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Learning Objectives Animal Identification and Food Chain Information Asses food producing animals ID PH02 & PH03 Identify the most common fraudulent violations re...

Learning Objectives Animal Identification and Food Chain Information Asses food producing animals ID PH02 & PH03 Identify the most common fraudulent violations related to animal ID Describe producers responsibilities related to animal ID Describe FSA responsibilities Explain the role of the FCI Iñaki Deza-Cruz [email protected] Purpose of Animal Identification Legislation on Animal Identification Regulation (EC) 178/2002 – FBO must have systems and procedures in place to ensure traceability at all stages of production. Identify any person from whom they have been supplied with a food-producing animal, Identify the other businesses to which their products have been supplied. Regulation (EC) 853/2004 – All animals or batch of animals sent for slaughter must be identified for traceability purposes. Regulation (EU) 627/2019 – OV to verify FBO compliance, and – Ensure that animals whose identity is not reasonably ascertainable are killed separately and do not enter the human food chain. Identification of cattle Cattle ear tag requirements The Cattle Identification Regulations (CIR) 2015: All cattle born and imported into GB must be tagged in at least one – Keepers to ensure that cattle: ear. are registered, Cattle born: have correct ID (ear tag), have a valid Passport prior to slaughter for human consumption, – between 1 April 1995 and 31 December 1997 must be identified movements and/or deaths are registered, with at least one ear tag. – The Trading Standards is the enforcement authority for CIR. – from 1 January 1998 should have a tag in each ear (primary and Definition: Keeper: secondary). person responsible for the animals whether on a permanent or temporary basis (e.g. slaughterhouse operators, market operators, transporters). Cattle must be tagged properly to be moved 9 Primary ear tag Secondary ear tag distance readable yellow plastic two piece ear tag which requires Placed in the other ear to the primary tag (unless an ear is specific information: damaged, then can be fitted to the same ear). Info always Information: printed, – official (printed): Crown logo, letters ‘UK’ and the animal’s not hand unique number written – The secondary tag can also contain management information which can be printed or hand written If the ear tag is made from two pieces, both sides must be printed and bear the Crown logo Unacceptable Forms of Identification Cattle Tagging Age Requirements Hand written tags Apply First Tag Apply Second Tag Tattoos Dairy Herd within 36 hours of birth within 20 hours of birth Printed tags without a country code Other herd within 20 hours of birth Tags where the code appears to have been amended or tampered (other than bison) with (except the addition of management information to secondary when calf separated from dam or 9 months of birth, Bison ear tags) (whichever is the sooner) Tags with missing information (e.g. one tag missing one number) Unreadable ear tags Cattle must always be tagged to be moved!! Quiz Time: Cattle Ear Tag Requirements Quiz Time: Answer 10  Quiz Time: Cattle Ear Tag Requirements Quiz Time: Answer Cattle Ear Tag Requirements Cattle Ear Tag Requirements Can we exchange ear tags? Are these ear tags valid? … Mine are too ugly, I like hers!!  Yes, a bar code may be included on an official tag, although it is NOT compulsory. Used mainly for animals that are being exported British ear-tag manufacturers can issue such No, it is an offence to identify an animal with ear tags if they are needed. tags which have already been used to identify and register on the CTS for a different animal. Passport Requirements Passport Requirements – Overview All cattle born in GB or imported in GB from an EU or TC (if not direct to slaughter or slaughtered within 15 days of arriving within the UK) must be registered with the BCMS and must be issued with a passport: – Cattle from the EU or TC must apply for a GB passport within 15 days of arrival to holding destination (if not direct to slaughter or slaughtered within 15 days of arriving within the GB), – Keepers are obliged to apply for replacement passports within 14 days of becoming aware of a missing passport. 11 Passport Requirements Imported cattle Information on a valid passport Imported cattle presented for slaughter within 15 days of complete registration details arrival from the EU or TC should have: an ear tag number matching the ear tags on the animal address details of the most recent holdings the animal has moved – Export Health Certificate (Issued by an official vet of the through (maximum of 6 holdings - full history available at CTS) exporting country) entries signed and dated by each keeper – Animal clearly identified (passport and eartags issued no sign of having been tampered with or amended in any way by the country of origin) a heat-sensitive diamond shape which will fade when held between finger and thumb (security feature in the bottom right- hand corner Imported Cattle (EU and TC) Passport types From EU – can retain its original double tag showing: There are five types of official cattle identification documents for – EU Country Code and Logo, cattle in GB depending on date of birth or import: – Official ID of no more than 12 digits (holding + animal), Date of birth / import Document – Can re-tag if tag lost but Country Code will change to UK. Before 1 July 1996 Certificate of CTS Registration (COR) or CHR3 (older cattle), From Third Countries (TC): 1 July 1996 to 28 September 1998 CCP1 (blue and green A4) – With is own TC tag and passport can only go directly for 28 September 1998 to 31 July 2011 chequebook-style passport with movement slaughter within 15 days from import + veterinary export health cards (CPP13) certificate. From 1 August 2011 single A4 sheet (CPP52) – Otherwise, re-tag with UK tag within 20 days of passing For cattle refused a cattle passport notice of registration (CPP35) veterinary checks if not going directly for slaughter, Note: such cattle are not eligible for slaughter – Farmer to apply for a UK passport within 15 days of arriving (the for human consumption – contact APHA - date of import will be shown on the UK passport). Animal ID CHR3 or COR CPP1 (Eartag N.) Born before 1 July 1996 Movement records Holding of death Death details Movement records Animal ID Breed, Birth, Sex, (Eartag N.) Date of death & kill number Breed, Birth, Sex, Dam ID Holding address Holding address 12  Born from 29 Sep 1998 to 31 July 2011 CPP13 Born from 2 July 1996 to COR 28 Sep 1998 CCP1 Born After CPP 52 1 Aug 2011 CPP13 Animal ID Movement records Breed, Birth, Sex, Dam and Sire ID Holding address Imported Cattle (NI) Valid Northern Ireland passport Cattle imported from Northern Ireland and presented for slaughter for human consumption must be identified with a printed ear tag in each ear showing the same official identity (double tagging) bearing: There are no passports in NI. Every animal is recorded on a central – unique NI logo (pictured) database: the Animal and Public Health Information System (APHIS). – UK prefix Print-out of the database containing the animal’s information, movement – official identity of 12 digits history and statuses of a particular animal is produced instead of a passport. Animals are moved within NI on what are called: ‘owners’ declarations’. Note: All cattle imported into GB from NI must be These can be either hand-written or electronic. However when animals registered with BCMS) (unless slaughtered within 15 days are moved ‘outside’ NI (and the APHIS system) the print-out will of arrival into GB). accompany the animal. This information will be produced directly from They must only be moved to an approved slaughterhouse APHIS for the purpose of moving the animal into GB. is accompanied by a cattle passport issued by BCMS. Single tagged cattle must be retagged within 15 days of arrival into GB. 13  Imported Cattle Imported Cattle Quiz Time: Passport Requirements Quiz Time: Answer Can you think of reasons which would Can you think of reasons which would deem a passport invalid? deem a passport invalid? If you are watching a recording of this lecture pause here and answer the question before moving forward. Quiz Time: Passport Requirements ReflectionTime: Passport Requirements Are photocopies instead of original Can you think of a situation when it may be acceptable documents acceptable? to slaughter an animal without valid documentation? If so, what additional requirements would need to be considered? Tip: You may consider reviewing Lectures PH11, PH14 and/or PH20 14  Animal ID Violations Cattle age requirements Source: The Lancashire Telegraph (2019) “Breaks, pleaded guilty Prohibition on older cattle: to 5 counts of failing to notifying the BCMS and – The sale of meat derived from cattle born in, or imported into, 1 count of changing a the UK before 1 August 1996 is strictly prohibited. cattle passport. Breaks was given a 12- Bovines requiring Bovine Spongiform Encephalopathy (BSE) month conditional testing: discharge and ordered – Different age requirements for removal of BSE Specified Risk Materials to pay £1745.36 in costs and a £15 victim (see Lecture PH26). surcharge”. – If emergency slaughter on the farm or a casualty in the lairage: Over 48 months need brain-stem tested for BSE. Producers Identification Responsibilities FBO Identification Responsibilities It is the farmer responsibility that all livestock sent for slaughter are To notify the BCMS of movements of: correctly identified. Animals onto their premises either electronically or using a movement card. Animals leaving their premises if the animals move off without being slaughtered (exceptional circumstances). It is the abattoir responsibility to ensure that all cattle slaughtered To complete the death details in the back of the passport. for human consumption: – The passport will be stamped at the slaughterhouse to indicate the date – Comply with the age criteria, and place of slaughter. – Are properly identified, and To keep an animal movement register. – Are accompanied by valid documentation. All cattle passports for slaughtered animals, must be returned to the BCMS within 7 days from slaughter. Dentition Check FSA Animal Identification Responsibilities Estimated ages of cattle and sheep based on dentition MHI or OV to verify abattoir compliance by checking 10% as CATTLE SHEEP minimum, increasing to 25% or 100% of bovine if necessary, and Permanent Estimated Estimated Permanent Estimated incisors minimum age maximum age incisors minimum age 100% of equine animals slaughtered for human consumption: (months) (months) (months) 1 erupted 18 26 1 erupted 12 – PASSPORT: Valid official documentation 2 erupted 18 27.5 – 34 (in 2 erupted 14 – EAR TAGS: Official ID in both ears wear) 3 erupted 23 3 erupted 23 32 – AGE: Dentition checks to ensure they comply with age criteria 4 erupted 24 4 erupted 24 42 – MATCH: Ear Tag = Passport = Animal 5 erupted 36 5 erupted 30 43 6 erupted 36 6 erupted 32.6 50 7 erupted 48 7 erupted 34.5 58 8 erupted 50 8 erupted 36.5 57 - 15 Quiz Time - Under or Over 30 months? Quiz Time - Cattle ID Requirements Can you think of reasons for identity discrepancies between the animal and its passport? If you are watching a recording of this lecture pause here and answer the question before moving forward. Quiz Time: Answer Can you think of reasons for identity discrepancies OV – Animal Identification Responsabilities between the animal and its passport? Animals not correctly identified should not be accepted for human consumption initially and kept alive in the lairage unless there is a welfare problem, in this case slaughter the animals and detain the carcases. Only when the identity has been ascertained, animals should be accepted for slaughter for human consumption, otherwise carcases should be destroyed. Discrepancies between ear tags and documentation to be reported to Trading Standards. Enforcement Identification of sheep and goats The OV should inform Trading Legislation: Standards of offences – EC Hygiene Regulations: 853/2004, 627/2019, regarding: Enforceable by the FSA – SAGRIMO (The Sheep and Goats (Records, Identification and Movement) Order 2009), enforcing transposed EC Reg 21/2004. Reported to the LA and report to Defra. Movement Suspect Animal ID fraud records Definition: Keeper: person responsible for the animals whether on a permanent or temporary basis (e.g. slaughterhouse operators, market operators, transporters). 16  Identification requirements Council Regulation (EC) 21/2004 has been transposed in to UK legislation by: What is ‘properly identified’? – FBO can only accept ‘properly identified’ SAGRIMO: – identified in such a way that the farm or holding from which a Statutory Instruments sheep or goat was sent for slaughter can be traced 2009 No. 414 Animals, Scotland – E.g.: Animal Health FCI The Sheep and Goats Ear tag showing the farm (Records, Identification and Movement) (Scotland) Order 2009 [And equivalent in England and Wales] Animals Born After 1st January 2010 Animals for slaughter: before 12 months of Electronic identification (EID) of age (no erupted adult tooth) - single EID UK sheep and individual recording is an tag. EU obligation and mandatory in the Any others (breeding stock): two eartags UK from 1 January 2010. (one in each ear) to be tagged: – Within 6 months of birth if animals are EID is not mandatory for goats housed overnight. because the UK goat population – Within 9 months of birth if animals are falls below the EU threshold. not housed overnight. However, goats will still need to be Animal movement before the specified minimum age individually recorded on a holding dictates earlier tagging register and movement documents. Lost or illegible tags should be replaced within 28 days 17  These animals are identified following the same rules as Click to edit Master title style described till now but with no EID tags. – Two eartags required in animals not intended for slaughter before reaching 1 year of age. – One eartag for animals going to slaughter before they reach 1 year of age. Movement of Sheep From EU: Sheep and goats must be identified with two ID Animal Movement Licence (AML) tags with the same individual ID number. Address, including postcode and CPH of the holdings from, and to From TC: Sheep and goats must be double tagged with which the sheep are being two identical UK tags: moved – within 14 days of their arrival Date of movement – before they leave the holding of import. Number of sheep moved FBO must retain for 3 years FBO must send a copy to the LA within 3 days of the arrival of a sheep. Responsibilities at the Slaughterhouse No movement documents are needed Abattoirs should have in place a system to check that: when: All sheep accepted for slaughter are properly identified. Moving animals within the same All sheep are tagged and correspond to the movement farm where animals are moving document (AML). across land under the same farmer The movement document is completed and shows the management of control. correct number of sheep in the batch and where required, Moving animals within the same records animals’ individual identities and that FCI details business to or from the main site, have been completed appropriately. where the distance moved is within 5 miles of the main site. A system to notify the OV of arrival of animals in the slaughterhouse. Any movement to or from a vet. 18  What is “Reasonably Ascertainable”? Difficulties may arise from loss of a tag or Under Regulation (EC) 853/2004, the abattoir may accept for slaughter only animals that are “Properly Identified”. from lack of correlation with the information – Means identified in such a way that the farm from on the movement document or in the FCI. which a sheep was sent for slaughter can be traced. – Sheep can be deemed to be properly identified within the requirements of SAGRIMO if the animal bears an The identity of a sheep may be considered ear tag showing the farm where the animal was born. “Reasonably Ascertainable” if it can be – This information should be shown on the movement traced back to its last holding. document which accompanies the animals, – Animals are ID in such a way that it enables them to be FSA staff may refer to: – Batch homogeneity (does this sheep related to the food chain information (FCI). – Movement documentation “fit” into this batch? - Farm checks carried out by Trading – Tagging Standards OV Responsibilities Pigs Identification Animals not correctly identified should not be The Pigs (Records, Identification and Movement) Order 2011 accepted for human consumption initially and kept All pigs arriving at the slaughterhouse should be identifiable alive in the lairage unless there is a welfare problem, in this case slaughter the animals and by means of an identification mark, this can be: detain the carcases. – Slapmark, or – Eartag, or Only when the identity has been ascertained, animals should be accepted for slaughter for human – Tattoo. consumption, otherwise carcases should be destroyed. Discrepancies between ear tags and documentation to be reported to Trading Standards. Pigs Movement Pigs imported from outside the EU must be identified at the destination holding with an ear tag containing the letters "UK" followed by the herd mark and the letter "F" unless the pigs are delivered directly to slaughter. Pigs moving to slaughter require an Animal Movement Licence form completed. In Scotland, details of pigs moving to slaughter should be notified to the ScotEID movement reporting database. 