et al., 2015; Ha et al., 2016). The degree of support for the use of irradiation as a post-processing intervention specifically for the control of STEC in raw milk cheese was medium. 5.3.3. Bacteriophage In a limited study, bacteriophages were found to be not very effective in reducing STEC levels in finished cheese products. The study used 16 cm2 pieces of cheese seeded with STEC O157:H7. Treatment with phages only showed < 0.15 log10 CFU/g reduction, suggesting that phages were not very effective in controlling STEC in the finished cheeses (Hong, Pan and Ebner, 2014). It was suspected that perhaps the low pH of cheese (~5.5 to 6.8) inhibited phage function, but ineffectiveness of the phage treatment is most likely due to other undetermined factors. The degree of support for the use of bacteriophage as a post-processing intervention specifically for the control of STEC in raw milk cheese was low. CHAPTER 5 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESES 91 References Al-Moghazy, M., Mahmoud, M. & Nada, A. A. 2020. 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97 6 Primary production and processing control strategies for STEC in other animal species 6.1. SMALL RUMINANTS: PRIMARY PRODUCTION Similar to cattle, other ruminant animals such as sheep and goats can harbour STEC, which can be transmitted through faecal contamination to food products derived from these animals (e.g. lamb, mutton, milk, and cheese). Although some STEC serotypes associated with human disease have been detected in sheep, goats and wild game they often carry a wide variety of non-O157 STEC serotypes that are well adapted for colonizing these specific animal species (Furlan et al., 2019; Jacob et al., 2013). While the carriage of STEC O157:H7 is less frequent, small ruminants, sheep and goats are considered to be the second most important source of human STEC infection after cattle (FAO and WHO, 2019). Because of their adaptability and low maintenance costs, sheep and goats are raised in diverse environments and conditions around the world, which creates challenges when applying STEC control interventions globally or systematically. Relative to the amount of literature available for cattle, there is much less information on the application of interventions during primary production, processing and post-processing of products from sheep and goats. It is predicted that most GAP and GHP that are used in cattle milk or meat production will be similarly impactful in goats and sheep. For example, mixed species (sheep, deer and cattle) in the same pasture resulted in STEC transfer and increased carriage of STEC O157:H7 in sheep as well as in cattle (Section 2.2.1), and ionophore feeding CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 98 did not impact on STEC O157:H7 populations in sheep – a similar result to that observed in in cattle (Section 2.4.4) (Edrington et al., 2003). While there are expectations that different farm level interventions used to reduce STEC during cattle production may also be effective in sheep and goat at the primary production stages, there are few studies that have validated their effectiveness on a commercial scale for reducing STEC in small ruminants, hence the low degree of support for most interventions in small ruminants. Interestingly, many of the studies examining interventions meant for cattle utilized (at least initially) sheep and goats in experimental infection studies with promising results. 6.1.1. Diet composition and feeding strategies As observed in cattle, dietary components and changes in ration can influence STEC shedding in sheep. For example, sheep fed a grass hay diet shed STEC O157:H7 for twice as long a period as sheep fed a ration low in protein and digestible energy (Kudva et al., 1997). Largely because of the limited amount of available data, the degree of support for the use specific diets as an intervention specifically for the control of STEC in small ruminants was low. 6.1.2. Feed additives Probiotics A variety of probiotics, composed of lactic acid bacteria previously described for cattle (Section 2.3.4.1), have also been used in research trials in lambs and sheep to reduce STEC O157:H7 and non-O157 STEC excretion and prevalence (Rigobelo et al., 2015). The degree of support for the addition of probiotics to feed as an intervention specifically for the control of STEC in small ruminants was low. Bacteriophage A number of studies have investigated the potential of bacteriophage therapy to reduce STEC O157:H7 in sheep. Most of the reports have shown that bacteriophage can reduce intestinal carriage of STEC O157:H7 (Wang et al., 2017; Raya et al., 2011). In the rumen, reductions in STEC O157:H7 counts was not statistically different between phage treated and untreated sheep. However, bacteriophages applications significantly reduced STEC O157:H7 counts in the lower intestinal tract (up to 4 log10 CFU) with no adverse effects (Callaway et al., 2008). The degree of support for the addition of bacteriophage to feed as an intervention specifically for the control of STEC in small ruminants was low. CHAPTER 6 – PRIMARY PRODUCTION AND PROCESSING CONTROL STRATEGIES FOR STEC IN OTHER ANIMAL SPECIES 99 Lactoferrin The use of lactoferrin, an immunomodulatory protein found in milk, in the prevention of STEC O157:H7 colonization and excretion in sheep has also been studied. In one study, lactoferrin, administered orally to sheep, reduced STEC O157:H7 counts (up to 5 log10 CFU) and the duration of excretion as compared to untreated controls (Yekta et al., 2011). The degree of support for the administration of lactoferrin as an intervention specifically for the control of STEC in small ruminants was low. Sodium chlorate Feeding sodium chlorate to sheep before transport to the abattoir (a period of 24 h prior to slaughter) reduced inoculated STEC O157:H7 levels in the rumen, caecum, and colon by 1-4 log10/g digesta (Callaway et al., 2003). Results showed that treatment reduced STEC populations throughout the gut; yielding results that were very similar in scope and scale to those found from cattle (Section 2.3.4.4). The degree of support for the addition of sodium chlorate to feed as an intervention specifically for the control of STEC in small ruminants was low. 6.1.3. Vaccination Previous experimental challenge studies of vaccinated goats with STEC O157:H7 showed promising results, but only small numbers of animals were used, so it is still experimental. In another study, goats inoculated with a STEC vaccine based on Stx2B-Tir-Stx1B-Zot protein and recombinant H7-HCP-Tir-Intimin proteins, significantly elicited Stx2b-Tir-Stx1b-Zot-specific serum IgG antibodies. When these vaccinated goats were challenged with STEC O157:H7, they showed reduced STEC O157:H7 excretion (Zhang et al., 2014). The degree of support for the use of vaccines as an intervention specifically for the control of STEC in small ruminants was low. 6.1.4. Feed withdrawl prior to slaughter In the United States of America, sheep and goats can be held off feed for up to 24 h to reduce gut fill and hide/pelt contamination; but fasting is thought to cause an increase in STEC population in sheep and goats similar to what occurs in cattle (Section 2.6.1). Withdrawing feed from sheep and goats for 12 h prior slaughter reduced NTS E. coli, Enterobacteriaceae and total coliform levels in the rumen CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 100 as compared to animals that were fasted for 24 h (Gutta et al., 2009; Pointon, Kiermeier and Fegan, 2012). The degree of support for the use of feed withdrawal prior to slaughter as an intervention specifically for the control of STEC in small ruminants was low. 6.2. SMALL RUMINANTS: PROCESSING (MEAT AND DAIRY) Similar to cattle, the main sources of STEC in sheep and goat carcasses are hides/fleeces contaminated with intestinal faecal material. Controlling faecal contamination by ensuring that only clean animals are slaughtered while at the same time preventing or reducing faecal content transfer to sheep and goat carcasses during slaughter, are acceptable GHP that can also reduce STEC and other bacteria (EFSA, 2013). Wool/pelt treatments have been applied to reduce carriage of STEC into the processing facilities, but the mechanism of spraying is different than that used on cattle due to the differences between cattle hides and sheep pelts for carriage of faecal material. When sheep carcasses are processed, the pelt is removed by a slightly different process than that of cattle. Through a process known as "fisting", which may be performed by a machine. The pelt is separated from the felt membrane which is left on the carcass to prevent shrinkage due to dehydration. In commercial sheep and goat processing, typically the carcasses are not split or ribbed as done for cattle. There were no other significant differences identified in the procedures utilized in sheep and goat meat processing, but some studies on carcasses treatments during pre-chill were evaluated. 6.2.1. Pre-chill carcass treatments Hot water, lactic acid, and other organic acids have been used as sprays on sheep and goat carcasses to effect STEC reductions broadly similar to cattle (Section 3.4) and it is possible to achieve acceptable E. coli log10 CFU reductions. Hassan et al. (2015) reported a mean reduction in NTS E. coli of 1.1 log10 CFU/cm2 on sheep and lamb carcasses treated with steam vacuum pasteurization (>82 °C for 10 s) after trimming. Another study achieved greater than 3 log10 CFU/cm2 reductions of NTS E. coli on uninoculated and inoculated sheep carcasses by submersion or spraying hot water and steam (80 °C) or a combination of hot water and antimicrobials (Dorsa, Cutter and Siragusa, 1996). Furthermore, using antimicrobials such as lactic acid, peroxyacetic acid, a hydrochloric and citric acid blend, and levulinic acid plus 0.5 percent sodium dodecyl sulfate, on goat carcasses during slaughter CHAPTER 6 – PRIMARY PRODUCTION AND PROCESSING CONTROL STRATEGIES FOR STEC IN OTHER ANIMAL SPECIES 101 and subsequent chilling was effective in reducing STEC with mean reductions between 0.47–2.26 log10 CFU/cm2 (Thomas et al., 2019). The degree of support for the use of pre-chill carcasee treatments as interventions specifically for the control of STEC in small ruminants was low. 6.2.2. Dairy processing Harvesting and processing milk from small ruminants is not markedly different from cattle, other than the physical differences in the processes depending upon the number of teats and the usual reliance on hand milking in small ruminants rather than automated systems. The use of raw milk for the production of cheese and other dairy products is similar to that for raw cows’ milk. 6.3. OTHER SPECIES: PRIMARY PRODUCTION AND PROCESSING 6.3.1. Reindeer Reindeer (Rangifer tarandus) are primarily used for meat production, as the use of milk from reindeer is uncommon. STEC and the presence of stx genes have been identified in reindeer (Zweifel et al., 2017; Magwedere et al., 2013; Miko et al., 2020). Processing of reindeer occurs at specific reindeer slaughter facilities or by traditional field methods. Most reindeer are slaughtered at 6–7 months of age and are transported long distance via specialized trucks. The process and hygiene practices of reindeer slaughter are similar to that of cattle or sheep. In Europe, the slaughter and processing of reindeer and other farmed wild game animals is legislated (Reg. [EC] No. 852/2004 and Reg. [EC] No. 853/2004) (Laaksonen et al., 2017). GAP and GHP are suggested during reindeer primary production or meat processing; however, there was limited available evidence to support interventions specific for the control of STEC in reindeer or reindeer meat processing. 6.3.2. Yaks Yaks (Poephagus grunniens or Bos grunniens) live at high altitude (above 3 000 m) in China, India, Nepal and other countries and are used for both meat and milk production (Rehman et al., 2017; Bandyopadhyay et al., 2009; Bai et al., 2013). Yaks are a natural source of STEC when raised under migratory or free ranging CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 102 systems. The transmission of STEC O157:H7 and non-O157 STEC from yak happens through meat, unpasteurised milk, or direct and indirect contact with humans. This occurs primarily in the nomadic herdsmen who often consume raw or undercooked yak meat, milk and milk products (e.g. "churpi", a dried and smoked hard cheese made from yak milk) (Bandyopadhyay et al., 2012). Primary production control strategies are focused on the production of meat and dairy products from the farm through transport to processing facilities, or until milk reaches the bulk tank for pasteurization or for inclusion into making raw milk cheeses. GAP and GHP are suggested during yak primary production or meat and milk processing; however, there was limited available evidence to support interventions specific for the control of STEC in yaks and yak meat and dairy processing. 6.3.3. Camel Camel production is focused mostly in arid regions and knowledge of their production parameters is quite limited compared with other domestic animals. Camels can carry STEC, and the animals are used for both meat and raw milk production (Baschera, Cernela and Stevens, 2019; Salehi et al., 2012). The life pattern of camels in the desert minimizes the contact of camels with other animal species that can be STEC reservoirs. However, during the dry season, camels can be reared along with cattle, sheep, and goats (along with exposure to birds and other wildlife) which poses increased STEC transmission risk to the camels. Camels shed STEC in their faeces (Tabatabaei et al., 2013), and shedding is the highest during the wet season when feed intake (and faecal output) is increased (Adamu et al., 2018). GAP and GHP are suggested during camel primary production and meat and milk processing; however, there was limited available evidence to support interventions specific for the control of STEC in camels or camel meat and dairy processing. 6.3.4. Water buffalo Water buffalo (Bubalus bubalis) are typically used as draft animals and also used for milk production - primarily for making cheeses (including raw milk cheeses) and yogurt. Water buffalo are also used for meat production in Europe and Asia. Several studies have demonstrated that domestic water buffalo are common STEC reservoirs and can be colonized by a diversity of STEC serogroups possessing stx subtypes associated with severe disease in humans. STEC in buffalo can be CHAPTER 6 – PRIMARY PRODUCTION AND PROCESSING CONTROL STRATEGIES FOR STEC IN OTHER ANIMAL SPECIES 103 transmitted to humans via meat, and unpasteurised milk and cheeses (Lorusso et al., 2009; Vu-Khac and Cornick, 2008). Currently, no specific STEC interventions are applied or suggested to reduce STEC in water buffalo during primary production through to product consumption. However, to control STEC contamination in raw buffalo milk intended for the production of mozzarella cheese, some limited studies demonstrated that heating curd during stretching produced a reduction of STEC (Trevisani, Mancusi and Valero, 2014). Other studies have shown that use of hot wash water reduced initial bacterial load on carcasses substantially and improved the microbiological quality of buffalo meat (Sachinda, Sakhare and Rao, 1998). Similarly, aerobic plate counts on water buffalo meat product was reduced by lactic acid spray (Manzoor, Jaspal and Yaqub, 2020). GAP and GHP are suggested during water buffalo primary production and meat and milk processing; however, there was limited available evidence to support interventions specific for the control of STEC in water buffalo or water buffalo meat and dairy processing. 6.3.5. Bison Bison (Bison bison) are large ruminants that typically roam freely mainly in the United States of America and Canada. They are primarily fed on hay or grass and are slaughtered at about 18 months of age. In the United States of America, bison are considered an exotic species, so the federal inspection of the slaughter process is voluntary. There are some commercial feedlots for finishing bison in Canada, but little information exists on STEC carriage in these animals. Similar to other ruminants, bison can be colonized by STEC O157:H7 and non-O157 STEC (O121, O145) (Reinstein et al., 2007; Magwedere et al., 2013). In the United States of America, ground bison meat has been implicated in a multistate outbreak of STEC non-O157 (O103 and O121) (FDA, 2019). GAP and GHP are suggested during bison primary production and meat processing; however, there was limited available evidence to support interventions specific for the control of STEC in bison or bison meat processing. 6.3.6. Wild game Wild game (deer, wild boar and hare) are reservoirs for STEC serotypes linked to human illness. Currently, no specific interventions against STEC have been validated for wild game small ruminants. However, it is thought that GHP and various processing/post-processing interventions that have been used to reduce CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 104 NTS E. coli and STEC levels on farms and along the cattle food chain are most likely to be effective against STEC that colonise wild game (Miko et al., 2020). Use of GHP is suggested during wild game meat processing; however, there was limited available evidence to support interventions specific for the control of STEC in wild game meat processing. 