Cefas contract report C7416

FSA Reference: FS616025

Summary technical report for the UK National Reference Laboratory for – April 2020 to March 2021

July 2021

Summary Technical Report for the UK National Reference Laboratory for Anisakis – April 2020 to March 2021

Final

20 pages

Not to be quoted without prior reference to the author

Author: Cefas Laboratory, Barrack Road, Weymouth, Dorset, DT4 8UB

Cefas Document Control

Submitted to: FSA Valerie Mcfarlane Date submitted: 05/05/2021 Project reference: C7416 Project Manager: Sharron Ganther Report compiled by: Alastair Cook Quality controlled by: Michelle Price-Hayward 29/04/2021 Approved by and date: Sharron Ganther 04/05/2021 Version: Final Classification: Not restricted Review date: N/A Recommended citation for NRL technical report. (2021). Cefas NRL Project this report: Report for FSA (C7416), 20 pp.

Version Control History

Author Date Comment Version Alastair Cook 26/04/2021 First draft V1 Draft V1 for internal review Michelle Price- 30/04/2021 Technical Draft V2 Hayward Review V2 Alastair Cook 30/04/2021 V3 for approval V3 for internal approval Sharron Ganther 04/05/2021 Final V1 Draft for FSA review Valerie Mcfarlane 07/07/2021 Final

Contents 1. Introduction ...... 3 2. Ongoing maintenance of general capacity ...... 4 3. Completion of 2021 EURL proficiency test ...... 5 4. Official control laboratory (OCL) engagement ...... 6 5. Maintenance of collection of reference material ...... 6 6. EURL Annual meeting (2020)...... 7 7. Representation on working groups ...... 7 8. Standard operating procedures ...... 8 9. Industry engagement ...... 8 10. Literature review, calendar year 2020 ...... 10 11. References...... 14 12. Annexe: Legislative standards for parasites in fishery products ...... 16

1. Introduction

During the reporting period, the Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth was designated as the UK National Reference Laboratory (NRL) for the foodborne and potentially zoonotic parasite Anisakis. Anisakis is a genus of parasitic which have life cycles involving fish as intermediate hosts and marine mammals as the final host. When fish containing live Anisakis larvae is consumed the nematodes can burrow into the walls of the human digestive tract causing pain, nausea and other symptoms. They can also cause allergic reactions.

The roles and responsibilities of the NRLs have not changed significantly in recent years. The services required to be delivered under the scope of the current Cefas/Foods Standards Agency (FSA) agreement include the following basic duties of the National Reference Laboratory, based on Articles 100-101 of Regulation (EU) 2017/625 (retained from January 1st 2021 in UK law subject to amendments through Statutory Instrument 2019 No. 665: The Official Controls for Feed, Food and Health and Welfare (Amendment etc.) (EU Exit) Regulations 2019):

(a) cooperate internationally in their area of competence (and where possible the relevant EU-RL);

(b) collaborate with international laboratories (where possible with the relevant EU-RL) and participate in training courses and inter-laboratory comparative tests organised by these laboratories;

(c) coordinate the activities of OCLs responsible for the analysis of samples to ensure the verification of compliance with feed and food law;

(d) where appropriate, organise comparative tests between OCLs and ensure an appropriate follow-up of such comparative testing;

(e) ensure the dissemination of any information required by the competent authority;

(f) provide scientific and technical assistance to the Competent Authority for the implementation of MANCPs in accordance with Article 109 and of co-ordinated control plans adopted in accordance with Article 112 of Regulation (EU) 2017/625;

(g) where necessary, conduct training courses for OCL staff;

(h) upon request by the appropriate authority, actively assist in relevant emergency situations and in cases of non-compliance of consignments, carry out confirmatory analysis;

(i) be responsible for carrying out other specific duties as required by the competent authority, where appropriate and by prior agreement.