19  Horse Identification Horse Identification The Horse Passports Regulation (EC) No 262/2015 and the Foals born on or after 1 July 2009 must be mandatorily implanted Equine Identification (England) Regulations 2018 require: with a microchip transponder (usually at the neck) and have a – Any equidae presented for slaughter for human consumption single lifetime identification document known as ‘passport’. must be accompanied by a valid passport which does not show that the animal is not intended for slaughter for human Any new passport issued on or after 1 January 2016 must be in the consumption (i.e. Section IX Part II is not signed if old passport format as described in Commission Regulation (EC) No 262/2015. or Section II if new passport). – There are no other stamps or markings within the passport Horses not in possession of a passport and a transponder after 1 which indicate that the horse is not intended for human July 2009 will be signed out of the food chain. consumption. – There is no record of any veterinary treatment not allowed to This also applies to any older horse which had not been identified use for food producing animals (e.g. Phenylbutazone) or by 1 July 2009. veterinary treatment whether the withdrawal period has not being complied with. Horse Identification Central Equine Database (CED) Horses born before 31 December 2015 and not identified The CED is a centrally managed database of all equine identification data in the UK. in accordance with Regulation (EC) No 504/2008 should be identified in accordance with Regulation (EC) No 262/2015 and should be classified as NOT INTENDED The purpose is to secure the human food chain, and to help agencies deal with lost, fly-grazed, stolen or abandoned FOR SLAUGHTER FOR HUMAN CONSUMPTION. horses and combat criminal activity. Horses born on or after 1 January 2016 should be The FSA and Defra have agreed that OVs and MHIs use the identified no later than 12 months following the date of CED as a platform to: birth. – Assist with the equine identification verification checks, – Report the slaughter of horses and the outcome of the carcase – Exception: horse populations living under wild or semi-wild (either in or out of the human food chain). conditions in designated areas. Use of Veterinary Drugs in Horses Prohibited Substances A private veterinary surgeon (PVS) or other person The annex to Regulation (EU) 37/2010 contains 2 lists of administering any veterinary medicinal product to a horse pharmacologically active substances: must first check the passport to ascertain whether the horse – Table 1 lists allowed substances, which may be administered to is intended for human consumption. food-producing animals. – Table 2 lists prohibited substances, which may not be administered The PVS must record details of any substance administered to food-producing animals. (including vaccines) which appears in the list of medicines In the event that a medicine containing any of the considered essential for horses, as required by Regulation substances listed in Table 2 is administered to a horse, the (EC) No. 122/2013 in Section IX of the passports of horses animal can never be slaughtered for human consumption. intended for human consumption (Section II where the passport was issued from 1 January 2016 onwards). These The owner or PVS must sign Part II of Section IX of the substances have a set minimum six month withdrawal passport or Section II of the new passport to declare the period before horses can enter the food chain. horse as “not intended for human consumption”. 20  The Case of Phenylbutazone (Bute) Phenylbutazone Toxicity in Humans Phenylbutazone is neither listed in Table 1 nor has been included in Table 2 of Regulation (EU) 37/2010. Therefore, it Oxyphenbutazone in horse meat is a metabolite of phenylbutazone. The myelotoxic cannot be used in food producing animals (although it is not effects of phenylbutazone (including its metabolites) are analogous to banned). chloramphenicol and other cytotoxic drugs. In veterinary medicine, both chloramphenicol and phenylbutazone are banned from VMD has authorised the use of products containing food-producing animals because of a rare but potentially fatal risk of aplastic phenylbutazone as an active ingredient to non-food horses anaemia in humans. only. However, for chloramphenicol it was shown that the reaction is idiosyncratic, not Horses that have been treated with phenylbutazone must not dose-related, whereas in the case of phenylbutazone is not. enter the food chain and their passports must be signed by The aplastic anaemia caused by phenylbutazone is dose-related and, hence, in the the owner or PVS at Part II of Section IX (Section II where body, concentration dependent. the passport was issued from 1 January 2016 onwards) to indicate that the animal is not intended for human consumption. Is bute in Horses a Public Health Issue? Phenylbutazone Cross Contamination In all investigated species, phenylbutazone concentrations in muscle are least 10 Waste from animals treated with phenylbutazone, and spread on an area of times lower than the corresponding plasma concentrations; after an IV high dose of pasture, can contaminate untreated bovines grazing the pasture many weeks later. phenylbutazone (8.8 mg/kg), the possible ingested amount of phenylbutazone It was determined that this contamination, which can persist over a significant would be 40μg in total for a consumer eating 300g horse meat in one sitting, i.e. period, may be due to the ingestion of as little as 30μg phenylbutazone by a 500 kg less than 1/1000 of a phenylbutazone therapeutic dose in man (Lees and Toutain, bullock (Barnes et al., 2017). 2013). Food-producing animals should be kept away from any vessels used to administer Since phenylbutazone cannot be classified as a carcinogenic substance in phenylbutazone to horses; from the treated horses themselves for at least two humans, and noting that blood dyscrasias in humans are likely to be dose and weeks, from housing used during or after treatment (until thoroughly cleaned) and treatment duration-dependent, the illegal and erratic presence of trace amount from pasture that was previously occupied by treated animals (Fodey et al., 2014). residues of phenylbutazone in horse meat is not a public health issue (Lees and Toutain, 2013). Food Chain Information Food chain information (FCI) is used by slaughterhouse FBOs to assess any potential hazards presented by the animals intended for slaughter as part of their HACCP-based Food Chain Information and Collection food safety management systems. and Communication of Inspection FCI is required for every animal intended for human consumption: Results – The producer must provide FCI to the FBO for all animals presented for slaughter. It is the FBO’s responsibility to evaluate the FCI and then make it available to the OV without delay. The OV must review the FCI before ante-mortem inspection to determine the inspection procedures required. 