6.3.7. Swine Several studies have demonstrated that domestic swine can carry and shed STEC and it may serve as vectors in human STEC outbreaks linked to fresh produce and other row crops (e.g. lettuce and spinach). Many studies worldwide have identified a low prevalence of STEC O157:H7 in swine, however results are conflicting (Tseng et al., 2014; Magwedere et al., 2013). In some studies, the prevalence of non-O157 STEC was high and included serogroups associated with severe disease in humans. However, many of these STEC carried the stx2e subtype which may cause edema disease in swine, but so far, has not been associated with severe disease in humans. Pork products have occasionally been confirmed as vehicles of STEC transmission (Mughini-Gras et al., 2018), but it remains unknown whether the STEC contamination on pork was natural or came from processing or via cross- contamination from other foods (Colello et al., 2016). In swine production, the entry of STEC into swine herds may be limited by appropriate biosecurity measures (e.g. good feed hygiene, keeping swine herds separate from wild and production animals). During processing and post- processing of pork, there are no practices specific to control STEC, but all of the GHP used in pork production are thought to be similarly effective against STEC as they are against Salmonella (Colello et al., 2016). General interventions throughout the pork production chain described in the FAO/WHO report; “Interventions for the control of non-typhoidal Salmonella spp. in beef and pork” (FAO and WHO, 2016) are also expected to be effective against STEC. 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111 Laboratory testing 7.1. LABORATORY TESTING FOR STEC DURING PRIMARY PROCESSING The expert committee concluded that the implementation of a monitoring plan at the cattle farm level to measure the impact on STEC prevalence in raw beef is impractical. This conclusion is based on several factors, including: • Excretion of STEC in faeces is highly intermittent, so the value of predicting risk from the collection of a single faecal sample is questionable. • Super shedders are likely responsible for the majority of STEC transmission within the herd, but currently, there is no reliable method for the rapid detection of super shedder cattle. • The degree of STEC excretion varies substantially among individual animals, hence, in order to gain a true measurement of prevalence, it will be necessary to sample single animals individually as opposed to collecting composited faecal samples from the pen. • Detecting all the possible STEC serogroups simultaneously in cattle has proven to be extremely challenging. At present, there is no test that can detect all the STEC serogroups other than the “top seven” serotypes that may be present in a faecal sample collected from an animal. • Some available STEC detection methods are relatively sophisticated and not easily implemented on a farm. Consequently, a significant investment for a centralized laboratory infrastructure and trained personnel will be required to support on farm monitoring. 7 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 112 • Testing a single faecal sample for STEC is inadequate and testing multiple faecal samples from a single animal would be extremely costly. The need to repeatedly restrain the animal to collect multiple faecal samples would raise animal welfare concerns. • If an animal was found to be STEC positive, it would be difficult to manage it within the production chain, as delaying slaughter would substantially increase production costs and will likely require isolating the STEC positive animal by housing it away from the main herd to reduce the risk of STEC transmission. Such a practice could become costly and adversely impact animal health and productivity and meat quality. • Even if the animals were found to be STEC negative at the time of sampling, it would not be advisable to reduce processing and post-processing intervention strategies, as many of these practices are broad-based and intended to prevent a variety of pathogens from entering the food chain. 7.2. LABORATORY TESTING FOR STEC DETECTION ACROSS THE BEEF PROCESSING CHAIN It is clear that in general, the occurrence of STEC in meats is lower for intact meat products than in trim or ground/minced beef (Kintz et al., 2017; Devleesschauwer et al., 2019). Intact meats originate from a single animal, whereas ground/minced beef is composed of meat pieces from many animals, so one piece of meat contaminated with STEC can spread the contamination to the entire batch. However, the overall occurrence of STEC in these products can vary considerably due to the differences in primary, processing and post-processing conditions and the interventions applied. As a result, it is difficult to set STEC criteria for laboratory analysis for all possible conditions. Laboratory testing to monitor for STEC at beef processing and post-processing stages depends on a country’s regulation or export requirements and the testing methods used may also vary. For example, there are methods that use a stepwise approach to first screen for Shiga toxin genes (stx) and the E. coli attaching and effacing gene (eae), followed by testing for major O serogroup–specific genes (e.g. USDA, 2021 ISO/TS 13136-2012). A separate method is used to only test for STEC O157:H7 (e.g. ISO 16654:2001). According to the JEMRA report on STEC risk characterization, testing for STEC virulence genes (stx, eae and aggR), regardless of the serogroup, was recommended as the serotype does not necessarily predict the virulence profile (FAO and WHO, 2018). For reliable STEC detection in food, methods accredited or validated by independent or official organizations are required. The many different STEC CHAPTER 7 – LABORATORY TESTING 113 detection methods include microbiological culture enrichment, selective/ chromogenic media, immunological Enzyme linked immunosorbent assay (ELISA), Immunomagnetic separation (IMS), Lateral flow, Latex agglutination, Microplate enzyme immunoassay (EIA), Optical immunoassay, Reverse passive latex agglutination (RPLA), and molecular (polymerase chain reaction [PCR], Real-time PCR, Loop-mediated isothermal amplification PCR [LAMP], digital droplet PCR [ddPCR]) methods. Many, but not all of these methods have been validated. Culture enrichment is a critical step in most STEC detection methods and an indispensable requirement in order to detect the low numbers of STEC that maybe present amidst the high-level of background cells in meats and other foods. Improved selective media formulations that facilitate STEC enrichment can decrease the sample-to-result time and improve sensitivity. Improved enrichment procedures coupled with screening tools (e.g. Shiga toxin EIA, PCR, LAMP) can be used to obtain definitive negative or presumptive positive results more rapidly. At present, more culture media to enhance the enrichment of STEC O157:H7 have been developed (Bai and Xiong, 2019) than for non-O157 STEC strains (Conrad et al., 2016; Parsons et al., 2016). Further work is required to develop and validate enrichment procedures for all STEC serogroups of health concern (Brusa, Piñeyro and Galli, 2016; Castro et al., 2017; NACMCF, 2019). The use of assays, such as PCR, that target STEC virulence gene in foods is recommended to, as they demonstrate both high sensitivity and specificity, and are also fast, low cost and commercially available. Currently, real-time PCR, LAMP and dd-PCR that target stx and/or eae genes are commercially available and some have been validated. Most stx PCR assays can detect stx subtypes often associated with severe disease, but will miss genetically more distant stx subtypes, some of which have been associated with severe human disease (Paton and Paton, 1998; Feng et al., 2011; Reischl et al., 2002; Beutin, Jahn and Fach, 2009; Scheutz et al., 2012). It is also important to realize that bacteria other than STEC may harbour some of these virulence genes. Furthermore, the mere presence of a virulence gene may not be reflective of health risk due to differential or lack of stx gene expression. Subtyping the stx gene variants is also important in order to discriminate subtypes most often associated with human disease from those that may not cause human infections (Scheutz et al., 2012; Staten Serum Institute, 2014; FAO and WHO, 2018). Isolation of the STEC organism from presumptive positive samples is a requirement for confirmation and this is often performed with traditional culture-based methods. Not all presumptive positive samples can be confirmed, but the use of IMS techniques have greatly improved the isolation of STEC from enrichment CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 114 cultures. However, IMS is currently available only for the eae positive STEC serogroups commonly associated with human diseases. Furthermore, IMS are very serogroup-specific, but some IMS assays lack target specificity, and may capture strains from more than one serogroup or fail to identify other STEC serogroups (Kraft et al., 2017). Immunoblot and colony hybridization protocols have recently been developed that can enhance and improve STEC isolation from a variety of food matrices. Testing food for STEC as part of monitoring programmes is of limited use due to the low level of STEC found in foods. Hence, the quantitative detection of NTS E. coli (ISO 16649-2) as a process hygiene indicator, is proposed as an alternative approach to monitor hygiene during processing and post-processing. NTS E. coli counts can vary considerably from sample to sample or plant to plant, so internal company standards or specific criteria recommendations are not possible for all meat products. The criteria for decision making based on hygiene indicator levels can vary depending on the pre-defined limit set and the sampling plans implemented, but they could still be useful for trend analysis with regard to STEC surveys and for STEC baseline studies (ICMSF, 2011). Several novel technologies (e.g. LAMP, recombinase polymerase amplification [RPA], PCR-mass spectrometry, whole genome sequencing [WGS]), are being explored for STEC detection (Pierce et al., 2012). WGS for STEC detection and typing uses different high throughput sequencing platforms (Illumina, Oxford Nanopore technology, Ion Torrent and Pacific Biosciences [PacBio]) but is becoming increasingly common and accepted (Worley et al., 2017; Allard et al., 2018; Wilson et al., 2018; Mylius et al., 2018; González-Escalona et al., 2019; Franz et al., 2014). However, issues associated with the implementation and long-term sustainability of WGS, such as cost, equipment, maintenance, supplies, IT, training, are limitations for many countries. Further work is needed to develop rapid and standardized analysis protocol for the various WGS methodologies so that the data are comparable and these methods also need to be validated. Additional experimental data are also needed on these (and other) novel technologies to support their potential application as methods for the identification and characterization of STEC. Biosensors (Subramanian et al., 2012; Pandey et al., 2017), mass spectrometry (MALDI-TOF MS; Pierce et al., 2012), and nanotechnology (Jyoti et al., 2010) are examples of emerging future technologies that may be explored for detection and characterization of STEC from foods. CHAPTER 7 – LABORATORY TESTING 115 7.3. LABORATORY TESTING FOR STEC DETECTION ACROSS THE DAIRY PROCESSING CHAIN Sampling and analysis of raw milk and raw milk products are important steps within the verification plans to confirm that the practices and procedures implemented in the food safety program have been met. 7.3.1. Raw milk Although STEC has been isolated from raw milk, STEC testing of milk is uncommon and most sampling and testing protocols target indicator organisms such as E. coli. Whereas the presence or concentration of NTS E. coli or other indicator organisms in raw milk is not indicative of the presence of STEC, they remain useful hygienic markers of the quality of raw milk (Metz, Sheehan and Feng, 2019). Sampling and testing plans for raw milk are highly dependent on consumption practices, the scale of production, local vs regional regulations, and as such, are highly variable between countries and across regions. At the farm level, given the impracticality of sampling milk from each producer or from every animal daily, it is essential to use a well-designed sampling program that can reduce cost and time but also provide sufficient data to adequately assess the hygiene of milk in bulk tanks. Depending on the size of the milk collection operation (small-, medium- or large-scale) different sampling approaches are available, including periodic, random, composite and universal sampling. Sampling may be done once a week or once every two weeks or done periodically at irregular intervals. Raw milk can also be sampled and tested on a random basis (random sampling), or samples may be pooled together over a period of time (composite samples) and tested. For large-scale operations, a universal sampling system is used by bulk milk haulers every time raw milk is picked up at the farm and collected. Aliquots of the universal milk sample are sent to an approved laboratory for analyses (FDA/USPHS, 2017). Entry of STEC via contaminated raw milk into dairy food processing plants can lead to persistence of pathogens in biofilms, exposure of consumers to STEC in unpasteurized dairy products as well as subsequent contamination of other processed milk products (Oliver, Jayarao and Almeida, 2005). Thus, samples from valves, equipment, filters and environmental sources are routinely collected at plants for microbiological analysis. The universal sampling system permits the competent authority, at any given time and without notification to the industry, to analyze samples collected by the bulk milk hauler/sampler and/or industry plant sampler at the farm and milk processing plant, respectively (Oliver, Jayarao and Almeida, 2005). CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 116 Although most testing has focused on sampling of bulk tank milk, studies have also assessed the utility of sampling milk filters. Jaakkonen et al. (2019) conducted a 1-year longitudinal study on the presence of STEC and Campylobacter jejuni on Finnish dairy farms and in raw milk. STEC O157:H7 was isolated from 17 percent of the cattle, but from only 2 percent of milk filters and not from any samples of raw milk. However, the stx gene was detected at a higher frequency from milk filters (37 percent) than in raw milk (7 percent), suggesting that the filters may be a more effective sampling point than raw milk. Artursson et al. (2018) also observed that in-line milk filters were a better sampling point for the presence of pathogens in general than sampling the milk in the bulk tanks. Some of the logistical limitations to bulk tank and plant sampling include: the duration of agitation to ensure adequate mixing, the need to use aseptic sampling techniques and the need to use disinfected sampling equipment and containers. Additional factors to consider include, sample preservation methods, storage temperatures (raw milk should be cooled to 7 °C or less within 2 h after the completion of milking) and the collection of appropriate information, which are necessary to ensure proper sampling, storage, transportation and identification/tracing of the samples. 7.3.2. Raw milk cheeses The microbiological safety of raw milk cheese is managed by the effective implementation of control measures that have been validated, where appropriate, to minimize contamination from the milking process through to the maturation of the cheese. This preventative approach is more effective than relying solely on microbiological testing of individual final product lots for market acceptance. However, setting microbiological criteria may be appropriate for verifying that food safety control systems were implemented correctly (FAO and WHO, 2013). The established microbiological criterion should be based on risk assessment taking into considerations such factors as epidemiological evidence. The criterion should be meaningful for consumer protection, so if a criteria is not met, the cheese in question may represent a significant public health risk. Consideration should also be given to the behaviour of STEC during the cheese- making and maturation process, because STEC prevalence changes over the course of manufacturing, distribution, storage, marketing and preparation. There are strain to strain variations, and some pathogenic strains of STEC may be more acid tolerant than NTS E. coli and persist when the indicator has died off. So, any microbiological criterion set must be established at a specified point in the food chain (FAO and WHO, 2013). Consideration needs to be given to sampling plans that can effectively remove highly contaminated lots and result in continuous improvement without CHAPTER 7 – LABORATORY TESTING 117 completely disrupting the food supply (ICMSF, 2001). Perrin et al. (2015) conducted a risk assessment of soft cheeses made from raw milk and considered the effect of applying various microbiological criteria at the end of ripening. They found that various criteria (e.g. differing in terms of sample size, the number of samples that may yield a value larger than the microbiological limit, and the methods for STEC detection) could reduce the risk of STEC induced hemolytic uremic syndrome (HUS) in human patients by 25 to 89 percent. Increasing the sample size of the end- product for analysis from 25 g to 100 g for STEC testing was also predicted to reduce HUS risk (ANSES opinion, 2018). Risk managers must balance risk reduction with economics so that the products remain available for sale at a reasonable cost. Consequently, microbiological criteria should be considered to be part of a food safety control system and should also include ongoing monitoring of the system. Although there is strain to strain variations, some pathogenic strains of STEC may be more acid tolerant than NTS E. coli and persist when the indicator has died off. There are general recommendations in microbiological sampling programs to assure the hygiene and safety of cheeses, but none are specific for STEC. In cheeses made from pasteurized milk, the International Commission on Microbiological Specifications for Foods (ICMSF) (2011) recommends the use of E. coli limits that are established under a 3-class sampling plan, where n (sample number) = 5, c (allowable number of samples between m and M) = 3, m (acceptable level) = 10 and M (unacceptable level) = 102. Raw milk cheeses are tested for Staphylococcus aureus only, which is consistent with the European Union recommended sampling criteria. The ICMSF provides a suggested sampling plan for Salmonella in raw milk cheese (medium or low importance) where n = 5 (25 g samples), c = 0, m = nd (not detected), but makes no STEC recommendations. As STEC prevalence in raw milk and raw milk cheeses is low, STEC testing in these products is uncommon and challenging, and most sampling and testing protocols target indicator organisms such as E. coli. It is notable that for the European Union microbiological criteria for cheese, no limits were established for E. coli in raw milk cheese. It is regarded that E. coli does not offer a meaningful hygienic index in raw milk cheese as its presence is expected, consistent with guidance from ICMSF. However, the Health Protection Agency (the United Kingdom) recommends that raw milk cheese be tested routinely for E. coli, and if detected, the source of contamination investigated, particularly if an upward trend is noted since STEC may also be present (Donnelly, 2018). Although the presence of NTS E. coli or other indicator organisms in raw milk does not indicate the presence of STEC, they remain useful hygienic markers of the quality of raw milk and raw milk cheeses and many other countries in the world have also established NTS E. coli limits to monitor the sanitary quality of raw milk cheeses (Metz et al., 2019). CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 118 Monitoring of raw milk cheese quality is not a true intervention step, however, it can contribute to the safety of raw milk cheeses. Systematic selection of quality raw milk with < 50 CFU/mL of E. coli for use in the manufacturing of uncooked, pressed cheese was predicted to reduce the risk of HUS from STEC (ANSES opinion, 2018). Depending on the cheese types and the technology used, quality guidelines for the manufacturer of raw milk cheeses, which though may not fully eliminate STEC from the product, may include STEC relevant microbial criteria that contribute to the safety of raw milk cheese products. However, setting such criteria is complex, as there is a large variety of cheese types and wide diversity in behaviour among STEC strains. As a result, challenge tests with various STEC strains in various raw milk cheese types need to be performed to investigate the survival of these pathogens under those specific manufacturing conditions. In the absence of official criteria for STEC or E. coli (in some countries) in raw milk cheeses, small and medium enterprises that make a specific cheese type or a group of similar products, and where real-time monitoring of E. coli in raw milk is not feasible, a specific limit for E. coli in young 24-hour old cheese (after pressing, before brining) should be defined. 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125 8 Conclusions The expert committee performed a review of accessible scientific evidence on the efficacy and utility of physical, chemical and biological control measures effective against STEC during the primary production and processing of raw beef, raw milk and raw milk cheeses. The quality of evidence varied greatly depending on study design, method of analyses, STEC serogroup used, and the scale of each study (e.g. laboratory, farm or processing plant). Many results were from laboratory or small-scale studies, which may not be scalable to meet commercial demands under a myriad of diverse production and processing conditions. There are, therefore, uncertainties as to whether these studies are truly representative of production and processing conditions, and whether the observed STEC reductions will occur in actual situations. Scientific evaluations of intervention treatments for STEC are ideally as representative as possible of the scenario in which they would be applied; however, these studies are frequently prohibited due to the health risk associated with the introduction of a pathogen into the food manufacturing facility. Consequently, surrogate bacteria (e.g. NTS E. coli) are used as substitute and the results extrapolated, which means that the evidence on the effects of interventions specifically on STEC may not be available currently, or in the future. Furthermore, molecular techniques are increasingly refining current evidence, hence, existing data may be subject to future revision. Implementing STEC monitoring plans at the farm level to measure their impact on STEC presence in raw beef and dairy products may or may not be practical because of the nature of beef and dairy production being composed of many small-scale, independent producers, with little or no integration between production phases, as well as variability in cattle STEC excretion dynamics. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 126 Intervention strategies have generally been examined individually at specific points in the food chain. The use of multiple control measures has also been implemented sequentially on farms and in beef, ground beef and dairy processing plants. Although it is uncertain to be cumulative, the additive effect of multiple interventions applied sequentially to reduce STEC transmission in the meat or dairy production chain remains unknown, and it is almost certain that they will not completely eliminate STEC. • Perhaps more important than the effectiveness of the intervention measures, the producers and processors making the decision to select a specific control measure must also consider the ability and logistics to install or implement the measure, its practicality, occupational health and safety concerns, environment resource management and cost. • Beef and dairy producers and processors typically follow GAP and/or GHP to reduce the spread of pathogenic and spoilage organisms. While these practices likely reduce STEC as well, specific evidence of STEC reduction is lacking or limited. • In the processing plant, data on the impact of interventions on quantitative reduction of STEC is limited or lacking as product inoculation studies with STEC cannot be performed in a commercial facility. As such, in-plant evidence for impact of interventions on STEC is generally based on data from prevalence studies. However, evidence on the impact of interventions on STEC obtained from research laboratories or pilot plants, can be combined with in-plant data on surrogate generic NTS E. coli or other microorganisms to make efficacy assessments. • Farm based practices and interventions can reduce STEC carriage, excretion and transmission/recirculation within a herd. But these reductions can be negated at later stages of the processing chain as a result of mixing with other animals during transport and lairage. Mixing animals that have not been similarly treated will result in cross-contamination during processing. • Good animal management and production practices include hygienic housing and bedding, low animal density, clean drinking water, biosecurity, safe and effective sanitation, and manure management. All of these will contribute to reducing faecal-oral transmission of pathogens, including STEC, among cattle. • The impact of several dietary management and nutritional strategies on reducing STEC populations in meat and dairy animals have been explored, but with varying degree of effectiveness. There is little evidence supporting these interventions for the control of STEC. • The use of numerous feed additives to manage STEC levels were examined. The reported effects of using probiotics, colicins, bacteriophage (in feed), and sodium chlorate in vivo were highly variable, depending on the agent and the CHAPTER 8 – CONCLUSIONS 127 animal host. At present and based on the available evidence, these are not recommended for consideration for the control of STEC. • Some vaccines have been shown to reduce faecal excretion of STEC O157:H7, but the efficacy is dependent on the type of vaccine and also the number of doses required. • In the processing plant, data on the impact of interventions on quantitative reduction of STEC is limited or lacking as product inoculation studies with STEC cannot be performed in a commercial facility. As such, in-plant evidence for impact of interventions on STEC is generally based on data from prevalence studies. However, evidence on the impact of interventions on STEC obtained from research laboratories or pilot plants, can be combined with in-plant data on surrogate NTS E. coli or other microorganisms to make efficacy assessments. • Long distance cattle transport increased faecal excretion and cross- contamination between animals. The exact role of lairage in spreading STEC among animals is unclear and is likely dependent on facility design, duration, stress, animal density, and cleanliness. In lairage, clean animal scoring can be used to classify clean and dirty animals, but the association between clean animal scores and reduced STEC prevalence on carcass is unclear. • GHP measures used during processing include lairage hygiene, optimized dressing and evisceration procedures to minimize carcass contamination from the hide and gut, trimming to remove visible contamination, minimizing handling cross-contamination, and effective cooling systems to prevent microbial growth. All of these interventions contribute to reducing contamination of pathogens, including STEC, in raw beef. • Treatments to decontaminate hides include washes, dehairing, and bacteriophage, applied before or after stunning. The reported effects were highly variable and there were practical and logistical issues for in-plant application. At present, there is limited evidence of their effectiveness in reducing transfer of STEC to carcasses • Processing measures that specifically reduced STEC prevalence on carcasses included: steam vacuuming of visible faecal contamination on carcasses, carcass wash using hot potable water, steam pasteurization, and 24-h air chilling and combinations of these. In-plant studies showed these measures to have significant reductions in STEC prevalence. • Despite the widespread commercial use of organic acids and other chemical agents to decontaminate pre-chill carcasses, there is wide variation in the reported reductions of STEC levels and prevalence in both research and commercial applications, depending on the trial parameters used. CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 128 • The comparative efficacy of available and putative control measures (e.g. bacteriophage, lactic acid treatments, irradiation) for reducing or eliminating STEC on primal cuts, trim and cheek meats is wide ranging and most of the studies are laboratory-based with none of those examined being performed in commercial production conditions. • The comparative efficacy of available and putative control measures for reducing or eliminating STEC in ground beef and in retail packs was wide ranging and only high-pressure processing, gamma irradiation, and eBeam were identified as most efficacious. • The process of grinding beef and comingling raw milk results in a broader distribution of STEC throughout the product. STEC levels in raw beef, raw milk and raw milk cheeses can vary considerably depending on primary- production, processing, and post-processing conditions, and the interventions applied. • For raw milk, interventions using bactofugation, microfiltration, bacteriophages, eBeam and high pressure reduced bacteria, E. coli and/or STEC levels. But all of these interventions presented logistical issues such as the need for sub-pasteurization temperature heating, costly equipment, and may be associated with potential organoleptic changes to the product. • For the manufacturing of raw milk cheeses, the cooking, acidification, and ripening steps, or a combination of these may be associated with STEC or E. coli reductions; however, the magnitude of reduction varied by STEC serotype and the type of cheese. Thus, the quality of raw milk used in cheese making along with manufacturing hurdles are crucial to reducing the risks associated with the end products. • Some studies showed that combinations of interventions are more efficacious than individual treatments in reducing STEC levels, but the results can be inconsistent and varied depending on study parameters. Even if proven to be effective, the added cost and time for the application of combined treatments may render them impractical for use in plants. Annexes

131 Annex 1 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 2.1 Animal Factors STEC genomics Distinct genetic lineages of STEC are associated with human illness and colonizing cattle. Determines likelihood of strain causing human illnesses. Super shedder strains/phage types may have differences. No specific genomic linkages are known that provide targets for intervention strategies to reduce STEC carriage by cattle or transmission. No known STEC targeting capacity. Feng et al., 1998; Whittam et al., 1988 2.1.1 Cattle genetics Host genetics Impact both the phylogenetic diversity and the relative abundance of members of the intestinal microbiome. Innate and acquired immunity as well as other host-microbiome communication channels may influence the establishment of STEC within the host and STEC excretion. Wang et al., 2016; Munns et al., 2014, 2015 Animal bread: Occurs in both dairy and beef cattle Some evidence that hosts immune status at the recto- anal junction may influence shedding, but mechanisms are unclear. Investigation of host genomic- STEC. interactions are worthy of continued investigation. Other factors such as animal management and diet are likely to be more important in determining shedding status than host genetics. Wang et al., 2016; Munns et al., 2014, 2015 Primary production control strategies for STEC in beef and dairy CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 132 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Super shedder strains may have a host genetic component Cattle that shed > 104 CFU/g of faeces, play a significant role in the transmission of STEC O157:H7 within the production environment. Significant source of STEC O157:H7 during primary production. Shedding is intermittent and not clear with regard to what controls it; could be related to sloughing of biofilms from intestinal epithelium. Wang et al., 2016; Munns et al., 2014, 2015 2.1.2 Cattle intestinal microbiome Non-O157 STEC serogroups can be carried in ruminants as commensal-type organisms Limited studies have indicated that STEC O157:H7 is typically the most prevalent STEC in human cases because it was the earliest to recognize and to test for. However, we now know that the prevalence of non-O157 STEC is broadly similar in cattle. Colonization with multiple serogroups simultaneously occurs. Non-O157 STEC are theorized to behave similarly to STEC O157:H7. While it appears that there are individual differences in serotype capacity to colonize the gut of cattle, there are no differences between serotypes that can be exploited by specific intervention strategies. Regions have variable levels of non-O157/O157:H7 ratios. Insufficient information exists on carriage of O157:H7 vs non-O157 world-wide. Influence of geography, management practices, as well as animal genetic background affects prevalence. There are significant physiological differences within STEC O157/non-O157 strains that play a role in the ecological niche, and the resultant prevalence of each serotype. Arthur et al., 2002; Bonardi et al., 2004; Cernicchiaro et al., 2014; Bergholz and Whittam, 2007; Cull et al., 2017; Dewsbury, 2015; Fan et al., 2019; Free et al., 2012; Mellor et al., 2016 Evidence that phylogenetic composition of intestinal microbiota differs between super- shedders and non-shedders No specific profiles have been attributed to super- shedders, nor have specific microbiota been shown to be associated with non- shedders. Interactions among members of the microbiome within the gastrointestinal tract merit further investigation. Cattle gut microbiome can be altered by use of other interventions. Zaheer et al., 2017; Xu et al., 2014 133ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 2.1.3 Cattle demography Calf rearing practices (including bob veal) Randomized controlled trial investigated 3 intervention packages to reduce STEC O157:H7 in young-cattle. Feeding colostrum from the calf’s dam, a decrease in serum IgG conc. and high temperature-humidity index increased the likelihood of STEC O157:H7 in pre-weaned calves. Calves for veal have increased risk of shedding and carriage of STEC compared to older calves. Veal calves positive on hide and carcasses for STEC O157:H7 and non-O157 STEC. Keeping young cattle in the same groups was one the most important measures. Strategies that mitigate the effect of temperature could be advantageous. Immunity and overall health of the calf GI tract are important factors. Questions regarding colostrum administration route (amount of colostrum ingested, passive transfer of immunity, transmission of STEC O157:H7 via colostrum, etc.). Calves housed independently or in small groups to prevent disease transmission is a GAP. Future research needed. Ellis-Iversen et al., 2008; Stenkamp- Strahm et al., 2018; Bosilevac et al., 2017 Stage of production Grouping cattle based on age and production status to prevent disease spread and maintain appropriate animal nutritional status. Grouping cattle based on age and production status is a GAP. Ekong, Sanderson and Cernicchiaro, 2015; Edrington et al., 2004; Venegas-Vargas et al., 2016 Animal age Calves shed STEC O157:H7 more