21  Food Chain Information Information Cycle (FCI and CCIR) The FCI contains all the information regarding a batch of animals with relevance to the FBO and the OV. FBO uses the FCI to: –Reduce risk of hazards to the consumer. –Minimise cross contamination during slaughter. OV uses the FCI to: –Properly assess the health, welfare and inspection requirements of a batch of animals intended for slaughter. Food Chain Information Example of FCI for Cattle Legislation states must arrive no less than 24 hours before animals: –Ideally for poultry – plan slaughter schedule –For other species – can come with animal Animal Health restrictions Guidelines available for what FCI should include: –Health status of the farm Withdrawal periods –Animal health status –Any vet products/treatments Analysis of samples –Any occurrence of disease –Any test results OV Use of FCI Animals presented with No FCI OV checks and analyses FCI and may decide: –If animals have disease/condition transmissible to humans or animals OV may permit slaughter, on welfare grounds: through handling / eating meat, MUST not be sent for slaughter. –In such cases the OV must detain carcases of animals slaughtered in the absence of FCI, and their related offal, –If presented at the abattoir should be killed separately under pending receipt of FCI. conditions so no cross-contamination with fit meat, then disposed of as animal by-product. FCI should arrive within 24 hours of slaughter: –Change slaughterhouse process: –If not – declared unfit, and carcases rejected. - decrease line speed, - increase number of inspectors. Inform Trading Standards. –Slaughter particular batch last. –Detain for further testing (animals or carcases). 22  Collection and Communication of Take Home Messages Inspection Results (CCIR) All food producing animals must be correctly To provide feedback from slaughterhouse to farmer and identified according to the legislation. private vet to allow action to be taken on farm to improve animal health and welfare. Animals that are not correctly identified cannot be transported to another farm or to slaughter. At abattoir FSA only collection of “quantitative” data. Conditions of possible animal health and animal welfare Animals that are not correctly identified or relevance (e.g. fluke, pneumonia, mastitis, etc.) are not without the FCI must not enter the food chain. scored as to give a more useful aid to the vet at the farm (qualitative data). Any breach of the animal identification Other conditions may be passed undetected (e.g. prevalence regulations must be reported to Trading of parasitic infection in the gut) if carcase/offal not Standards. compromised. References FSA Manual for Official Controls (MOC) Volume 1, Chapter 2.5 Acknowledgments: Alex Seguino 23  Food Microbiology Learning Objectives At the end of the lecture you should be able to: List the main foodborne pathogens Differentiate the three types of statutory microbiological testing put in place to protect public health. Discuss the biology of the main food borne pathogens Apply the biology of foodborne pathogens to measures intended for their control Distinguish between the ability of different laboratory methods to isolate and identify food microorganisms. Clinical skill intended to be developed: formulate control plans to ensure food safety, and appraise the effectiveness of microbiological contamination control, in food business operator & processing establishments. Food-borne pathogens & disease 1. Introduction Food microbiology is concerned with the interaction of various micro-organisms and their metabolites with food products. This includes bacteria, fungi, viruses and the metabolites and toxins which are associated with foods of animal origin. The interaction of food and micro-organisms focuses on the following areas: 1. Food spoilage 2. Preservation of food 3. Food–borne disease (covered here) 4. Food processing Food-borne disease is a major cause of illness and mortality worldwide. Foodborne disease includes diseases caused by pathogens and toxins. Non-infectious foodborne disease (e.g toxins from either from microorganisms or chemical contamination) can occur without live pathogens. Infectious food-borne pathogens include bacteria, viruses, parasites and fungi. Presentation is most commonly enteric disease but it can also be systemic, e.g. Salmonella typhi Incidence of food-borne disease is difficult to estimate due to under-reporting and sometimes the difficulty in linking cases to food. 2. Which pathogens are we worried about? Viruses (especially norovirus) are the most common causes of food-borne disease. Although food is only one of the vehicles for norovirus transmission, foodborne norovirus is still the top of the list in the UK, second in the EU. Generally (and certainly in developed countries), bacteria have the largest cost and impact on health. They are also easier to detect and identify than viruses. Good practices that minimise bacterial infection, also minimise the chances of viral transmission. In some developing countries, parasitic infections play a major role. In developed countries Toxoplasma gondii is a concern. A big survey of foodborne disease was carried out in the UK in 2018 (see tables 1, 2 & 3). It estimated that there were ~2.4 million (1.8 - 3.1 million) cases in the UK. Of those they were able to attribute to a particular pathogen, foodborne Norovirus was the most common (~383,000 cases). Campylobacter was the most common of the bacterial pathogens (~299,000 cases). Norovirus also caused the most deaths (~56), followed by Salmonella (~33), Listeria monocytogenes (26), Clostridium perfringens (~25) and Campylobacter (~21). The UK survey provided a snapshot, a particular outbreak can change the picture from year to year. In the EU, for example, pathogenic Salmonella enterica strains are the most frequent cause of outbreaks, and is responsible for the highest number of cases and hospitalisations. Listeria monocytogenes doesn’t cause many cases, but it has a very high fatality rate (12-30%) and causes the most deaths from bacterial foodborne disease in the EU. Cost of food-borne disease: Cost to individual infected (treatment, loss of earnings), healthcare costs, food industry (increased testing, product recalls, decreased sales), indirect costs to the economy (loss of working days by infected people). The 2018 analysis estimated the UK’s burden to be approximately £9 billion (~£4000 per case). 24 Table 1 - Estimates of Bacterial and Parasitic Foodborne Diseases in the UK in 2018. The Burden of Foodborne Disease in the UK 2018 FSA report 2020 Table 2 – Estimates of Viral Foodborne Diseases in the UK in 2018 The Burden of Foodborne Disease in the UK 2018 FSA report 2020 Table 3 – Total attributed foodborne disease and unattributed foodborne illness UK 2018 The Burden of Foodborne Disease in the UK 2018 FSA report 2020 25 Legislation Legislation regarding microbiological food safety was introduced by the European Council in 2004. Regulation (EC) 852/2004 Article 4 stipulates that food business operators shall, as appropriate, comply with microbiological criteria for foodstuffs and Regulation (EC) 2073/2005 outlines the microbiological criteria for foodstuffs. Regulation (EU) 2017/625: updated the Official Controls Regulation (OCR). Regulation 2017/625 Article 18 “... official controls performed in slaughterhouses, cutting plants and game-handling establishments, by an official veterinarian, under the supervision of the official veterinarian or, where sufficient guarantees are in place, under the responsibility of the official veterinarian, to verify compliance with the requirements applicable to: (i) the hygiene of meat production; (ii) the presence of residues of veterinary medicinal products and contaminants in products of animal origin intended for human consumption; (iii) audits of good hygiene practices and procedures based on HACCP principles; (iv) laboratory tests to detect the presence of zoonotic agents and animal diseases and to verify compliance with the microbiological criterion as defined in point (b) of Article 2 of Commission Regulation (EC) No 2073/2005(54); (iv) the handling and disposal of animal by-products and of specified risk material; (v) the health and welfare of the animals.” Brexit: Currently in the UK, the EU laws are still the relevant legislation, this may change as the legislation is updated (in either the EU or the UK). Microbiological criteria are limits on presence or number of microbes or metabolites in food. - Why are they necessary? o to interpret results of tests in a consistent manner o ensure food is safe and produced hygienically o BUT cannot guarantee food safety – hence HACCP o microbiological testing verifies HACCP procedures The FBO must ensure product meets requirements and the competent authority, i.e. the official veterinarian (OV), is responsible for compliance. Categories