frequently than older cattle. Cull dairy cattle often come from herds based on disease status or age, which is linked with increased carriage of STEC O157:H7. Grouping cattle together in age-specific groups is a GAP and allows higher shedding calves to be separated from older cattle. Herriott et al., 1998; Ellis- Iversen, 2008 2.2 Environmental Factors 2.2.1 Biosecurity Fly control Association of STEC O157:H7 with filth on flies and experimental transmission of STEC O157:H7 by flies. While these effects are probably minimal in their direct impact on food safety within a farm, they represent vectors that can transfer pathogens between “clean” groups of cattle or farms. No intervention study to date. Hancock et al., 1998; Talley et al., 2009; Ahmad, Nagaraja and Zurek, 2007 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 134 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Rodent control STEC O157:H7 in feedlot cattle and in Norwegian rats from a large-scale farm. Čížek et al., 1999 Bird control Wild birds as source of clonal dissemination of STEC O157:H7 among dairy farms, both migratory and native. Wild bird density & farm management are important issues. Wetzel and Lejeune, 2006; Cernicchiaro et al., 2012; Callaway, Edrington and Nisbet, 2014 Open vs closed herd (cattle population) Randomized controlled trial that investigated three intervention packages on reduction of STEC O157:H7 in young-stock cattle farms in England & Wales. Maintaining the animals in the same groups is one of the most important measures (48 percent reduction in STEC O157:H7). Introduction of STEC O157:H7 through incoming animals should be prevented by reducing entry to the farm. Farms with animals at pasture with water supply from natural source and with higher numbers of finishing cattle had lower prevalence. Ellis-Iversen et al., 2008; Gunn et al., 2007; Garber et al., 1999; Smith et al., 2001; Sanderson et al., 2006 2.2.2. Animal density Animal density Animal density linked with an increased risk of carriage of STEC O157:H7, and stocking density increased both shedding and horizontal spread of STEC O157:H7. Density is especially important when super shedding animals are present, as density increases contact between animals. Increased density reduces environmental footprint. Frank et al., 2008; Strachan et al., 2006; Vidovic and Korber, 2006; Haus-Cheymol et al., 2006 2.2.3. Environmental hygiene Pen scaping Effective in reducing presence of STEC. Pen scraping avoids use of water flush which increases STEC prevalence. Garber et al., 1999; Smith, et al., 2002 Pen floor Avoid muddy pen floors which favour STEC survival and spread. Effective in reducing presence of STEC. Pen floors are more significant source of STEC O157:H7 infection than feed and water. Smith et al., 2001; Bach et al., 2005a Animal handling facilities Animal handling facilities including squeeze chutes (crushes) and other contact points can horizontally spread STEC on hides. GAP that can limit cross- contamination Amount of faeces present on hide is directly related to transmission risk. Mather et al., 2007 ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 135 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 2.2.4 Manure management issues Solid waste composting Composting at 55 °C to 65 °C; Covering with finished compost.Highly effective, recommended as a post treatment for manure prior to land application. Proper carbon: nitrogen ratio required to achieve kill temperatures; temperature used as validation of the process. GAP. LeJeune et al., 2004 Slurries Anaerobic digestion at 30–35°C with 20 days retention. Addition of lime (pH 12 for at least 2 h). Highly effective, recommended as a post treatment for manure prior to land application. Care must be taken to monitor nutrient level and ensure that other contaminants are not released into the environment from bio- digestion of sludge. GAP. Blaustein et al., 2015 Manure applied to fields Can serve as an STEC contaminant of both ground and surface water. Secondary treatment is recommended to reduce risk of application to both crop and forage land. Not a specific STEC intervention. Care must be taken to monitor nutrient level and ensure that other contaminants are not released into the environment from bio- digestion of sludge. GAP. Ongeng et al., 2015 Grazing practices Limit grazing in pastures shared with other ruminants, which can transfer STEC. Ensure that shared pasture usage between ruminant species is limited is a GAP. Stacey et al., 2007; Duffy, 2003; Callaway et al., 2013 2.2.5 Seasonal variability and temperture Summer peak in excretion and prevalence Correlation of STEC O157:H7 shedding, human cases and seasonality, weather and water. No intervention study to date. Clear seasonality (summer peak), multiple causes: e.g. growth of STEC, growth of other vectors (protozoa), day length affecting faecal shedding, water troughs, and animal aggregation in shade and near water sources. Money et al., 2010; Ekong, Sanderson and Cernicchiaro, 2015; Besser et al., 2014; Gautam et al., 2011; Dawson et al., 2018 Temperature STEC O157:H7 outbreak in humans after heavy rainfall (vector: sheep faeces). Manure run-off contaminating different waters such as retention ponds in feedlots, exposed surface waters, streams. No intervention study to date. See also heat/cold stress Ogden et al., 2002; Tymensen et al., 2017; Tanaro et al., 2014; Johnson et al., 2003; Cook et al., 2011 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 136 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Heat / Cold stress Heat stress has shown limited impact on STEC shedding. Sprinkler use to alleviate heat stress demonstrated no impact on STEC O157:H7 populations. Not an intervention, but important for animal welfare and productivity. Sprinklers can increase mud in pens which increases STEC survival and the amount of coat tag. Alleviating heat/ cold stress is animal welfare issue. Brown-Brandl et al., 2009; Edrington et al., 2009a 2.3 Water and feed management strategies 2.3.1 Drinking water quality and hygiene Cleaning of water troughs Water troughs are sources of STEC O157. Higher cleaning rate predicted as being particularly efficacious at reducing the load of STEC at the farm and at increasing death rate of STEC O157:H7. Cleaning of water troughs predicted to reduce STEC population and dissemination of STEC in cattle production. No effect found on chlorinated versus non chlorinated water on prevalence on STEC O157:H7. And no effect of improved water hygiene (randomized controlled trial). Some limitations to its effectiveness based on environmental conditions. VosoughAhmadi et al., 2007; Ayscue et al., 2009; Ellis- Iversen et al., 2008; LeJeune et al., 2004 Water-to-cattle ratio (automatic refilling water troughs) Keeping water levels high in water troughs suggested to reduce prevalence of STEC O157:H7. Association between reduced water level and increased STEC O157:H7 prevalence. Dilution of pathogens; dependent on water availability/cost and can increase water utilization by a farm. Beauvais et al., 2018 2.3.2 Drinking water treatment Chlorine Chlorine is an effective disinfectant and can be used to clean water troughs, but troughs quickly become re- contaminated after cleaning. Antimicrobial activity of chlorine is reduced if high levels of organic matter come in contact with chlorinated water or if it is exposed to UV light. LeJeune, Besser and Hancock, 2001, 2004; Smith et al., 2002; EO water An effective disinfectant and possibly more active than just chlorinated water, but subject to same inactivation by UV light and organic matter. Has been used in processing and postprocessing environments. Bosilevac et al., 2005a; Stevenson et al., 2004 ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 137 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 2.3.3 Diet composition, feeding strategies and feed hygiene Cleaning of feed troughs Feed hygiene to reduce STEC faecal contamination. No relationship between pens shedding STEC O157:H7 and recovery from feed (and water), but modelling suggests an influential source. Providing clean water and feed is a GAP and is critical to ensuring good animal health and productivity. Smith et al., 2001; Dodd et al., 2003; Sanderson et al., 2006; Ayscue et al., 2009; Berry and Wells, 2010 Forage: concentrate ratio: Variation in diet composition in regard to forage to concentrate ratio E. coli populations (both generic and STEC) are generally higher in grain-fed cattle than in forage fed. Experimentally inoculated calves were fed high grain or high forage diets on the duration or shedding of faecal STEC O157:H7 populations in experimentally inoculated calves have found that low quality forage feeding caused a faster rate of death of STEC O157 populations in manure. Faeces from grain fed cattle had higher VFA concentrations and lower pH allowing STEC O157:H7 populations to survive longer than in faeces from grass-fed cattle. Controlled studies have been few and mostly observational. Feeding forage diets to all cattle would reduce the availability of animal protein and is difficult to implement in feedlots and arid regions. Effect also appears to be linked to forage quality (tentative). However, the host/dietary/ microbial factors underlying the “super shedder” status of cattle remains unknown, as do factors that allow simple gut colonization by STEC O157:H7. A better understanding of microbial populations and physiology of the gastrointestinal tract of cattle will allow reduction of STEC O157:H7 at pre- harvest through diet. Callaway et al., 2009, 2013 Rapid Dietary shift from grain to forage A rapid shift from a high grain to hay diet resulted in a 3 log10 reduction in NTS E. coli populations. Dietary shifts are difficult to implement in feedlots and in arid regions, with logistical challenges abound. Callaway et al., 2009, 2013; Diez Gonzalez et al., 1998 Grain type Barley feeding is linked with increased shedding and survival of STEC O157:H7 in faeces compared to corn. Barley doubled the survival time of STEC O157:H7 in faeces and shedding concentration of STEC O157:H7 < 0.5 log10 vs corn. Barley feeding also resulted in an increase in excreted STEC populations. Barley is a grain that is often fed to cattle which is more rapidly fermented in the rumen than corn, and so, little barley passes to the hindgut and little starch reaches the lower gut. Bach et al., 2005a, 2005b, 2002; Berg et al., 2004 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 138 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Processing of grain (cracking, steam flaking, etc.) Dry rolling of grain increases starch flow to the hind gut and reduces STEC O157:H7 populations compared to Steam flaked grain. Steam flaking corn increased STEC O157:H7 shedding in heifers compared to whole corn. Faecal starch concentration and pH are not linked to STEC O157:H7 shedding. Location of fermentation of starch in cattle is shifted by grain processing, which impacts animal health and growth efficiency. Faecal starch concentration and pH were not linked to STEC O157:H7 shedding, yet post-ruminal starch infusion increased NTS E. coli populations in the hindgut. Fox et al., 2007; Depenbusch et al., 2008 Dried Distiller's Grains with solubles (DDGS); Wet Distiller's Grains with Solubles (WDGS) Feeding 40% WDGS increased faecal shedding of STEC O157:H7 (10% in 0 vs 70%), but 15% DGS did not increase excretion. Experimentally inoculated cattle showed that STEC O157:H7 manure populations were decreased from 6.28–2.48 log10 CFU/g by 40% WDGS. DGS fed at 20, 40, and 60% corn WDGS increased STEC survival in manure from 1-3 log10 CFU/g of faeces. Feeding 40% DGS increased faecal STEC populations by > 3 log10, and increased survival time in manure. Evidence supports recommending feeding Distillers or Brewer’s grains at levels < 15% is thought to not increase STEC O157:H7 populations. Impact of including drieddistillers grains is highly variable due to poor QC/QA. Effects of feeding >40% DGS increases risk of faecal STEC shedding. Jacob et al., 2008, 2009; Wells et al., 2011; Paddock et al., 2013; Berry et al., 2017 Tannins Feeding of Tasco-14 (Phlorotannin-containing) displayed anti-STEC activity higher than other terrestrial tannin sources. Complete or partial elimination of different STEC-Serotypes, with STEC O157:H7 reduced by up to 36%. Zhou et al., 2018; Braden et al., 2004 Essential oils A large variety of diverse feed additives can be fed to ruminants (e.g. feeding orange peel to sheep; thyme). Breadth of category limits the ability to determine effectiveness of individual essential oils. Conflicting or not repeatable study results done in small scale challenge studies. Callaway et al., 2011; Jacob, Callaway and Nagaraja, 2009 ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 139 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 2.3.4 Feed additives Priobiotics/Direct fed microbials (DFM) Some DFM are effective in reducing prevalence of STEC O157:H7 faecal shedding in beef cattle. Most efficient: Lactobacillus acidophilus (NP51) and Propionibacterium freudenreichii (NP24); at doses of 109 CFU/animal/ day, for 137 days reduced prevalence of STEC O157 faecal sheding in beef cattle. STEC O157:H7 isolation was 74% less likely on hides and in faeces. DFM (eubiotic and post- biotic) effectiveness vary widely based on the active organism in the product as well as dosing level. Wisener et al., 2015; Stephens et al., 2007 Competitive exclusion cultures (calves) Oral administration of probiotic E. coli to calves. Effectiveness shown against STEC O26 & O111, but not O157. Zhao et al., 2003 Phage hijack cellular metabolic machinery of bacteria and cause bacterial lysis Efficacy in primary production has been mixed, with either little change in STEC numbers or the establishment of a cyclic response where STEC counts decline with phage numbers increase, but phage numbers decrease and STEC counts increase. Phage show promise for use in processing and post- processing phases. Phage are often highly specific with most having a narrow host range, which is limited to one or a few STEC serotypes. STEC can also become resistant to individual phage. Sabouri et al., 2017; Liu et al., 2015; Arthur et al., 2017; Wang et al., 2017 Colicins kill STEC O157:H7 Colicins reduced STEC O157:H7 strains in vitro and in small ruminants but do not differentiate between generic and STEC. Colicin production by plants allows for production scale up for inclusion in rations. Colicin producing probiotic cultures have been used to reduce STEC O157:H7 in cattle in experimental studies. Evidence of colicin anti-STEC activity has been more promising in ground beef and in cleaning processing facility surfaces. Callaway et al., 2004; Schulz et al., 2015; Schamberger et al., 2004 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 140 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Sodium chlorate E. coli, including STEC can respire anaerobically using the enzyme nitrate reductase, which also reduces sodium chlorate to chlorite, an antimicrobial compound. Feeding Sodium chlorate to sheep reduced inoculated STEC O157:H7 in the rumen, cecum, and colon by 1-4 log10. Treatment in cattle reduced inoculated STEC O157:H7 populations by 2-3 log10 throughout the gut with no impact on meat quality. Product has been developed but not on the market pending on the approval process, which can be lengthy. Edrington et al., 2004; Callaway et al., 2002, 2003 2.4 Vaccines and clinical antimicrobials 2.4.1 Vaccines Novel bivalent vaccine against STEC infection via Clostridium perfringens enterotoxin (CPE)-based protein engineered for vaccine design and delivery system Administration of C-terminus of CPE (C-CPE) alone to mice induces C-CPE-specific IgM, but not IgG response due to its low antigenicity. In contrast, administering Stx2B–C- CPE, sufficient IgG immune responses with neutralizing activity against CPE were induced. Formulations against STEC strains are both protective in mice. However, mice are not an effective model for ruminants. The C-CPE is non-toxic and is the part of the toxin that binds to epithelial cells via the claudins in tight junctions; however, C-CPE has low antigenicity. Low to high, depending upon vaccine Lan, Hosomi and Kunisawa, 2019 Genetically inactivated recombinant Shiga toxoids (rStx1MUT/ rStx2MUT) A group of 24 calves was passively (fed colostrum from immunized cows) and actively (intra-muscularly at 5th and 8th week) vaccinated. Another 24 calves served as unvaccinated controls (fed with low anti-Stx colostrum or injected with placebo). Each group was divided according to the vitamin E concentration (moderate and high supplemented) they received by milk replacer. The effective transfer of Stx- neutralizing antibodies from dams to calves via colostrum was confirmed by Vero cell assays. Serum antibody titers in calves differed significantly between the vaccinated and the control group until the 16th week of life. Low to high, depending upon vaccine Schmi dt et al., 2018 ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 141 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle The experiment used two conventionally raised bull calves aged 11 months that tested negative for Stx- specific antibodies (16 and 4 weeks before the trial by VNA). In cattle, Stx suppresses the immune system thereby promoting long-term STEC shedding. First infections of animals at calves’ age coincide with the lack of Stx-specific antibodies. Antibodies in sera of cattle naturally infected with STEC recognized the rStx mut toxoids equally well as the recombinant wild type toxins. Low to high, depending upon vaccine Ker ner et al., 2015; Schmi dt et al., 2018 Immune response in calves vaccinated with type three secretion system Antigens and Stx 2B Subunit of STEC O157:H7 Calves were tested for STEC shedding 16 weeks before the trial then vaccinated with two doses of different vaccine formulations: two antigens (IntiminC280, EspB), three antigens (IntiminC280, EspB, BLS- Stx2B), BLS-Stx2B alone and a non-vaccinated group as control. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titer increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seemed to have a stimulated the humoral response to IntiminC280 and EspB after the booster. Low to high, depending upon vaccine Martorelli et al., 2017 Outer membrane vesicle (OMV)- based vaccine formulations against STEC are both protective in mice and immunogenic in calves Fifteen calves from a beef producing brand and between six and eight months old were allocated to a single pen and randomly divided into three groups of five. Group 1 was assigned a 50 µg OMVi plus aluminum adjuvant per dose, group 2 a 100 µg OMVi plus aluminum adjuvant per dose, and group 3 was treated with aluminum adjuvant in saline (control). Each group was vaccinated subcutaneously on days 0, 21, and 42. OMV obtained after detergent treatment of gram-negative bacteria have been used for decades for producing many licensed vaccines. These nanoparticles are not only multi-antigenic in nature but also potent immunopotentiators and immunomodulators. Formulations based on chemical inactivated OMV (OMVi) obtained from a STEC O157:H7 strain (was found to protect against pathogenicity in a murine model and to be immunogenic in calves. These initial studies suggest that STEC-derived OMV has potential to be developed as both human and veterinary vaccines. Low to high, depending upon vaccine Fingermann et al., 2018 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 142 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Commercial vaccine products Econiche™, designed to reduce the shedding of STEC O157:H7 by cattle, has received full licensing approval from the Canadian Food Inspection Agency (CFIA). Econiche is now available for unrestricted use by Canadian cattle growers but there are resistance concerns and no proven efficacy has been demonstrated in scientific studies. Due to valid concerns regarding the use of antimicrobials in animal husbandry, some countries have banned the use of antimicrobials growth promoters in cattle farming. Although this vaccine induces antibody responses effective in significantly reducing colonization, they are only partially protective. Low to high depending upon vaccine Snedeker, Campbell and Sarge ant, 2012 2.4.2 Clinical antimicrobials • Ionophores, tylosin, chlor- tetracycline, and oxytetra- cycline-feedlot cattle (subther- apeutic levels) • Tylosin-prevent hepatic ab- scessation and promote growth • Chlortetra- cycline and oxytetracycline: used at thera- peutic levels • Other The efficacy of these antimicrobials against STEC has not been shown in vivo or in vitro studies. But one would expect that the use of broad spectrum or bacteriostatic and/or bactericidal antimicrobials against gram negative bacteria, would most probably reduce STEC colonization and shedding. But the use of antimicrobials remains controversial because of antimicrobial resistance concerns and they can induce Stx bacteriophage which may spread Stx-encoding genes to naive E. coli. No proven efficacy of use has been demonstrated in scientific studies. Due to valid concerns on their use, some countries have banned the use of antimicrobials growth promoters in cattle farming. Key considerations: These antimicrobials 1) may induce Stx bacteriophages that are able to transduce stx-encoding genes and antimicrobial resistance genes to naive E. coli, thereby expanding the STEC pool in individual animals and at the herd level. (in vitro studies). 2) Some antimicrobials may exert selective pressure on intestinal microbiota, thereby favouring the survival of antimicrobial resistant STEC (no studies). 3) Antimicrobials against gram positive bacteria may favour the dominance of gram-negative bacteria including STEC or vice versa. Allison, 2007; Köhler, Karch and Schmidt, 2000; Kimmit et al., 2000; Herold et al., 2004; Colavecchio et al., 2017; USDA/FSIS, 2014 Neomycin sulfate Reduced STEC shedding in cattle. Antimicrobial Resistance issues are significant and preclude recommendation. Elder et al., 2002 ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 143 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Ionophores Ionophores are fed to improve ruminant feed efficiency. It alters the microbial population of the rumen through inhibition of gram-positive bacteria, resulting in a change in fermentation end products and reduction in methane production. Highly successful in improving feed efficiency and reducing environmental pollution. Theoretical competitive advantage to Gram negative species like STEC, butnotshown to occur in sheep or cattle. Percent of animals shedding STEC O157:H7 was greater for monensin fed steers than for controls, yet the presence of monensin, did not affect the percentage of animals in the pen shedding STEC O157:H7. Antimicrobial usage concerns. Not an intervention directly against STEC. Inclusion of ionophores in cattle feed is included to improve animal growth efficiency. Edrington et al., 2003; McAllister et al., 2006; Hales et al., 2017 2.4.3 Beta-agonists/hormones Ractopamine and zilpaterol Theorized to increase STEC shedding as a result of increased stress on the animal and possibly promoting the growth of STEC, however no impact has been demonstrated. Zilpaterol is no longer used, as no evidence from research studies showed that beta- agonists alter shedding of STEC O157:H7. Wells et al., 2017; Paddock et al., 2011; Edrington et al., 2009b 2.5 Dairy production specific interventions Milking environment Certain hygiene interventions on the milking equipment and the environment in a Flemish dairy herd were associated with a decrease in bacterial counts. The consistent application of a few hygiene practices could significantly improve the microbiological quality of milk. Verbeke et al., 2014 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 144 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Udder hygiene Washing teats with a sanitizer compared with no treatment reduced microbial load by 44%, whilst washing with a sanitizer and drying decreased microbial load by 85%. Washing of teats with an effective disinfectant (chlorine) and then drying was the most effective in another study. Using an automated teat scrubber with chlorine dioxide disinfection and drying also effectively reduced bacterial loads on teats. Overall, pre-dipping teats into disinfectant followed by drying has been shown as effective means of teat skin sanitation. Lack of correlation between cleaning regime and total viable counts, Enterobacteriaceae or E. coli levels in milk were reported. The environment, milking equipment and water are also contributors of milk contamination. Galton, Petrsson and Merrill, 1986; Fremaux et al., 2006; Gibson et al., 2008; Elmoslemany et al., 2010; Baumberger, Guarín and Ruegg, 2016 Hygienic storage of milk Reducing contamination and opportunity for growth of spoilage and pathogenic organisms is an important GHP. Proper storage temperature and hygiene reduces or prevents growth of organisms, including STEC. 2.6 Animal transportation Feed withdrawal prior to slaughter Feed withdrawl for >8 h prior to animal slaughter reduces gut fill (and risk of gut rupture during subsequent evisceration) and reduces production of faeces that can be spread to the hide or carcass during processing. Reducing feed in the gut results in a reduction of volatile fatty acids (VFA) production in the gut. VFA concentrations inhibit the growth of STEC and Salmonella. Fasting increases prevalence and concentration of STEC in cattle; same observed in feeding low quality forages. Fasting cattle is a GHP that reduces gut rupture and faecal contamination of hides. Jordan et al., 1998; Buchko et al., 2000; Hovde et al., 1999; Pointon, Kiermeier and Fegan, 2012 ANNEX 1 – PRIMARY PRODUCTION CONTROL STRATEGIES FOR STEC IN BEEF AND DAIRY 145 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Transport distance and/or duration Association between STEC shedding with distance of transport have been inconsistent. Bach et al., 2004; Arthur et al., 2007a; Dewell et al., 2008; Stanford et al., 2011; Brown- Brandl et al., 2009; Schuehle- Pfeiffer et al., 2009 Trailer hygiene Potential source of contamination, similar to animal handling facilities. No association measured. Arthur et al., 2007a Plane of nutrition prior to slaughter Cattle undergoing negative energy balance are subject to metabolic disorders. The role of plane of nutrition on STEC colonization is not clear. No association measured. Callaway et al., 2009 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 146 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 3.1 Lairage Logistic scheduling for slaughter Younger animals have a higher risk for STEC carriage. Calves for veal have increased risk of shedding and carriage of STEC compared to older calves. Keeping young cattle in the same groups was identified as one the most important measures. GHP; keep higher shedding calves to be separated from older cattle. Ellis-Iversen et al., 2008 Logististical slaughter based on hide cleanliness Association between hide cleanliness scores and prevalence of pathogenic bacteria has not been consistently reported. Hide cleanliness scores have been significantly associated with STEC O157:H7 after controlling for season; specific seasons and regions have been associated with hide contamination of different STEC serotypes in a specific locality. GHP; supplemental pre- harvest interventions could be considered in seasons or regions of known higher prevalence. FAO and WHO, 2005; Cernicchiaro et al., 2020; Schneider et al., 2018; van Donkersgoed et al., 1997; Brown et al., 2000; Keen & Elder, 2002; Smith et al., 2005b; Nastasijevic, Mitrovic and Buncic, 2008; Antic et al., 2010a, 2010b; Blagojevic et al., 2012 Lairage cleanliness Pressure washing with water, quaternary ammonium chloride or steam resulted in 0.9–5.8 log10 CFU/cm2 reduction in E. coli and Enterobacteriaceae. GHP; no specific data on STEC. Small et al., 2007 Processing control strategies for STEC in beef Annex 2 ANNEX 2 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 147 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Livestock cleanliness The cleanliness of the lairage environment is important in the maintenance of coat cleanliness. Measures include washing trailers, cattle handling facilities, holding pens between uses, regularly removed pen floor faecal material. Different cleaning and disinfection procedures might be used, and a key issue might be drying the lairage pens after cleaning and disinfection. Lots of cattle held in STEC O157:H7-positive lairage pens had 8 times greater risk of having positive slaughter hide samples (RR=8.0; 95%CI =1.6–38.8). Transport and lairage do not cause an increase in the prevalence of STEC O157:H7 faecal shedding in cattle, as demonstrated by a higher prevalence of STEC O157:H7 shedding in cattle sampled on the farm than during post-transport or lairage. GHP; increased water usage; water used should be fit-for-purpose. Avery et al., 2002; Arthur et al., 2008; Mather et al., 2008; Dewel et al., 2008; Small et al., 2003; Minihan et al., 2003; Walia et al., 2017; FAO and WHO, 2005 Holding animals in lairage Withdrawal feed up to 12 h while in lairage pens could reduces faecal output and soiling of environment and hide. Water misting animals in holding pens. GHP; improves dressing percentage. 3.2 Hide decontamination 3.2.1 Bacteriophage Bacteriophage Lab experiment on hide treated with two phages (e11/2, e4/1c) showed a significant reduction of STEC O157:H7 after 1 h. Spray application in lairage – did not produce a significant reduction in levels or prevalence of STEC O157:H7. Lab experiment on hide treated with phage cocktail to several STEC serotypes for 1 h. A reduction was observed, but not at a high efficacy. Efficacy studies are lacking; Highly adopted in USA in warm months;varying regulatory issues; cost relatively low. Coffey et al., 2011; Arthur et al., 2017; Tolen et al., 2018 3.2.2 Hide wash with ambient or hot water, organic acid and other chemicals Ambient water Washing with power hose for 3 min removed faecal contamination and STEC O157:H7 inoculated onto hide. Conflicts with the need for dry animals. Extra water usage can be costly. Conflicts with the need for dry animals. Byrne et al., 2000 Ozonated and electrolyzed oxidizing water Reduced EB counts by 3.4 and 4.3 log10 CFU/100 cm2, respectively. Not done in commercial conditions. Bosilevac et al., 2005a CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 148 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Other chemicals A hide wash cabinet (water and chlorine 100–200 ppm spray at the end) to be used in small and medium size plants Reduced STEC O157:H7 prevalence on hides from 35 to 13%. Cattle prewashed with water a day before harvest, then immediately before stunning, they were sprayed twice with 1% CPC for 3 min and then 1 min; CPC reduced prevalence of STEC O157:H7 by 18%. The use of an in-line hide-wash cabinet that used a sodium hydroxide wash and a chlorinated (1 ppm) water rinse. Hides sampled before entering and after exiting the cabinet had APC and EB counts that were reduced by 2.1 and 3.4 log10 CFU/100 cm2, respectively, and the prevalence of STEC O157:H7 on hides was reduced from 44–17%. Whole beef hides were inoculated with STEC O157:H7 and decontaminated with spray solutions of sodium hydroxide (1.5%) followed by high-pressure washing with chlorinated (0.02%) water (SHC; both applied at 23°C), potassium cyanate (PC; 2.4%, 30oC) or sodium sulfide (SS; 6.2%, 30oC).Resulted in the greatest reductions of STEC O157:H7 (P < 0.05), by 5.1, 4.8 and 5.0 log10 CFU/cm2, respectively. Hide pieces were treated with 1% caprylic acid (CA) and 1% β-resorcylic acid (BA) applied at 23°C and 60 °C sampled after 2 and 5 min. All treatments more effective at 60 °C, but in general 3-4 log10 reduction. No “real world” studies at slaughter houses. Study on pre-slaughter wash; I) Single water wash (1,325 l); II) Lactic acid (0.5 ± 0.2%); III) Double water wash; IV) Chlorine (50 ppm). Each wash lasted for 30 s. Increase in aerobic plate counts, coliforms, and E. coli. Animal welfare issues for use on live animal hides. Potential environmental issues with disposal and practical use in plant. Brown et al., 2000; Bosilevac et al., 2004; Bosilevac et al., 2005b; Bosilevac et al., 2006; Carlson et al., 2008; Mies et al., 2004 ANNEX 2 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 149 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Washing using water Hide was cabinet - washing with water (cold) + chlorine spray (100-200 ppm). Wash with water followed by water rinsing with subsequent vacuuming reduced bacterial load. Ozonated and electrolyzed water - systematic literature review and meta-analysis. Effect size or intervention effectiveness was measured as raw log10 reduction, least- squares means were calculated. Only slightly decreased prevalence of STEC O157:H7. But also, reduced STEC O157:H7 load, meaning the enumeration data indicated that the hide cabinet was effective. Least-squares mean reductions in log10 CFU/cm2 on hide surfaces (n = 47), 0.08 [95%Cl, 0.94–1.11] for water wash. Redistribute microbial contamination (forequarter sites). Arthur et al., 2007b; Bosilevac et al., 2005a; Bosilevac et al., 2005b; Zhilyaev et al., 2017 Organic acids Least-squares mean reductions (log10 CFU/ cm2). On hide surfaces (n = 47), least-squares mean reductions were 2.21 [95%CI, 1.36– 3.05] for acetic acid, 3.02 [95%CI, 2.16–3.88] for lactic acid. Systematic literature review and meta- analysis. Effect size or intervention effectiveness was measured as raw log10 reduction. Least-squares means were calculated. May select for acid-resistant bacteria; Increase equipment corrosion; Environmental and safety of employee issues. Other chemicals 1.6% Sodium hydroxide or 4% trisodium phosphate or 4% chlorofoam or 4% phosphoric acid. Rinse with water or acidified chlorine. Acidified chlorine (sodium hypochlorite with acetic acid) cetylpyridinium chloride (CPC). Hypobromous acid, reduced APC, TTC and EC by 2–3.8 log10. Least-squares mean reductions (log10 CFU/ cm2). On hide surfaces (n = 47), least-squares mean reductions were, 3.66 [95%CI, 2.60–4.72] for sodium hydroxide. May select for acid-resistant bacteria; Increase equipment corrosion; Environmental and safety of employee issues. Bosilevac et al., 2004, 2005b; Schmidt et al., 2012; Zhilyaev et al., 2017 3.2.3 Hide clipping, coating and chemical deharing Hide clipping, coating and chemical dehairing Food-grade resin in ethanol (Shellac) reduced hide STEC O157:H7 by 3.7 log10 CFU/cm2. Laboratory-based study of inoculated hides. In a small commercial abattoir under "worst- case" conditions (slaughtering dirty cattle, inadequate process hygiene), treatment of hides with Shellac reduced 1.7 log10 CFU/cm2, 1.4 log10 CFU/cm2 and 1.3 log10 CFU/cm2 of TVC, EC and GEC, respectively. Not commercially available and no data from commercial scale studies. Antic et al., 2012, 2010b CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 150 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Hide de-hairing Chemical dehairing; 10% sodium sulphide, water washes, 3% hydrogen peroxide reduced visible contamination but did not reduce total coliform counts, APC or EC. Study on small hide pieces (controlled lab conditions). Study of conventional and chemical dehairing Identified significant reduction of bacterial load (aerobic counts, coliforms, E. coli) and a reduction in STEC O157:H7 of 5 log10 CFU/cm2 on inoculated hides. Required cabinet; waste management (sodium sulphide); employees health and safety. Schnell et al., 1995; Castillo et al., 1998a; Nou et al., 2003 Clipping to remove faecal material Clipping hair from hides and singeing with handled blowtorch. Clipping followed by application of 1% CPC. Chemical dehairing resulted in lower bacterial load (~2 log10) and reduced prevalence of STEC O157:H7. Reduced total viable bacteria by 2.3 log10 CFU/cm2. Produced the greatest reduction of APC (3.8 log10) on the hide surface. Not conducted under commercial conditions. Small, Wells-Burr and Buncic, 2005; Baird et al., 2006 3.3 Slaughter and dressing Speed of processing The speed at which animals are moved along the processing line has been reported to have both positive and negative effects on TVCs on carcasses following dressing. Evidencs is inconsistent. Sheridan, 1998 Hide removal Either upward or downward hide pulling system. Total viable counts (TVCs) on forequarter (3 cm2) (n=15) indicated carcass contamination using a downward pulling system resulted in significantly lower TVC (0.4 log10 CFU/cm2) than an upward pulling system (1.2 log10 CFU/ cm2). No significant difference in total carcass contamination (8 sites, n=36) with TVC and Enterobacteriaceae on specific sites (flank, shin, brisket, neck); thought to be due to GHP and not direction of hide pulling. Evidence is inconsistent. Kang et al., 2019; Kennedy, Giotis and McKevitt, 2014 ANNEX 2 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 151 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Pre- and Evisceration Processes Bagging or tying of the bung: Intestines should not be severed from the stomach during evisceration and no other opening should be made into an intestine, unless the intestines are first effectively tied to prevent spillage; except in the case of poultry and game birds. A commercial facility study showed that bunging before as compared with after the pre-evisceration wash, resulted in lower contamination rates of carcasses with STEC (35% vs. 58.3%) and STEC O157:H7 (1.5 vs. 5%). GHP Greig et al., 2012; Sheridan, 1998; Stopforth et al., 2006 Removal of visible faecal material from carcass Knife and steel sterilized by immersion in a thermostatically controlled water-bath at 82 °C for at least 30 s or an equivalent combination to result in a 2 log10 reduction of E. coli. Using clean disposable gloves and sterilized knives and steels resulted in s ignificant differences between TVCs on the brisket though not on the hocks. Lower temperature and longer time combinations could provide equivalent log10 reductions to meet industry requirements and using a 2-knife system could overcome delays for workers. GHP; hot water use is a potential health and safety issues for employees. McEvoy et al., 2001; Eustace et al., 2007; Goulter, Dykes and Small, 2008 Trimming Knife trimming can result in higher log10 reductions than a water wash and these can be reduced by combinations with other interventions. Reductions in log10 CFU/cm2 in inoculation experiments were: TVC - 4.3, STEC O157:H7 - 3.1, Enterobacteriaceae - 4.1, E. coli - 4.1. In a commercial plant, reduction in TVCs was 3 log10 CFU/cm2. In 24 commercial plants, trimming alone reduced TVCs by 0.44 log10 CFU/cm2 and prevalence of E. coli by 29.1%; trimming plus another intervention (hot water, lactic acid, steam vacuum) reduced TVCs by 0.61 log10 CFU/cm2 and prevalence of E. coli by 36.8%. Additional interventions did not reduce Salmonella prevalence. Redistribution of bacterial contamination on carcass from and cross-contamination. Effectiveness can depend on employee skill. Horchner et al., 2020; Castillo, 1998b; Prasai, et al., 1995 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 152 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Steam vacuuming Steam vacuum applies steam or hot water (approx. 82 °C – 95 °C) using spray nozzles to loosen visible soil and inactivate bacteria and a vacuum to remove contaminants. Commercial steam vacuum systems have been reported to reduce E. coli between 3.0-5.5 log10 CFU/100 cm2. In a meta-analysis, the mean log10 reduction of E. coli was 3.09 log10 CFU/cm2 and thwas a higher reduction than from water or organic acid washes. On naturally contaminated carcasses under commercial conditions, steam vacuuming after carcass trimming was reported to reduce mean TVCs between 0.4-0.9 log10 CFU/cm2 and increased distribution of TVC < 3 log10 CFU/cm2 by 11.9%. Used on small areas or hot spots only prior to chilling. Effectivness depends on employee skill, equipment maintenance, exposure time, application temperature. Brashears and Chaves, 2017; Dorsa, Cutter and Siragusa, 1996; Hochreu- tener et al., 2017; Moxley and Acuff, 2014; Zhilyaev et al., 2017; Bacon et al., 2002 Head and cheek meat Head washing using water simulated plant conditions at pre-evisceration point using modified spray-wash cabinet; pre- evisceration wash (25 ± 20 °C) for 10 s at 3.2 kg/cm2, followed by water (74 ± 2 °C) for 10 s at 0.7 kg/cm2. Hot water applied for 26 s at 0.71 kg/cm2 and at 74 ± 20 °C. Rate of hot water spray not measured. E. coli O157:H7 (no Stx) on beef cheeks from inoculated beef heads (n=140) was reduced by ≥ 1.5 log10 CFU/cm2) after a pre- evisceration wash and a further 1.72 log10 CFU/cm2 using hot water at 74 °C. Targeted at cheek meat specifically; contamination can accumulate at head when vertical rail dressing; requires wash cabinet. Kal- chayanand et al., 2008 Head washing using chemicals Both lactic acid and FreshFx solutions were sprayed for 26 s at 1.75 kg/cm2 and at 25 ± 2 °C and sprayed at the rate of 14 L/ min. Acidic electrolysed water (EOI) was sprayed for 26 s at 1.75 kg/cm2 and 25 ± 2 °C; alkaline electrolysed water applied for 13 s at 1.75 kg/ cm2, followed by EO-I treatment for 13 s at 1.75 kg/cm2. Ozonated water (OZI) applied for 26 s at 1.75 kg/cm2 and 25 ± 2 °C; second treatment was a high-pressure water wash (HP; 0.2 kg/cm2 at 25 °C) applied for 6 s, followed by OZI for 20 s at 1.75 kg/cm2. Reductions (log10 CFU/cm2) were achieved relative to a pre-evisceration wash using lactic acid (1.52), FreshFXTM (1.06) washes while reductions using electrolysed and ozonated water washes were not significantly different. Targeted at cheek meat specifically; contamination can accumulate at head when vertical rail dressing; requires wash cabinet; GHP. Kal- chayanand et al., 2008 ANNEX 2 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 153 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 3.4 Pre-chilling Washing using cold or ambient water Inconsisten results form multiple studies. Spray beef carcasses 5.62 kg/cm2, 32°C for 15 s with tap water (pH 7.34). Initial wash with water reduced STEC O157:H7 by more than 1.5 log10 and reduced Listeria and Clostridium by 3 log10. STEC O157:H7 and Salmonella Typhimurium were reduced 2.3 log10. Apply carcass rinse of 1.5 L handwash (9 s at 69 kPa) and 5 L automated cabinet wash for 9 s. Meta-analysis of before and after effects conducted in commercial (large or small) slaughterhouses and pilot plants for 4 study consisting of 10 trials for prevalence and 3 studies comprising 9 trials for concentration estimates. Redistribute contamination on carcass. Milios et al., 2017; Dorsa, Cutter and Siragusa, 1996; Castillo et al., 1999; Greig et al., 2012; Gill, McGinnis and Badoni, 1996a, 1996b; McEvoy et al., 1999; Yalçin et al., 2004; Jericho et al., 1995; Jericho et al., 1996 Washing using hot water Hot water pasteurization is defined as sheets of water applied to a carcass at temperatures greater than or equal to 85 °C for 8 s to 15 s. Results based on systematic literature review and meta-analysis. Effect size or intervention effectiveness was measured as raw log10 reduction, least-squares means were calculated. May generate condensate and aerosols; pressure of spray, health and safety issues for operators; colour changes. Castillo et al., 2002; Dickson and Anderson, 1992; Gorman et al., 1995; Smith and Graham, 1978; Smith, 1992; Greig et al., 2012; Zhilyaev et al., 2017; Huffman, 2002; Phetx- umphou, 2018. Hot water and chilling Carcass wash using water greater than 50°C followed by 24 h of chilling. Meta-analysis of before and after effects conducted in commercial (large or small) slaughterhouses and pilot plants for 1 study consisting of 2 trials. Odds ratio of NTS E. coli carcass contamination = 0.02. Greig et al., 2012 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 154 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Steam pasteurization Steam pasteurization was defined as steam applied to a carcass at a temperature great than or equal to 82.2 °C for 6–11 s. Some observed reductions in aerobic plate counts of around 1.5 log10 cfu/cm2 and the reduction of coliforms to below detectable levels, following a 6-8 s treatment. Use of a similar system gave consistent results and showed that the reduction was uniform over the surface of the carcass. Meta-analysis of before and after effects conducted in commercial (large or small) slaughterhouses and pilot plants for 4 study consisting of 14 trials. Plus 1 controlled trial with natural pathogen exposure. Odds ratio of NTS E. coli carcass contamination = 0.13. Controlled trial demonstrated a standardized mean difference of 0.39 log10 Surface greying of carcasses, but after 24 h chilling, the meat returned to acceptable colour. Nutsch et al., 1997; Phebus, 1997; Greig et al., 2012; Huffman, 2002 Organic acids – Lactic acid In a laboratory study, lactic acid (2%) at 55°C was shown to reduce STEC O157:H7 on inoculated beef carcass tissue by 2.7 log10. Water gave a 1.6 log10 reduction 2.07 (95% CI 1.48, 2.65). Concentration and type of acids used; regulatory requirements. Zhilyaev et al., 2017; Ransom et al., 2003; USDA/FSIS, 2021; EFSA, 2011; FDA, 2003; FDA, 2021a; MLA, no date Organic acids - Acetic acid STEC O157:H7 on inoculated beef carcass - 2% acetic acid reduced levels by 1.4 log10 in a laboratory study. Water gave a 1.6 log10 reduction. Using 2% acetic acid on beef brisket fat for 12 s immediately after being inoculated with faecal matter. STEC O157:H7 was reduced by 3.69 log10. Ransom et al., 2003; Cabedo, Sofos and Smith, 1996; Zhilyaev et al., 2017 Oxidizer-type antimicrobials Peroxyacetic acid is approved by USDA FSIS for washing, rinsing, cooling, or otherwise processing fresh beef carcasses. Under laboratory conditions, researchers have achieved between 1-1.4 log10 reductions in STEC O157:H7 inoculated onto beef carcass tissue. In a commercial trial, the effect of a solution of 0.02% peroxyacetic acid on chilled beef quarters was investigated at two slaughter plants. The study found little effect on total bacteria or E. coli levels on meat from one of the plants, and no effect in the other plant. Regulatory requirements. Ransom et al., 2003; Gill and Badoni, 2004; FDA, 2021b ANNEX 2 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 155 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Electrolysed (EO) water Electrolysed (EO) water is produced by passing of electrical current through a dilute saltwater solution. One product of the reaction is sodium hydroxide (NaOH) and the other is hypochlorous acid, which has a low pH, contains active chlorine, and has a strong oxidation-reduction potential similar to that of ozone. Wheeler, Kal- chayanand and Bosilevac, 2014 Oxidizer-type antimicrobials – acidified sodium chlorite (ASC) Some studies have demonstrated a 1.9–2.3 log10 reduction in Salmonella and STEC O157:H7 on beef carcass tissue using a wash or spray of sodium chlorite activated with citric acid. One laboratory trial showed up to 4.6 log10 reduction in STEC O157:H7 and Salmonella using a water wash followed by an acidified sodium chlorite spray. Other studies indicated limited success, and found that spray treatment with acidified sodium chlorite was not as effective at reducing STEC O157:H7 on beef flanks as spray treatments with hot water, lactic acid or peroxyacetic acid. Method of activation and application and the contact time with the meat surface. Ransom et al., 2003; Castillo et al., 1999; Gill and Badoni, 2004; Kal- chayanand et al., 2012 Ozone STEC was reduced between 0.6-1.0 log10 on beef samples when exposed to 72 ppm of ozone. The results of that study indicated no difference in numbers of STEC O157:H7 and S. Typhimurium detected on the surfaces of a hot carcass after exposure to water wash containing 95 ppm ozone as compared to water alone. Oxidation of fat and muscle pigments. Coll Cárdenas et al., 2011; Castillo et al., 2003 Oxidizer-type antimicrobials - Sodium hypochlorite (NaOCl) Beef carcasses sprayed (4.22 kg/cm2; 4.2 L/ min) NaOCl solution with 50, 100, 250, 500, and 800 ppm of chlorine at 28 °C. E. coli was reduced from 0.5 - 1.28 log10 CFU/ cm2 by these treatments, but the reduction is not significantly different from that of water. Cutter and Siragusa, 1995 Trisodium phosphate Sprayed onto beef carcasses 5.62 kg/cm2, 32°C for 15 s with 12% trisodium phosphate (pH 12.31). Initial wash with water reduced STEC O157:H7 by more than 2.5 log10 and reduced Listeria and Clostridium by 3 log10. Dorsa, Cutter and Siragusa, 1996 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 156 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Combination of steam and lactic acid Steam + lactic acid is defined as steam pasteurization (steam applied to a carcass at a temperature greater than or equal to 82.2°C for 6–11 s.) followed by a rinse of 2% lactic acid. STEC O157:H7 was reduced between 1 to 1.5 log10 with rinse of 1%, 3%, or 5% acetic, lactic, or citric acid; STEC O157:H7 and Salmonella Typhimurium were reduced 3.8 log10; STEC O157:H7 and Salmonella Typhimurium were reduced 4.5 log10; odds ratio of NTS E. coli carcass contamination = 0.01. Cutter and Siragusa, 1994; Castillo et al., 1999; Greig et al., 2012 Dry chill Dry chill is defined as chilling following final carcass wash without the use of an acid or water spray chilling. Conventional chilling can reduce the microbial populations on carcasses by 0.3-0.7 log10, and can reduce E. coli counts by up to 2 log10 over 24-36 h. Some research shows APC loss and then recovery when simulated carcass conditions are used in a broth system. Meta-analysis of before and after effects conducted in commercial (large or small) slaughterhouses and pilot plants for 4 study consisting of 9 trials. Odds ratio of NTS E. coli carcass contamination = 0.17. Bacon et al., 2000; Nortjé, and Naudé, 1981; Thomas et al., 1977; McEvoy et al., 2004; Gill, 1986; Chang et al., 2003; Mellefont, Kocharunch- itt and Ross, 2015; Greig et al., 2012; Gill and Bryant, 1997 Carcass spray chilling Spray-chilling had only little effect on microbial populations when it is used. There was a higher likelihood of detecting E. coli after spray chilling. Greer and Dilts, 1988; Kinsella et al., 2006; Greig et al., 2012 ANNEX 2 – PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 157 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Carcass chilling In all experiments, the inactivating effects of oxidants were greatest on fat surfaces and much less effective on lean surfaces. ClO2 at 15 ppm, caused higher log10 reductions in E. coli numbers (approximately 3 log10 reduction) when applied during spray chilling than when applied immediately prior to "normal" spray chilling (approximately 1 log10 reduction). Abattoir trial: spray chilling treatments (with water alone, peroxyacetic acid, PAA at 200 ppm or chlorine dioxide, ClO2 at 50 ppm); water alone was effective at the hindquarters (hind legs and bung), indicator bacteria substantially reduced. Antimicrobial, either PAA (200 ppm) or ClO2 (50 ppm) was added to the spray chill water, the reduction in indicator bacteria was enhanced at all carcass sites, especially hindquarters, NTS E. coli eliminated. STEC might become more susceptible to oxidative damage when exposed to carcass chilling. (Chlorine dioxide, ClO2 or peroxyacetic acid, PAA) on beef meat during a simulated spray chilling process (sprayed for 4 s every 15 min for 36 cycles) and/or when applied (sprayed for 144 s) prior to spray chilling with water. King et al., 2016; Ko- charunchitt, 2020 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 158 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 4.1 Physical Interventions Air-drying heat treatment Treatment at 60 °C to 100 °C for 5 s to 600 s; beef cuts for catering; STEC O157:H7 reduction of 1.3 -6.1 log10 CFU/cm2. Impact on appearance and colour; selection of resistant subpopulation; laboratory based study. McCann et al., 2006 Condensing Steam Treatment at 75 °C for 10 s at 38.6 Kpa; reduction of STEC O157:H7 by 1.5 log10 CFU cm2 using meat slices. Laboratory study. Logue, Sheridan and Harrington, 2005 Hot water Water at 82 °C sprayed for 20 s; STEC O157:H7 reduction of 1.0 log10 CFU/100 cm2 on sub-primals before mechanical tenderization-blade tenderization. Can cause temperature change. Heller et al., 2007 Water at 82 °C, aerobically or anaerobically (559 mm/Hg vacuum) for 3 min; Escherichia coli (ATCC 11775; EC); hot water treatment of beef trimmings before grinding did not reduce any microorganism. Laboratory study. Stivarius et al., 2002 Inoculated trimmings were exposed to hot water treatment, using about 23 L of water at 95 °C for 3 s (3 s was required for the surface to reach 82 °C). Beef trimmings from young or mature cattle were treated with hot water and challenged with STEC O157:H7 had a reduction of 0.9 log10 CFU/g (5.2-4.3 log10). Laboratory study. Ellebracht et al., 1999 Surface trimming The external surface was trimmed away using a sterile knife. Subprimals with antimicrobial interventions before mechanical tenderization/blade tenderization had a STEC O157:H7 reduction of 1.1 log10 CFU/100 cm2. Loss of products. Heller et al., 2007 Post-processing control strategies for STEC in beef Annex 3 ANNEX 3 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 159 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Surface trimming Partial-surface trimming, full-surface trimming; STEC O157:H7 inoculated on vacuum packaged sub-primals. High- inoculum level: reduction of 4.0 log10 CFU/ cm2 (from 4.8 ≤ 0.7). Low-inoculum level: reduction of 2.0 log10 CFU/cm2 (from 2.9 ≤ 0.7). Laboratory based. Jacob et al., 2011 Dry chilled ageing All samples were suspended in a cold room (3 °C), with four defrost time periods of 20 min, an air velocity of 0.25 m/s, and a relative humidity of 80%. STEC O157:H7 on beef had a reduction of 4 log10 CFU/cm2. Laboratory based. Packaging aerobically, stored for 5 d at 7 °C. Beef cuts inoculated with STEC O157:H7; Reduction between 1.9-2.2 log10 (if previously vacuum packed at 12 °C). Sensory changes not assessed. Ashton et al., 2006 Blast Freezing: Bovine bulk manufactured cartons of beef; in frozen storage (minimum -18 °C) for a minimum period of 6 weeks. Seven STEC serogroups (O157, O26, O103, O111, O121, O145 and O45) showed a reduction by 70% of positive lots. Plant based study. Koh, 2020 High pressure processing (HPP) Vacuum packaged sub-primals, 1 to 2 °C, 120 days. Neither carcass nor intervention treatment had any significant (P > 0.05), beneficial effect on the microbiological quality of sub-primal cuts. HPP treated ground beef. Single-cycle. 