of microbiological criteria: o Process Hygiene Criteria, o Food Safety Criteria, o Meat and Working Surface Testing Where to find the criteria: Annex 1 of Regulation (EC) 2073/ 2005 – Look out for updates: o UK: https://www.legislation.gov.uk/eur/2005/2073 o EU (Eire): https://www.fsai.ie/legislation/food_legislation/hygiene_of_foodstuffs/microbiological_criteria.html 1. Process hygiene criteria: They are not designed to assess the fitness of individual carcases or processed meat for human consumption but to provide an indication of performance and control of the slaughter, dressing and production processes at the time of sampling. To comply with process hygiene criteria for meat and processed meat producers have to demonstrate that they meet certain limits within the following categories: o Aerobic colony count and Enterobacteriaceae on cattle, sheep, goats, horses and pig carcases; o Salmonella on cattle, sheep, goats, horses, pig, broiler and turkey carcases; o Aerobic colony count and E. coli in minced meat and mechanically separated meat; o E. coli in meat preparations; 26 2. Food safety criteria: Food safety is ensured through implementation of HACCP rather the microbiological criteria in themselves but food safety microbiological criteria are in place to monitor the efficacy of HACCP implementation. Food safety criteria have been set for fresh poultry meat, minced meat, meat preparations, meat products and mechanically separated meat. Exceeding these criteria indicates that the batch tested is unsatisfactory and should be not placed on the market or has to be removed from the market. Demonstration of compliance with food safety criteria for meat and processed meat requires monitoring of E. coli, Salmonella and Listeria. Total enterobacteriaceae count and specific E. coli counts have to be below a specified threshold in order for the food to be marketable. In addition, the criteria outlined below have to be met. Salmonella is required to be completely absent in the following products: o minced meat and meat preparations intended to be eaten raw; o minced meat and meat preparations intended to be eaten cooked; o mechanically separated meat (MSM); o meat products intended to be eaten raw o meat products made from poultry meat intended to be eaten cooked o fresh poultry meat (S. typhimurium and S. enteritidis) L. monocytogenes is required to be completely absent in the following food products (all ready-to-eat (RTE) foods): o RTE foods intended for infants. o RTE able to support the growth of L. monocytogenes other than those intended for infants, o RTE unable to support the growth of L. monocytogenes other than those intended for infants, 3. Meat and work surface testing Further verification of the HACCP plan is achieved through the microbiological testing of working surfaces and utensils. Listeria monocytogenes microbiological testing of working surfaces and utensils are a legal requirement in a cutting plant. The exceptions are if they do not produce Ready-to-Eat products or, if the Food Business Operator can provide other forms of verification of cleaning procedures (e.g. rapid test systems). 27  Table 4 - Meat category micro-organisms (taken from Chapter 13, Microbiological Critieria, Meat Industry Guide, Food Standards Scotland) Microorganisms Description Test indication Enterobacteriacae The name given to a group of The presence of these (ENT) bacteria that live predominantly organisms on the surface of in the intestines of animals. The carcases is an indicator of group includes most of the faecal and environmental major food borne pathogens of contamination. animal origin such as Salmonella, Yersinia and E.coli O157. Generic E.coli (EC) A group of bacteria that live in The test procedure does not the intestines and are shed in specifically recover E.coli the faeces of man and food O157 but does indicate the producing animals. Presence of risk of contamination with E.coli is an indicator of faecal this and other dangerous contamination. faecally-derived bacteria. Salmonella species A group of bacteria that (Sal) includes several pathogens of Further analysis of the type significance in human food of Salmonella can be useful poisoning disease. They mainly in investigating and arise from faecal contamination preventing the reoccurrence but can also arise from the of positive results as well as processing environment. providing information that Salmonella Types of Salmonella that have can be Salmonella used in a Typhimurium been associated with frequently risk analysis. Salmonella causing disease in humans. Enteritidis Known as Salmonella with public health significance (SPHS). * Listeria A pathogenic bacterium that The presence of the bacteria monocytogenes occurs in the environment. Able in ready to eat food that can to survive and grow at chill support the growth can be a temperatures. problem. *Also includes monophasic Salmonella Typhimurium with the antigenic formula 4 5 12 i. Bacterial food-borne pathogens – Biology & Control. 1. Campylobacter a. Biology of pathogen Gram-negative curved bacillus More than 20 species identified; approximately 12 species associated with human disease C. jejuni 80% of human cases C. coli 10% of human cases; remaining 10% caused by other species Microaerophilic organisms; grow at reduced oxygen tensions (5% O2 in the lab) Thermophilic campylobacters (C. jejuni, C. coli, C. lari, C. helveticus and C. upsaliensis) only grow at temperatures in excess of 300C and show optimal growth at 420C Non-thermophilic species are not important cause of disease Sensitive to heat, UV light, dryness and salt concentration 28  Reservoir is intestine of domestic and wild birds and mammals; in chickens grow up to 109 bacterial cells/g of caecal contents Can survive but not grow in environment; presence of moisture required for survival b. Disease caused Most common zoonotic disease in EU 63,946 laboratory-confirmed cases in UK in 2017; IID2 study indicated under-reporting in the UK by a factor of 9 so actual figure may be around 600 000; estimated total cost to the economy of food-borne disease is around 2 billion pounds with Campylobacter accounting for £500 million. Incubation period 2-5 days Symptoms include fever, headache and myalgia initially then diarrhoea, dysentery, abdominal pain Usual course 80% reported. NB method of detection may affect results. Henry et al. (2017) reported 6% in England and Wales, and 10% in Scotland using immuno-magnetic separation (IMS) as the method. Spread between animals by contaminated feed or water; hide contamination and grooming; wild animal and bird sources; spread by flies. Shedding by cattle may be intermittent and may only shed low numbers of bacteria Greater prevalence of STEC in younger animals ‘Super-shedders’ – animals that shed large numbers (>103 cfu/g faeces) are especially important and may account for over 80% of STEC load and transmission in a group of animals Seasonal effect on shedding - higher shedding in summer months Cattle are also source of environmental contamination of soil and water directly and indirectly via slurry spread. Non-meat sources of EHEC are of increasing importance; direct contact with animals (e.g. petting farms), contact or consumption of contaminated water, contamination of fruit and veg via slurry have all been implicated in outbreaks of O157 in humans d. Control measures On farm: good biosecurity, identify and target super-shedders, vaccination Vaccine based on siderophores and porins licensed in the USA by Zoetis; new vaccine produced by Roslin and Moredun Research Institute recently but not commercially available. In the food chain: good hygiene and a HACCP system in place, slaughter clean cattle only or clean them before slaughter, careful de-hiding and evisceration, carcass washing, avoid mixing batches of products. 4. Listeria monocytogenes a. Biology of pathogen Gram-positive facultative anaerobic bacillus 31  13 serotypes, all potentially pathogenic, but 1/2a, 1/2b and 4b are most frequent in human disease Capable of growth over a wide temperature range (2 – 450C), including refrigeration temperatures Tolerates a relatively broad pH range, salt concentrations and changes in osmolarity Widespread in soil, vegetation and throughout environment Intestinal carriage in animals b. Disease caused 2 distinct syndromes, depending on the immune status of the host: o Acute febrile gastroenteritis – fever, nausea, vomiting, diarrhoea; lasts 7 – 10 days o Invasive disease – septicaemia and meningitis; abortion in pregnant women Infective dose: varies depending on individual but can be less than 1000 CFU Incubation period: o Gastroenteritis: few hours to few days o Invasive disease: days to months c. Epidemiology Cases are usually sporadic but outbreaks also occur generally associated with ready-to-eat, refrigerated foods, and often involves the post processing recontamination of cooked foods. Incidence of listeriosis is relatively low (162 UK cases in 2017) but high case-fatality rate (~16% in UK) UK – 8 incidents between 2015- 2023 (31 cases, 8 deaths) – smoked fish EU (2020 data) - 7 outbreaks associated with raw, preserved and smoked fish Has also been isolated from: – raw and pasteurised milk, cheeses (particularly soft-ripened varieties), ice-cream, butter – fermented raw-meat sausages – raw and cooked poultry other raw and processed meats ~ 40% deaths in the EU from foodborne disease due to L.monocytogenes d. Control measures Good hygienic practices in food production Strict temperature control throughout food chain Where necessary, re-formulation of food to prevent growth of Listeria 5. Other bacteria that can cause food poisoning Bacillus Disease is caused by three species: cereus, subtilis and licheniformis. Bacillus cereus is more common – it causes two clinical syndromes: o Emetic syndrome: nausea and vomiting o Diarrhoeal syndrome: diarrhoea and abdominal pain Disease can be caused by ingestion of toxin or by live bacteria. Bacillus spp. form spores that can survive for a long time and contaminate food; they can germinate and start growing in warm conditions, e.g. in the kitchen. Common sources of infection for Bacillus spp. are: o environmental: soil, sediment, dust, vegetation o food sources: cereal products, herbs and spices, dried foods, milk and dairy products, meat and meat products Food sources of Bacillus cereus: mostly rice dishes; occasionally: pasta, meat, vegetables, dairy, soups and sauces Control is through stringent temperature control for cooked foods during refrigeration (under 4 oC) and by ensure appropriate cooking/reheating – over 18 minutes at 1200C in dry heat Brucella spp. This pathogen is most commonly transmitted through undercooked meat or unpasteurised milk Can also be acquired by aerosol or through open wounds 32  Initial symptoms: fever, sweats, malaise, anorexia, headache, pain in muscles, joints and/or back, fatigue Long-term symptoms: recurrent fevers, arthritis, swelling of the testicle and scrotum area, endocarditis, neurologic symptoms (in up to 5% of all cases), chronic fatigue, depression, swelling of the liver and/or spleen. Can cause abortion in humans; but unclear if the rate of abortion is higher than with other bacteraemias. The best way to prevent brucellosis infection is to be sure you do not consume undercooked meat or unpasteurized dairy products (milk, cheese, ice cream). GB has been declared free of Brucella abortus in 1985 and NI in 2015. Brucella ovis, B. melitensis, B. suis and B. microti were never detected in the UK Clostridium perfringes Spore-forming gram positive bacterium It is found in the environment and in the intestines of humans and animals It requires anaerobic conditions to grow Disease is caused by enteric toxin production Common sources are: beef, poultry, gravies, pre-cooked foods that are not reheated adequately Diarrhoea and abdominal cramps can develop within 6 hours from toxin ingestion Fever or vomiting are not usually part of the symptoms Prevention: reheat food to at least 74 oC or ensure stringent temperature control during storage under 4 oC Staphylococcus aureus Food poisoning is caused by toxins (haemolysins, leukotoxins) rather than bacterial infection It can happen even if the food is cooked – bacteria are killed by heat but toxins are thermo-stable; bacterial lysis during cooking can even release more toxin Foods at highest risk of transmitting Staphylococcus toxins are those that people handle and then do not cook: sliced meat, puddings, pastries, and sandwiches It is salt tolerant – can grow in ham Symptoms usually develop within 30 minutes to 6 hours Patients typically experience vomiting, nausea, stomach cramps, and diarrhoea It is non-contagious and typically lasts for 1 day only. Control measures: o Wash hands thoroughly with soap and water before handling and preparing food. o Do not prepare food if you are ill. o If you have wounds or infections on your hands or wrists, wear gloves while preparing food. o Keep kitchens and food serving areas clean. o If food is to be stored longer than two hours, keep hot foods hot (warmer than 140°F) and cold foods cold (40°F or colder). Yersinia enterocolitica There were 141 documented cases in the UK in 2017 It is most commonly acquired from undercooked pork; occasionally from unpasteurised milk Incubation period: 4 to 7 days Symptoms: o fever, abdominal pain, and diarrhoea, which is often bloody. o In older children and adults, right-sided abdominal pain and fever may be the predominant symptoms and may be confused with appendicitis Sequelae: o reactive arthritis, most commonly in the knees, ankles, or wrists; usually develops about 1 month after yersiniosis illness begins and generally goes away after 1 to 6 months. o a skin rash, called "erythema nodosum," on the legs and torso; the rash is more common in women and usually goes away within a month. Control: cook pork thoroughly; drink pasteurised milk only. 6. Viral food-borne pathogens Norovirus Most common cause of infectious intestinal disease in the UK; NB! Not zoonotic. Highly infectious and easily transmissible 33  Symptoms: vomiting, diarrhoea, headaches, abdominal pain, fever. Recovery 2-3 days Can be transmitted through food, but majority of cases are not foodborne Control: heat treatment and good hygiene, especially with RTE food that is not heat-treated before consumption Hepatitis viruses 5 etiological agents of hepatitis: hepatitis A, B, C, D and E viruses Two separate entities are identified: infectious and serum hepatitis. Serum hepatitis are those parenterally transmitted. The infectious type corresponds to those viruses transmitted through the faecal-oral route; also called enteric hepatitis, Only the enteric hepatitis will be discussed below and it includes virus types A and E. Hepatitis A virus Hepatitis A infection is mainly propagated via the faecal-oral route; person-to person contact is the most common mode of transmission. Food can be directly contaminated by infectious individuals during processing. HAV can survive long enough in water, between a contamination event and the use of the water for crop irrigation or during food processing, to constitute a risk to health. Once on foodstuffs such as vegetables, HAV can persist under normal storage conditions over the periods usual between purchase and consumption. Incubation period: ranges from 15 to 50 days and clinical illness usually does not last longer than 2 months. Clinical findings: acute illness with moderate onset of symptoms (fever, malaise, anorexia, nausea, abdominal discomfort, dark urine) and jaundice, and elevated serum bilirubin and aminotransferases levels later. US Food and Drug Administration: infectious dose is presumably around 10-100 virus particles. It often induces jaundice, or yellowing of the skin, and in rare cases leads to chronic liver dysfunction. Source of infection: Foods of primary importance are those susceptible to be contaminated at the pre-harvest stage such as: bivalve molluscs (particularly oysters, clams and mussels), salad crops (lettuce, green onions and other greens), soft fruits (raspberries and strawberries). One of the most effective treatments to reduce viruses from any food product is to cook the food thoroughly; however this may not be applicable to commodities like shellfish that become unpalatable. Heat treatment to an internal temperature of 85°C - 90°C for 90 seconds, may destroy viruses in molluscs but careful control is necessary to achieve this without toughening of the shellfish flesh. Hepatitis E virus In contrast to NoV and HAV, HEV has been identified as a zoonosis. Although rare, its importance is increasingly recognised in the EU. The diagnosis of HEV infections in humans is not routinely done in most laboratories, and therefore, there