400 MPa 12 °C for a 1- to 20-min cycle. Multiple-cycle four 1-min cycles at 400 MPa and 12 °C and three 5-min cycles; 4-7 °C, 450 MPa and 15 min. Laboratory data. Kenney et al., 1995; Morales et al., 2008; Hsu et al., 2015 Irradiation Irradiation (eBeam) 1 KGy. Sub-primals prior to mechanical tenderization inoculated with STEC and STEC O157:H7 - 4 log10 reduction. Irradiation (eBeam) on ground beef of 1 KGy. Irradiation (Gamma) of 2.5 KGy and of 0.5 or 2 KGy on trim, prior to grind. Laboratory data, costly equipment; no effect on sensory quality below 5 KGy. Kundu et al., 2014; Arthur et al., 2005; Arthur et al., 2005; De la Paz Xavier et al., 2014; Cap et al., 2020 Irradiation and packaging Ground ground beef packaged at 4°C; < 2 KGy. MAP treatment of ground beef meat balls. Irradiation (gamma) at 1.5 KGy in MAP (3% O2+ 50% CO2+ 47% N2) or aerobic packages. MAP/vacuum packaging of Ground beef patties. Irradiation (eBeam) at 0.5, 1, or 1.5 KGy in MAP (99.6% CO2, 0.4% CO) or vacuum. Lab data, costly equipment. Sommers et al., 2015; Gunes et al., 2011; Kudra et al., 2011 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 160 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Irradiation and organic acid Trim treated with radiation (eBeam)/lactic acid; Lactic acid (LA) – 5% (55 °C), 1 KGy in aerobic or vacuum packages (4 °C). Trim treated with radiation (gamma)/lactic acid/caprylic acid: Lactic acid (LA) 0.5% (50 °C); Caprylic acid (CA) – 0.04% (50 °C); 3. 0.5 - 2 KGy (12 °C). Laboratory data; costly equipment. Li et al., 2015; Cap et al., 2020 4.2 Chemical Interventions Organic acids Lactic acid treatment of sub-primals, trim and cheek meat; various studies using STEC O157:H7 strains and/or NTS E. coli. Range of reductions achieved - STEC O157:H7, 0.2-2.8 log10; NTS E. coli 0.2-3.4 log10. Review of 20 studies (18 involved artificial inoculation, 1 controlled trial and 1 before and after trial). Antic, 2018 Hydroxypropanoic acid: Thin slices from sub- primals and primal cores inoculated with a 4- strain cocktail of STEC O157:H7; incubated for 1h-14 d at 4 °C. STEC O157:H7 reduction: 1 h -0.67 log10; 1 d - 0.89 log10; 7 d -1.47 log10. Pilot plant tenderization; No sensory evaluation: Residual antimicrobial activity questions. Muriana et al., 2019 Lactic acid dip; Beef trim for ground beef. Two 4 strain cocktails - STEC O157:H7 and non O157 STEC. Treatment used 4.4% lactic acid (ambient temperature) dip and spray treatments – 5 s dip or 13 s spray - samples tested after 1 h and after 20 h vacuum packed at 4°C then later ground to produce ground beef. Sensorial quality could change depending on the exposure time. Wolf et al., 2012 Other chemical treatments Sulfuric acid-sodium sulfate blend (SSS): Mixtures of STEC O157:H7 (5 strains), non-O157 STEC (12 strains). 2 spray treatment levels on pre-rigor beef resulted in 0.6–1.5 log10 CFU/cm2 reduction. Laboratory study, safety implications. Scott-Bullard et al., 2017 ANNEX 3 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 161 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Other chemical treatments Hypobromous acid, neutral acidified sodium chlorite, and two citric acid-based compounds used in treatment of ground beef: Cocktail 1 was STEC O26, O103, O111, O145, and O157; Cocktail 2 STEC O45, O121, O157 and Salmonella seeded at high cfu (1.5x107) and low cfu (1.5x104) inoculum as a spray for 15s and then quantified after 10 min and 48 h at 4 °C with 48 h storage. The treatments resulted in 0.7- 2.3 log10 reductions of STEC. Reductions of 2 log10 or more were achieved for O26, O103 and O145 but only with the high inoculum and after chill storage following citric acid or acidified sodium chlorite. One of the citric acid products tested gave a reduction of 2 log10 for O103 following treatment. In low inoculum study none of the treatments eliminated the 7 STEC strains tested. Laboratory study Kal- chayanand et al., 2015 Disodium metasilicate: Thin slices from sub- primals and primal cores, 1 h - 14 d at 4 °C. 4 strain cocktail of STEC O157:H7. Surface spray 6%, 18-20 °C; pH 13. STEC O157:H7 reduction: 1 h - 1.06 log10; 1 d - 2.07 log10; 7 d - 3.61 log10. Pilot plant tenderization; no sensory evaluation; antimicrobial activity concerns. Muriana et al., 2019 Thin slices from sub-primals and primal cores inoculated with a 4 strain of STEC O157:H7 cocktail, incubated for 1 h-14 d at 4 °C. Treated with Lauric arginate and Peroxyacetic acid. Surface spray 5000 ppm (LA) and 220 ppm (PA). STEC O157:H7 reduction: 1 h - 1.16 log10; 1 d - 1.95 log10; 7 d - 2.18 log10. Pilot plant tenderization; no sensory evaluation; antimicrobial activity concerns. Muriana et al., 2019 Ozone Exposed to ozone (72 ppm) in a continuous ozonation chamber at 0 °C and 4 °C during 3 h and 24 h incubation. Impact on E. coli (STEC not specified) was 0.6-1.0 log10 reduction. Impact on colour and lipid oxidation. Coll Cárdenas et al., 2011 Minced beef, 500-5000 ppm dry ozone gas. Laboratory data, costly equipment, impractical to work with these concentrations. McMillin and Michel, 2000 Lactoferricin B Ground beef - Lactoferricin B (100µg/g) added and stored at 4 °C and 10 °C for 3 days to control a 5 strain STEC O157:H7 cocktail inoculated at 107 cfu/ml. Laboratory data, no sensory assessment of impact on TVC. Venkita- narayanan, Zhao and Doyle, 1999 Essential oils Thyme oil treatment of minced beef - 0.6% thyme oil then stored at 4 °C and 10 °C for up to 12 days; 2 strains of STEC O157:H7. Laboratory data; sensory changes were acceptable. Solomakos et al., 2008 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 162 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 4.3 Biological Interventions Bacterio- phages Primals treated with phage, 7 log10 PFU/4 cm2, STEC O157:H7 had a 2.7 log10 reduction (37 °C). Primals treated with a 6-phage cocktail - ~9 log10 PFU/mL. STEC and STEC O157:H7 seeded 9 log10 cfu/ml had a reduction of 0.5 - 1 log10 (4 °C) and 3-3.8 log10 (37 °C) in 48 h. Sub-primals treated with a phage cocktail - 10 log10 PFU. STEC and STEC O157:H7 reduced by 0.77 log10 (3 h), 1.15 log10 (6 h). Laboratory data; not suitable for food processing; emergence of phage insensitive mutants; only tested on small pieces of meats. Hudson et al., 2013; Tomat et al., 2013a, 2018 During packaging, 3 phage cocktails. Laboratory data, takes too long. Hong, Pan and Ebner, 2014 Lactic acid bacteria (LAB) Lactic acid bacteria (LAB) with vacuum packaging; LAB inoculum - log10 8.7 cfu/ml, 4 LAB strains - inoculum log10 7 cfu/ml. LAB inoculum with ageing strips of meat – log10 8.7 cfu/ml; STEC and STEC O157:H7. 0.4 log10/cm2 reduction (4 °C), 14-28 days. Laboratory data, takes too long; no effect on sensory quality. Smith et al., 2005a; Kirsch et al., 2017 Colicins Sub-primals of pork prior to tenderization, 3 mg colicin M + 1 mg colicin E7/kg. STEC O157:H7 seeded at 5 log10; reductions of 2.3 log10 in 1 h and 2.7 log10 in 1 d (10 °C). Laboratory data; economically made in tobacco tissue; limited data for pork. Schulz et al., 2015 Combinations of post-processing interventions Combinations of Steam/ Vacuum Meat slices treated with condensing steam at 75 °C, following vacuum packaging and stored in air or under vacuum at 0 °C; STEC O157:H7 reduction of 1.5 log10 CFU/g. Laboratory data Logue, Sheridan and Harrington, 2005 Combinations of MAP or vacuum packaging/ lactic acid Steaks treated with 10% lactic acid; STEC O157:H7 reduction of 2 log10 CFU/g. Some loss of colour Salim et al., 2017 Combinations of lactic acid and hot water Raw beef treated with 4% lactic acid, 80 °C, 20 s; STEC O157:H7 reduction of 3 log10 CFU/g. Cost Buncic et al., 2014 Combinations of vacuum packaging, ambient water and organic acids Treatment of meat inoculated with STEC O157:H7, non-O157 STEC (O26, O103, O111, and O145) treated with ambient water, 200 ppm Hypobromous acid, 200 ppm peroxyacetic acid, and 5% lactic acid. Spray and stored vacuum packed at 4 °C for 14 d. STEC O157:H7 reduction of 1.6-2.1 log10 CFU/50 cm2; non-O157 STEC reduction was smaller at 0.4 -0.3 log10 CFU/50 cm2. Laboratory study Liao et al., 2015 ANNEX 3 – POST-PROCESSING CONTROL STRATEGIES FOR STEC IN BEEF 163 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Combinations of Lauric arginate, water or other chemicals Beef trim - Spray treatment with lauric acid (LA) 5% or followed by 0.4% cetylpyridinum chloride (CC), 4% sodium metasilicate (SM), 0.02% peroxyacetic acid (PA), 10% trisodium phosphate (TSP) or sterile water (SW) prior to grinding. Incubated at 4 °C for up to 3 d under simulated retail conditions. Inoculated with cocktail including 1 STEC O157:H7 and 6 non-O157 STEC strains. Laboratory testing storage simulated retail conditions, no impact on colour of ground beef. Dias-Morse et al., 2014 Combinations of essential oil and colicin (nisin) Minced beef treated with thyme oil (essential oils) and colicin (nisin). 4 °C and 10 °C for up to 12 d; 0.6% thyme oil; 2 strains 4°C and 10 °C for up to 12 d; 0.6% thyme + nisin (500 IU/ml). Laboratory data; sensory changes were acceptable. Solomakos et al., 2008 Combinations of irradiation (eBeam) and lactic acid Trim treated with radiation (eBeam)/lactic acid; Lactic acid (LA) – 5% (55 °C); 1 KGy in aerobic or vacuum packages (4 °C). Laboratory data; costly equipment. Li et al., 2015 Combinations of irradiation (eBeam) and packaging Combination of radiation (eBeam)/MAP/ vacuum packaging of ground beef patties; 0.5, 1, or 1.5 KGy in MAP (99.6% CO2, 0.4% CO) or vacuum. Laboratory data, costly equipment Kudra et al., 2011 Combinations of irradiation (gamma) and organic acids Trim treated with radiation (gamma)/lactic acid/caprylic acid; Lactic acid (LA) 0.5% (50 °C); Caprylic acid (CA) – 0.04% (50 °C); 0.5 - 2 KGy (12 °C). Laboratory data; costly equipment. Cap et al., 2020 Combinations of irradiation (gamma) and MAP Radiation (gamma)/MAP treatment of ground beef meat balls; 1.5 KGy in MAP (3% O2+ 50% CO2+ 47% N2) or aerobic packages. Laboratory data, costly equipment. Gunes et al., 2011 Combinations of HPP and vacuum packaging Frozen ground beef vacuum-packaged, pressure-treated at 400 MPa for10 min at -5 °C or 20 °C and stored at -20 °C or 4 °C for 5–30 d; four, 60 s cycles, 400 MPa, 17 °C. Laboratory based Black et al., 2010; Jiang et al., 2015 High Pressure Processing (HPP) combinations HPP and freezing of ground beef 25 °C, 400 MPa at five pressure cycles of 3 min. GAP; additional studies to assess further sensory changes in ground beef; cost. Zhou, Karwe and Matthews, 2016 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 164 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 5.1 Raw milk processing Bactofugation Milk heated to 55 °C to 60 °C, Enterobacteriaceae 72% removal; double bactofugation at 54.4 °C, removed 95% of E. coli. Cost, volume that can be processed at one time. Laboratory-based data. No STEC data. Faccia et al., 2013 Microfiltration 1.4 µm ceramic filter; skim milk only and at 50 °C. Effectively removed 3-5 log10 CFU/ml to be non-detectable. Cost, volume that can be processed at one time. No E. coli or STEC data. Laboratory-based data. Some data on cold (6 °C) microfiltration. Elwell and Barbano, 2006 High Pressure Processing (HPP) 400 MPa at 50 °C for 15 m; 5 log10 CFU/mL reduction of STEC O157:H7 in 15 m in UHT milk. Earlier study observed significant strain to strain variations. 400 MPa at 21 °C to 31 °C for 50 m; 6 log10 CFU/mL reduction of E. coli in 30 m in human milk but 8 log10 CFU/mL after 10 m in peptone solution. E. coli ATCC 25922 (Stx negative) was inactivated by 8 log10 after 10 m in peptone solution and by 6 log10 after 30 m in human milk. Temperature used may not be acceptable for definition of “raw milk”. Laboratory- based data. Cost, volume that can be processed from bovine or ruminant milk. Limited data for NTS E. coli only. Laboratory based data. Patterson and Kilpatric, 1998; Viazis, Farkas and Jaykus, 2008 Irradiation (cold pasteur- ization) eBeam at 1 to 2 kGy; 1 kGy eliminated coliform; 2 kGy - 4 log10 CFU/mL reduction in aerobic count; STEC O157:H7 D10 value - 0.062 kGy. Off-flavors in dairy products in laboratory-based studies. Ward, Kerth and Pillai, 2017 Bacteriophage 6 phage cocktail,- 9 log10 PFU/ml used in inoculated challenge studies with NTS E. coli and STEC O157:H7. E. coli and STEC O157:H7 - seeded at 4 log10 CFU/mL. 2 log10 CFU/mL reduction in 1 d at 4 °C. Takes too long for practical applications; sensory attributes not affected. Laboratory based data. Tomat et al., 2018 Processing and post-processing control strategies for STEC in raw milk and raw milk cheese Annex 4 ANNEX 4 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESE 165 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 7.3 Laboratory testing for STEC detection across the dairy processing chain 7.3.1.Raw milk STEC and stx gene in raw milk and milk filters In study over 1-year STEC O157:H7 recovered from 2% of milk filters but not from raw milk. Stx gene detected in 37% of milk filters and 7% of raw milk. Filters are more effective for detection of pathogens than reliance on sampling from the bulk tank. It has been used on farm, so is technically feasible. Filter type, duration, location flow rate of milk. Milk filters can be optimized for bacterial detection. Jaakkonen et al., 2019 Sampling plans Large-scale milk collection: universal sampling plan (bulk tank samples); small- scale milk collection: periodic, random or composite sampling all have roles but depending on local conditions. In Germany, STEC are included in the testing of bulk tank milk from cattle, sheep and goats.roles but depending on local conditions. Suggested indicator criteria: APC - n=5, c=2 m=2x104, M=5x104; Enterobacteriaceae- n=5, c=1, m=10, M=102; S. aureus - n=5, c=2, m=10, M=102 In developed and certain developing countries, testing raw milk filters can offer a better sampling point that bulk tank sampling. In Italy, researchers identified the presence of STEC O157:H7, O45, O103, O121 and O145 in bulk tank milk and raw milk filter samples collected at commercial dairy plants. Limitations of bulk tank and plant sampling; sampling and testing plans vary by country and region; under unfavourable hygiene and temperature conditions in the supply chain; microbial limits should be adjusted as the situation improves. Most testing plans target E. coli and other indicator organisms. Only a few countries (e.g. Germany) test for STEC; other do so based on risk assessment. FDA/ USPHS, 2017; Draaiyer et al., 2009; ICMSF, 2011; FAO/ WHO, 2018; Albonico et al., 2017 5.2 Raw milk cheese processing 5.2.1 Milk fermentation Milk fermentation Autochthonous strains of LAB: The bacteriocins produced by isolates showed antimicrobial activity towards different spoilage and pathogenic microorganisms. Laboratory data for Listeria monocytogenes, Staphylococcus aureus, Clostridium tyrobutyricum and Brochothrix thermosphacta. Dal Bello et al., 2010 5.2.2 Protective cultures Protective cultures Addition of a consortium of H. alvei, Lactobacillus plantarum and Lactococcus lactis - Average of 2.8 log10 CFU/g reduction of STEC O26:H11 in uncooked pressed cheeses made from different raw milk batches. Variation in reduction depending on the milk microbiota (positive/ negative interactions between the microbiota, consortium and STEC); in pilot plant challenge studies. Fretin et al., 2020 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 166 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 5.2.3 Bacteriophage Bacteriophage Phage cocktail - Inactivated E. coli and STEC O157:H7 in 8 h. Without phage treatment, NTS E. coli grew to 4-6 log10 cfu/g in 6 h. Starter culture - not affected; under laboratory-based conditions. Tomat et al., 2013b 5.2.4 Acidification, salting and cooking Lactic acid - dry salting on surface (for lactic goat cheeses) Temperature/time 24 °C/24 h; decrease of pH (4.21 at day 2) versus variety of STEC. No increase (except 1 log10 CFU/g for STEC O26 during the first hours). Reduction of STEC O26, O103, O145 and O157 to levels below enumeration limit (i.e. <10 CFU/g) (but still detectable by enrichment). Effective in pilot plant challenge studies. Acidification (pH < 4.3) is necessary for efficacy. Miszczycha et al., 2013 Rennet - pressing - brining (for uncooked pressed cheese with short ripening) Temperature/time 34 °C/30 m; decrease of pH (5.3 at day 1). Increase of 3.3 to 5 log10 CFU/g ue to growth and entrapment. Growth variations between serotypes; acidification must be below pH 5 for efficacy. Studies performed in pilot plants. Miszczycha et al., 2013 Acidification, salting and cooking Sharp increase of 2.2 to 3 log10 CFU/g, after 6 h, for cheeses inoculated with STEC O26:H11 at 0.05 and 0.5 CFU/mL respectively. pH must be < 5.0 for efficacy; in pilot plant challenge studies. Fretin et al., 2020 Rennet - pressing - dry salting of curd (for uncooked pressed cheese with long ripening) Temperature/time 32 °C/45 m decrease of pH (5.19 at day 1). Increase of 2 log10 CFU/g (STEC O157:H7), 4 log10 CFU/g (STEC O26) due to growth and entrapment. The pH must be < 5.0 for efficacy. Growth variation between serotypes (faster for STEC O26) during pilot plant challenge studies. Miszczycha et al., 2013 Rennet - dry salting on surface (for blue type cheese) Temperature/time 32.5 °C/1 h; decrease of pH (4.91 at day 3). Increase of 1 log10 CFU/g (STEC O157:H7), 2 log10 CFU/g (STEC O103), 3 log10 CFU/g (STEC O26) due to growth and entrapment. pH must be < 4.91 for efficacy. Growth variation between serotypes (faster for STEC O26) in pilot plant challenge studies. Miszczycha et al., 2013 Rennet - brining (for white mold cheese) Temperature/time 32.5 °C/1 h; decrease of pH (4.91 at day 3). Increase of 2 log10 CFU/g (STEC O157:H7) to 3 log10 CFU/g (STEC O26, O103, and O145), cdue to growth and entrapment. pH must be < 5.0 for efficacy. Growth variation between serotypes. Miszczycha et al., 2016 ANNEX 4 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESE 167 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Rennet - curd cooking - brining - pressing (for cooked pressed cheese) Temperature/time 54 °C/35 m; decrease of pH (5.38 at day 1). No increase. Reduction of STEC O26, O103, O145 and O157 to below enumeration limit at 1.75 h. Cooking step (> 53 °C) needed for efficacy. Pilot plant challenge test. Miszczycha et al., 2013 Curd cooking Temperature/time, 53 °C/20 m. > 4.5 log10 CFU/g reduction of heat-sensitive NTS E. coli. Cooking step (>53°C) needed for efficacy. 