is considerable under diagnosis of this infection and illness. HEV strains can be grouped into 4 genotypes: Genotype 3 is distributed worldwide in humans, pigs and other animal species (commonly isolated in Europe). Clinical features: o Incubation period (human volunteers): 4 to 5 weeks; 2 to 10 weeks reported during outbreaks. o Clinical symptoms of hepatitis E in humans same as other forms of viral hepatitis. o Hepatitis E is mostly self-limiting and in general does not progress to chronicity. o Faecal shedding of HEV occurs in most cases for approximately 2 weeks. o Epidemiological observations suggest that people previously infected with HEV are protected against further disease. Food as vector: o HEV is associated with large outbreaks of hepatitis E among humans in endemic countries. o This includes inhabitants from Asian and African countries, which are exposed to the virus due to poor sanitary conditions. o Sewage overflow that results from heavy rainfall may contaminate surface water that is used for drinking water production or as source for water used for household tasks. o As water is widely distributed and used, the number of people exposed is generally large, explaining the large-scale outbreaks of HEV in developing countries. Food-borne zoonosis: 34  o Several animal species have been identified as potential virus reservoirs: domestic pigs, wild boars and deer. o Direct evidence of zoonotic HEV transmission was obtained when cases of hepatitis E were linked directly to eating raw deer meat by the presence of identical HEV strains in the consumed deer meat and patients. o HEV can be transmitted through consumption of products of animal origin, especially through consumption of meat. o This virus may circulate in the blood at the time of slaughter and may be present in liver or meat. o No specific legislation for HEV is currently in place. Hepatitis E in animals: o Infected animals do not normally show clinical signs of disease. o HEV infection usually occurs at 8 to 12 weeks of age after the decline of maternal antibodies. o HEV is highly prevalent in pigs across Europe. o Viraemia at 3 months of age and faecal shedding between 10 to 16 weeks of age. o The immune response is between 14 to 17 weeks of age usually limits the infection. o HEV mainly replicates in the liver of infected pigs. Control: o No efficient method of detection at ante-mortem and post-mortem inspection – no visible changes in living animals or in their organs. o Presently the only efficient control option for HEV infection from consumption of meat or liver is sufficient heat treatment. o Biosecurity measures to prevent HEV introduction into pig farms is hampered by limited knowledge about transmission pathways of HEV in pigs. Laboratory Detection of Food-borne Pathogens Since food-borne pathogens, such as those discussed above, pose a threat to human health, it is important that reliable and standardised methods are available to detect these agents in food products. Numerous methods have been developed for the detection of various pathogens and these methods can be divided into 2 broad groups: conventional culture–based methods and rapid methods. Regardless of the method, there are several important criteria which must be considered when deciding which test to use. These include: Sensitivity of the test (proportion of true-positives identified as positive by the test) Specificity of the test (proportion of true-negatives identified as negative by the test) Limits of count detection for enumeration tests Time taken to carry out test Ease-of-use of the test Cost of test The last three are not considered validation criteria. The following points must be borne in mind when interpreting test results: The accuracy of the test – determined by sensitivity and specificity Result is only valid for one specific point in time. Bacterial numbers may increase or decrease subsequent to sampling Failure to detect specific organisms does not mean that metabolites or toxins of that organism are not present Results are only valid for the sample tested; bacteria may not be evenly distributed throughout a batch of product. To guarantee that a food is pathogen-free, it would require a test with 100% accuracy and testing of every individual lot of food at the point of sale. Obviously such an approach is not feasible and, even if it were, simply detecting pathogens at the point of sale does nothing to control the presence of pathogens in food. For these reasons, HACCP plans are used to ensure the safety and quality of the product at all points during production. In conjunction with a proper HACCP Plan, the FBO can determine the appropriate number of random samples which must be taken to ensure that the probability that the food is safe, is high. Detection, identification and quantification definitions Detection = can you find it? Qualitative methods Identification = what is it? 35 Quantitative methods Quantification = how much can you find? A: Conventional Microbiological Methods Classically these can be divided into two groups: Quantitative Methods Methods based on the detection & enumeration of microbes (usually in liquid and/ or agar media). Steps in Qualitative lab culture methods. 1. Sample collection followed by analysis on site or rapid transport to lab 2. Culture of samples in enriched broth 3. Culture on selective media 4. Confirmatory tests 5. Examine Identification tables 1. Sample Collection For example, FSA, CDC and SMI have defined procedures following similar protocols; the key points are: Preserve integrity of target microorganisms - may need to use a microbiological transport medium. Avoid contamination from yourself or equipment. Appropriate time, site and mode of collection Use proper equipment (swabs, filter paper, sterile bags). Commercial collection kits are available. Very careful, detailed labelling + cold-chain packaging. 2. Culture in enriched broth Pre-enrichment (if necessary): involves recovery of injured cells using 1:10 dilution in pre-enrichment broth (contains buffers, essential ions, growth nutrients and sodium pyruvate to stimulate injured cell metabolism). This is followed by enrichment: which supports growth of target & non-target microorganisms; e.g. tryptone-soy broth, nutrient broth, buffered peptone water. 3. Culture on selective media Selection: involves subculture on selective agar (or differential media that distinguishes target from non-target bacteria)*. The composition of selective media is designed so that only the bacteria of interest can grow or, in the case of differential media, turn a certain colour. They therefore have selected nutrients, substrates and selective chemicals (e.g. antibiotics) to achieve this goal. Purity Check: is a subculture onto nonselective media so you can see if there are dissimilar colonies. You then need to streak out a selected colony to get a pure culture for further testing (mixed cultures will give misleading results). 4. Confirmatory tests Biochemical, physiological, immunological, phage-lysis tests and nucleic acid testing (as needed) are employed to confirm the presumptive results from the selective media. A variety of tests are available we will look at some of these in the lab. 5. Examine identification tables For example : Historically “Bergey’s Manual of Determinative Bacteriology” is a great tome of a book first published in 1923. Companion book to “Bergey’s Manual of Systematics of Archaea and Bacteria”, which is a major classification reference encyclopaedia and is now available online; see https://www.bergeys.org/ ). There are also several searchable databases available online: e.g.http://www.tgw1916.net/bacteria_logare_desktop.html e.g. http://www.microrao.com/identify.htm e.g. http://www.identax.org/ 36 Quantitative Analysis Requires known quantities of sample and diluent to work out the number of bacteria (measured as colony forming units (CFU)) per gram or per millilitre. These quantitative results are required for the microbiological criteria in the food safety legislation Example Protocol 1. Add swab + 25mls of buffer/ 25g of sample to stomacher bag. 2. Add culture broth - make up to 250ml or 250g (i.e. 1 in 10 dilution). 3. Seal the bag, place in the stomacher machine and homogenise*. 4. Pipette a 1ml sample from the filter pocket and add it to 9ml of buffer. 5. Serially dilute the sample 1 in 10 (as above) up to 10 times. 6. Mix each dilution, then take 100µl from each and spread on an agar plate (or take 1ml for a pour plate) in triplicate. 7. Incubate at 37°C for 24h 8. Count the colonies on plates with 30-300 colonies. 9. Calculate the number of CFU-1 in the original culture/ sample Conventional culture methods Most commonly used tests Developed and validated over long period of time so now considered ‘gold standard’ tests Standardised methods for each test have been developed by agencies such as ISO and FDA to ensure accuracy and reliability of methods Relatively cheap Easy to perform with minimal training Should be carried out by accredited laboratories using standardised methods (or proven equivalent method) and good laboratory practice Slow; generally take few days for results which is a problem when dealing with perishable food products and quick developments during an outbreak. Utilise both broth and solid agar media; media may be non-selective, semi-selective or selective Non-selective media allows for growth of all organisms present in sample Semi-selective media selects for the growth of the pathogen of interest and some other bacteria but is also usually differential allowing the presumptive identification of the pathogen of interest Selective media selects for the growth of specific organism of interest and is inhibitory to the growth of others Chromogenic and fluorogenic media exist to allow for more rapid identification of pathogens Pre-enrichment step is often used to revive injured cells which are potentially pathogenic but are not detected in the injured state. B. Examples of Rapid Methods Of Detection Rapid Methods Of Detection - ELISA Enzyme Linked Immunosorbent Assay (ELISA) – Widely used in food testing – Rapid detection of specific organisms – Sensitivity varies – Can be specific for pathogen at species and subspecies / strain level – Can also detect toxins and allergens. – Specific Nucleic Acid Amplification / Detection e.g. PCR Types of Nucleic Acid Amplification Numerous types of PCR e.g. Reverse Transcriptase PCR e.g. Real time PCR Usually need thermocycler Non-thermal Enzymatic Amplification e.g. Recombinase Polymerase Amplification (RPA) e.g. Loop Amplification (LAMP) 37  Looking for cell RNA (let the cell do the amplification) Identification Rapid methods – serum agglutination Rapid methods – whole genome sequencing Latest generation sequencing machines are fast and cheap: – Can sequence a whole bacterial genome for £50-£100 in half a day – Can get long reads (e.g. 10kb for PacBio) – easier to assemble a genome de novo – Pocket sequencers now available for field use with laptops/tablets; MinION: direct and real time sequencing by passing DNA through a protein pore – Kind of rapid … – Analysis software is lagging behind! – Can take days to assemble a genome and find genes of interest, e.g. virulence factors, AMR 1. Rapid methods of detection (not a definitive list) Immunomagnetic separation (IMS) Magnetic particles coated with antibody specific to pathogen of interest are added to sample in pre-enrichment broth Target pathogen will bind to specific antibody; placement of magnet to the outside of the container will immobilise target pathogen allowing for removal of remainder of sample Target organism can then be plated onto selective agar or used in other rapid methods Aim of IMS is to concentrate target organism in as little as 10 minutes, removing need for overnight enrichment step thus reducing overall testing time IMS kits available for E. coli 0157:H7, Salmonella spp and Listeria spp ATP Bioluminescence This assay detects Adenosine Triphosphate (ATP) the energy molecule of cells. The underlying theory is that where there is ATP, there is life. The assays use the enzyme complex found in male fireflies, that makes them generate light. It is most widely used for monitoring cleaned surfaces. Can do real-time swabs to monitor effectiveness of Clean- in-place (CIP) systems etc. Firefly luciferin-luciferase, a two-step biochemical reaction takes place, which produces light: STEP 1: Luciferin + luciferase + ATP + Mg2+ ↓ (luciferin-luciferase-AMP) + pyrophosphate STEP 2: (luciferin-luciferase-AMP) + O2 ↓ oxyluciferin + luciferase + CO2 +AMP + light!! Direct Epifluorescence technique (DEFT) Sample is filtered through membrane filter: organism trapped on membrane Acridine orange stain added to membrane, membrane washed and dried Membrane observed under epifluorescence microscope RNA fluoresces red; RNA only present in live cells DNA fluoresces green; DNA present in dead cells Detects >6 x 103 cfu/ml Currently used to enumerate bacteria in milk and other samples Test takes 20-30 mins compared to 3 days for plate count Test only indicates the general presence of bacteria but does not identify individual species Hydrophobic Grid Membrane Filter (HGMF) technique Sample is first filtered to remove large particles but not organisms 38  Then filtered through HGMF to trap organisms Membrane contains 1600 grid squares Membrane transferred to agar plate and incubated to detect organisms or count bacteria. Enzyme Linked Immuosorbent Assay (ELISA) Commonly used in food testing Test plate coated with primary antibody specific to organism of interest If target organism is present in sample, it binds to primary antibody Washing to remove non-target organisms Secondary antibody then added; this is labelled with horseradish peroxidise Further wash step Substrate for enzyme then added, which will result in change of colour Stop solution added after a period of incubation to inhibit chemical reaction and enable reading Wide range of kits available for different pathogens Varying sensitivity of tests; usually require 106 cfu/ml Polymerase Chain Reaction (PCR) Detects specific sequence of DNA in organism The sequence is amplified to billions of copies, which can then be visualised on agarose gel PCR identifies both viable and non-viable organisms; also detects DNA in dead cells Commercial kits available for several pathogens Usually non-quantitative but can be quantitative if real-time PCR is run. Other nucleic acid amplification methods are available, such as non-thermal enzymatic amplification, and looking for the cell’s own RNA (high copy number). DNA sequencing and Whole Genome Sequencing Current machines can sequence an entire genome in half a day Cost has come down considerably in the last few years – can sequence a whole bacterial genome for under £50 It now takes longer to analyse a genome sequence than it does to produce it. Simple pathogen identification can be automated and quick though. Devices are becoming available for instant DNA sequencing and pathogen identification on a mobile phone, e.g. SmidgeION by Nanopore (Oxford). Useful Resources Modern food microbiology (7th ed.): J. Jay, M. Loessner & D. Golden; (electronic resource available) Food microbiology (4th ed): Adams & Moss; (electronic resource available) Microbiology of safe food (3rd ed.); S. Forsythe (electronic resource available) Meat Industry guide; Food Standards Scotland (FSS). http://www.ukmeat.org. Official Controls Regulation FSS The Burden of Foodborne Disease in the UK 2018 EU Foodborne Outbreaks Story map + EU Foodborne outbreaks dashboard FSA – Chief Science Advisor Reports Regulation (EC) 2073/2005 (as amended). EU 2021 Zoonosis Report https://www.efsa.europa.eu/en/efsajournal/pub/7666 Red Lion Code: British Lion Success Story | Egg Info. Red Tractor Salmonella control in pigs: https://www.thepigsite.com/articles/salmonella-control-plans- guidelines-for-the-veterinary-surgeon IFST (2017) – Foodborne Camplylobacteriosis https://www.ifst.org/resources/information- statements/foodborne-campylobacteriosis https://www.ecdc.europa.eu/en/news-events/increase-severe-food-borne-infections-reported-eueea-2022 39 PH06 Legislation 2024-24 10/09/2024 Learning Objectives By the end of this session you will be able to: Explain how the UK and EU legal systems work, Legislation and Enforcement Identify the main pieces of legislation that apply to [email protected] Animal Health, Animal Welfare and Food Safety, Gather the best evidences to support legal actions. --- With thanks to Alex Seguino -- Science UK Legal System Economy Tradition Law… a rule, usually made by a government, that is used Politics Religion Environment to order the way in which a society behaves or the whole Sociological system of such rules. Legisla