0.5 log10 CFU/g reduction of thermotolerant NTS E. coli. 2 NTS E. coli behaving similar to STEC in pilot plant challenge study. Peng et al., 2013b Acidification - brine -curd cooking Temperature/time/acidification/brine 53 °C to 40 °C/2 h and further steps/8 kg. > 4 log10 CFU/g reduction of thermotolerant NTS E. coli. Cooking step (> 53 °C) needed for efficacy. 2 NTS E. coli behaving similar to STEC in pilot plant challenge study. Peng et al., 2013b 5.2.5 Ripening and ageing Lactic goat cheese At 4 °C for 20 d then at 8 °C for 35 d. Increase of pH to 5.26 at day 25 (constant till day 60). The aw decreased in the core from 0.994 (day 2) to 0.967 (day 45). Further reduction for 4/8 strains (not detectable by enrichment at day 60). Ripening: efficient to prevent growth after acidification (but not for elimination). Effective in pilot plant challenge studies. Miszczycha et al., 2013 Cooked pressed cheese At 9 °C - 10 °C for 4 months; pH 5.38 stable till day 30 then increase to 5.82 at day 120. Core: aw 0.975 at day 120. Ripening: efficient to prevent growth after cooking (but not for elimination). In the core: not detected except for 1/4 replicate (STEC O157:H7); In the rind, detected by enrichment for 3/4 replicates. Variation in cell levels between core/rind. Effective in pilot plant challenge studies. Miszczycha et al., 2013 Uncooked pressed cheese with short ripening Uncooked pressed cheese with short ripening at 12 °C for 12 d - 20 d then 4 °C for 12-20 d then 8 °C for 8 d; increase of pH to 5.80 at day 40. The aw in the core remained constant (0.974). Ripening: not efficient for reduction. aw > 0.95 (minimum aw). No reduction (STEC levels remained constant till day 40). Effective in pilot plant challenge studies. Miszczycha et al., 2013 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 168 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Uncooked pressed cheese with long ripening Uncooked pressed cheese with long ripening at 9 °C -10 °C for 7 months. Small increase of pH to 5.52 in the core (constant till day 240). pH higher in the rind (7.34-7.64). In the core, the aw decreased slowly to reach 0.943 at day 240. In the rind, aw decreased to 0.922 at day 240. Reduction (>2 log10 CFU/g) from day 60–240. STEC O157:H7 and STEC O26 below enumeration limit (except for STEC O26, 3 log10 CFU/g core). Ripening: efficient for reduction (if duration > 60 d), but not for elimination Variation between serotypes (STEC O26 more persistent inside the core). Effective in pilot plant challenge studies. Miszczycha et al., 2013 Blue type cheese At 11 °C (16 d), -2 °C (125 d), 4 °C (30 d), 12 °C (60 d). Increase of pH to 6.69 at day 25, then decrease from day 100 to day 240 (pH 5.5). The aw gradually decreased to reach 0.898 at day 240. Reduction depending on time and serotypes: STEC O157:H7 and STEC O103 detectable at day 60 but not at day 240; STEC O26 detected at day 240 only after enrichment. Ripening: efficient (if duration >60 d) for reduction, but not for elimination. Combination of negative temperature, very low aw 0.898 (< 0.95 minimum aw), acidic pH and impact of Penicilium roqueforti. Variation between serotypes (STEC O26 more persistent). Effective in pilot plant challenge studies. Miszczycha et al., 2013 White mold cheese At 13 °C (14 d), 4°C (14 d), 8 °C (28 d). Increase of pH to 5.7-6.1 (core) or 6.5 (rind) at day 56. The aw gradually decreased but remained > 0.96 at day 56. Efficient for STEC O157:H7, not for non-O157 STEC. aw > 0.95 (minimum aw); high pH (rind). Reduction depending on serotypes: 1-2 log10 CFU/g (STEC O26, O103 and O145); 2-3 log10 CFU/g (STEC O157:H7; detected only after enrichment). Higher STEC level in rind compared to core. Variation between serotypes (STEC O157:H7 less persistent) and in core/ rind in pilot plant challenge studies. Miszczycha et al., 2016 Soft cheese Ripening at 11 °C for 2 weeks, efficient for STEC O157:H7, less efficient for non-O157 STEC. The survival probability after 2 weeks ripening was 1%, 34%, 37%, and 27%, respectively, for STEC O157:H7, O103:H2, O26:H11, and O145:H28. Variation between serotypes (STEC O157:H7 less persistent) in pilot plant challenge studies. Perrin et al., 2015 ANNEX 4 – PROCESSING AND POST-PROCESSING CONTROL STRATEGIES FOR STEC IN RAW MILK AND RAW MILK CHEESE 169 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH Hard cheese Cooking at 53 °C, 11 kg, up to 16 weeks ripening. Positive for thermotolerant E. coli in 2/8 samples by enrichment after 4 weeks. Positive for heat-sensitive E. coli in 1/12 samples by enrichment after 16 weeks. Temperature gradient from rind to core during first day, samples taken close to rind; effective in pilot plant challenge studies. Peng et al., 2013b Semi-hard cheese Cooking at 46 °C, 7.5 kg, up to 16 weeks ripening. Approx. 0.4 log10 CFU/g reduction/week in cheese core. Approx. 0.2 log10 CFU/g reduction/week in cheese rind low level of E. coli in raw milk (approx. 100 CFU/mL): < LOQ in cheese core after 8 weeks, but detectable by enrichment after 16 weeks. In rind, E. coli was quantifiable until end of ripening. 2 NTS E. coli behaving similar to STEC; effective in pilot plant challenge studies. Cooking at 46 °C, 250g, up to 16 weeks ripening. 0.25-0.79 log10 CFU/g reduction/week (depending on strain). 1 STEC showed fastest reduction: not possible to enrich after 16 weeks, others had no significant difference. 3 non-O157-STEC and 2 NTS E. coli, monitored separately in pilot plant challenge studies. Peng et al., 2013b Cooking at 40 °C, 250g, up to 16 weeks ripening. 0.23-0.58 log10 CFU/g reduction/week (depending on strain from NTS E. coli and 3 non O157 STEC). 1 STEC showed fastest reduction: not possible to enrich after 16 weeks, others had no significant reduction. 3 non-O157-STEC and 2 NTS E. coli, monitored separately in pilot plant challenge studies. 5.2.6 Cheese size Cheese Size Temperature gradient in hard and extra hard cheeses after cooking at first 24 h. For information: heat map of temperature gradient. Sampling of rind and core differed. Smaller size cheese differed from larger cheeses and impacted STEC survival in laboratory versus production scale cheese making. Ercolini et al., 2005 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 170 TYPES EVIDENCE TO SUPPORT INTERVENTION DETAILS OF RESEARCH STUDIES OTHER CONSIDERATIONS DEGREE OF SUPPORT CITATIONS LOW MED HIGH 5.3 Raw milk cheese post-processing Packaging Modified atmosphere packaging (MAP) can retard growth of pathogens in a hard cheese made of raw sheep milk, but a higher decrease in numbers of L. monocytogenes and S. aureus were observed than for STEC O157:H7. Active packaging is used the food industry mainly to extend shelf-life, but also to prevent growth of pathogens. Active packaging technologies applications in cheese are still very limited and further studies are needed to explore the potential and efficiency of these new technologies. Solomakos et al., 2019; Yildirim et al., 2018; Speranza et al., 2020; Al-Moghazy, Mahmoud and Nada, 2020 Irradiation (eBeam) 0.2 - 2 kGy, Brie and Camembert - 1.27–2.59 kGy, controlled growth. Tested with L. monocytogenes; no sensory defect in laboratory-based study Velasco et al., 2015 Bacteriophage 3-phage cocktail; multiplicity of infection - 1; at 24 °C - STEC O157:H7 seeded at 7 log10 CFU/g. No reduction in STEC in cheese slices at room temperature. Emergence of phage resistant STEC O157:H7 (and potentially other STEC). Studies performed in cheese slices under laboratory conditions. Hong, Pan and Ebner, 2014 7.3 Laboratory testing for STEC detection across the dairy processing chain 7.3.1.Raw milk Sampling plans Systematic selection of quality raw milk with E. coli counts < 50 CFU/mL for raw milk cheese manufacturing was predicted to reduce HUS risk. Various criteria could reduce the risk of HUS by 25% to 89% with probabilities of noncompliance between 3% and 79%. Increasing analysis sample size of end products from 25 g to 100 g for STEC detection was predicted to reduce HUS risk. In cheeses made from pasteurized milk The ICMSF (2011) recommended E. coli limits established under a 3-class sampling plan where n = 5, c = 3, m = 10 and M = 100. Raw milk cheese is tested for S. aureus only, consistent with EU recommended sampling criteria. There are general recommendations for microbiological sampling programs to assure hygiene and safety, but none are specific to STEC. It is notable that for EU microbiological criteria for cheese, no limits were established for STEC in raw milk cheese. Differences by type of cheese. ICMSF, 2011; Perrin et al., 2015; Anses opinion, 2018; Donnelly, 2018 171REFERENCES FOR ANNEXES 1-4 References for annexes 1-4 Ahmad, A., Nagaraja, T. G. & Zurek, L. 2007. Transmission of Escherichia coli O157:H7 to cattle by house flies. Preventative Veterinary Medicine, 80: 74–81. doi: 10.1016/j. prevetmed.2007.01.006 Albonico, F., Gusmara, C., Gugliotta, T., Loiacono, M., Mortarino, M. & Zecconi, A. 2017. 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A critical literature review to assess the significance of intervention methods to reduce the microbiological load on beef through primary production. University of Liverpool. Food Standards Agency Project FS301044. https://www. food.gov.uk/print/pdf/node/4506 Antic, D., Blagojevic, B., Ducic, M., Mitrovic, R., Nastasijevic, I. & Buncic, S. 2010a. Treatment of cattle hides with Shellac-in-ethanol solution to reduce bacterial transferability--a preliminary study. Meat Science, 85: 77–81. doi: 10.1016/j. meatsci.2009.12.007 Antic, D., Blagojevic, B., Ducic, M., Nastasijevic, I., Mitrovic, R. & Buncic, S. 2010b. Distribution of microflora on cattle hides and its transmission to meat via direct contact. Food Control, 21: 1025–1029. doi: 10.1016/j.foodcont.2009.12.022 Antic, D., Blagojevic, B. & Buncic, S. 2011. Treatment of cattle hides with shellac solution to reduce hide-to-beef microbial transfer. Meat Science, 88: 498–502. doi: 10.1016/j.meatsci.2011.01.034 Arthur, T. 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Food Microbiology, 58: 7–12. doi: 10.1016/j. fm.2016.02.010 FAO/WHO MICROBIOLOGICAL RISK ASSESSMENT SERIES 199 FAO/WHO Microbiological Risk Assessment Series 1 Risk assessments of Salmonella in eggs and broiler chickens: interpretative summary, 2002 2 Risk assessments of Salmonella in eggs and broiler chickens, 2002 3 Hazard characterization for pathogens in food and water: guidelines, 2003 4 Risk assessment of Listeria monocytogenes in ready-to-eat foods: interpretative summary, 2004 5 Risk assessment of Listeria monocytogenes in ready-to-eat foods: technical report, 2004 6 Enterobacter sakazakii and other microorganisms in powdered infant formula: meeting report, 2004 7 Exposure assessment of microbiological hazards in food: guidelines, 2008 8 Risk assessment of Vibrio vulnificus in raw oysters: interpretative summary and technical report, 2005 9 Risk assessment of choleragenic Vibrio cholerae O1 and O139 in warm-water shrimp in international trade: interpretative summary and technical report, 2005 10 Enterobacter sakazakii and Salmonella in powdered infant formula: meeting report, 2006 11 Risk assessment of Campylobacter spp. in broiler chickens: interpretative summary, 2008 12 Risk assessment of Campylobacter spp. in broiler chickens: technical report, 2008 13 Viruses in food: scientific advice to support risk management activities: meeting report, 2008 14 Microbiological hazards in fresh leafy vegetables and herbs: meeting report, 2008 15 Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formula: meeting report, 2008 16 Risk assessment of Vibrio parahaemolyticus in seafood: interpretative summary and technical report, 2011 17 Risk characterization of microbiological hazards in food: guidelines, 2009. 18 Enterohaemorrhagic Escherichia coli in raw beef and beef products: approaches for the provision of scientific advice: meeting report, 2010 CONTROL MEASURES FOR SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) ASSOCIATED WITH MEAT AND DAIRY PRODUCTS 200 19 Salmonella and Campylobacter in chicken meat: meeting report, 2009 20 Risk assessment tools for Vibrio parahaemolyticus and Vibrio vulnificus associated with seafood: meeting report, 2020 21 Salmonella spp. in bivalve molluscs: risk assessment and meeting report, in press 22 Selection and application of methods for the detection and enumeration of human pathogenic halophilic Vibrio spp. in seafood: guidance, 2016 23 Multicriteria-based ranking for risk management of foodborne parasites, 2014 24 Statistical aspects of microbiological criteria related to foods: a risk managers guide, 2016 25 Risk-based examples and approach for control of Trichinella spp. and Taenia saginata in meat: meeting report, 2020 26 Ranking of low-moisture foods in support of microbiological risk management: meeting report and systematic review, 2022 27 Microbiological hazards in spices and dried aromatic herbs: meeting report, 2022 28 Microbial safety of lipid based ready-to-use foods for management of moderate acute malnutrition and severe acute malnutrition: first meeting report, 2016 29 Microbial safety of lipid based ready-to-use foods for management of moderate acute malnutrition and severe acute malnutrition: second meeting report, 2021 30 Interventions for the control of non-typhoidal Salmonella spp. in beef and pork: meeting report and systematic review, 2016 31 Shiga toxin-producing Escherichia coli (STEC) and food: attribution, characterization, and monitoring; report, 2018 32 Attributing illness caused by Shiga toxin-producing Escherichia coli (STEC) to specific foods: report, 2019 33 Safety and quality of water used in food production and processing: meeting report, 2019 34 Foodborne antimicrobial resistance: role of the environment, crops and biocides: meeting report, 2019. 35 Advance in science and risk assessment tools for Vibrio parahaemolyticus and V. vulnificus associated with seafood: meeting report, 2021. 36 Microbiological risk assessment guidance for food: guidance, 2021 37 Safety and quality of water used with fresh fruits and vegetables, 2021 38 Listeria monocytogenes in ready-to-eat (RTE) foods: attribution, characterization and monitoring: 2022 39 Control measures for Shiga toxin-producing Escherichia coli (STEC) associated with meat and dairy products: meeting report, 2022

Although Shiga toxin-producing Escherichia coli (STEC) have been isolated from a variety of food production animals, they are most commonly associated with ruminants from which we derive meat and milk. Because of the widespread and diverse nature of ruminant-derived food production, coupled with the near ubiquity of STEC worldwide, there is no single definitive solution for controlling STEC that will work alone or in all situations. Instead, the introduction of multiple interventions applied in sequence, as a “multiple-hurdle scheme” at several points throughout the food chain (including processing, transport and handling) will be most effective. This report summarizes the review and evaluation of interventions applied for the control of STEC in cattle, raw beef and raw milk and raw milk cheese manufactured from cows’ milk, and also evaluates available evidence for other small ruminants, swine and other animals. The information is presented from primary production, to the end of processing, providing the reader with information on the currently available interventions based on the latest scientific evidence. This work was undertaken to support the development of guidelines for the control of STEC in beef, raw milk and cheese produced from raw milk by the Codex Committee on Food Hygiene (CCFH). Food Systems and Food Safety - Economic and Social Development jemra@fao.org http://www.fao.org/food-safety Food and Agriculture Organization of the United Nations Viale delle Terme di Caracalla 00153 Rome, Italy Department of Nutrition and Food Safety jemra@who.int www.who.int/health-topics/food-safety/ World Health Organization 20 Avenue Appia 1211 Geneva 27, Switzerland