This technical report summarises the activities carried out by the NRL for the financial year 2020-21 according to the requirements of (retained) Regulation (EC) No. 882/2004 and (retained) Regulation (EU) 2017/625 as

defined in the Service Level Agreement (SLA) between the FSA and Cefas over the time period April 2020- March 2021. This report constitutes the last report of the contract ending 31st March 2021.

It should be noted that because of the impacts of the current Covid-19 pandemic, some objectives associated with this contract were altered over the last year (with agreement of the FSA). These included a short delay in the delivery of certain laboratory tasks to meet Cefas Covid-19 secure requirements and attendance at meetings by virtual means only. Furthermore, attendance at EU meetings and opportunities for collaboration with EURLs was reduced in line with Government advice and the EU Transition negotiations.

The description of the activities included herein includes the ongoing maintenance of general capacity; the participation in the 2021 proficiency test scheme; progress made against the contract objective to engage with and evaluate the practices of the food production industry, and a brief report on relevant peer reviewed literature published in 2020.

Delivery of the responsibilities of the NRL has been brigaded into the following objectives in accordance with the SLA agreed between Cefas and the FSA. These include the following key responsibilities:

1. Provision of secretariat services (Objective 1) 2. Advice and representation within the UK and internationally (Objective 2) 3. Production of standard operating procedures, codes of practice and guidance documents (Objective 3) 4. Compliance assessment via audits and Proficiency Testing (PT) (Objective 4) 5. Co-ordination within the UK of international initiatives (Objective 5) 6. Communication of results and data use (Objective 6) 7. Provision of additional services, where requested by FSA (Objective 7) 8. Provision of NRL type activities post EU Transition

2. Ongoing maintenance of general capacity

2.1 Dissemination of information via NRL Website The NRL website (https://www.cefas.co.uk/anisakis) continues to deliver information on the responsibilities and function of the NRL to a public audience. A website analytical report was run on 25/03/2021. This determined that the NRL website has had 1994 visits since it was set up in 2014 (Figure 1) from approximately 1105 unique users. The sections of the website which experienced the most traffic were the annual reports, methodology, and regulations sections.

Figure 1 - NRL website visits

The website experienced much lower traffic during the 2020/21 reporting year, with only 32 visits recorded. All of these were visits to the pages hosting the annual reports.

2.2 Email enquiries A small number of email enquiries were received during the reporting period through the dedicated mailbox advertised on the website. These are summarised in Table 1. Table 1: Summary of external enquiries.

From Nature of query NRL response Government Lab, Non-fluorescent Sought advice from the EURL and Norwegian experts. Aberdeen anisakis larvae found It was considered that the non-fluorescing worms in some samples might be another species or loss of fluorescence could have occurred after an extended period of freezing’

Mudeford and District Request for advice on Advice provided, together with links to further Fishermen's safety aspects of information on the NRL website Association contaminated mackerel Government Lab, Request for advice on Some generic advice on pressing requirements was Aberdeen pressing equipment given. When the NRL identifies a suitable press supplier it will provide further details.

3. Completion of 2021 EURL proficiency test

3.1 Concerning the detection of larvae in fillets

Samples for the 2021 ‘Detection of Larvae in Fillets Proficiency Test’ (PT) were received 17th March 2021 and screened using the ultraviolet transillumination system. Our results were returned to the European Reference laboratory for parasites (EURLP) 25th March 2021. We received a negative evaluation – one positive sample was incorrectly diagnosed as negative. When this material was double checked it was found that an error had indeed been made. An internal investigation has been initiated. Initial findings have identified: • an unsuitable light box without top light as the light box normally used for Anisakis was being repaired. • double checking of results was not undertaken as usual, in part due to COVID-related access restrictions. Once the investigation is complete, a plan for addressing all identified remedial actions will be put in place and progressed as soon as possible.

3.2 Concerning the molecular identification of larvae to the species level Molecular samples were contained in the same package as the fillet samples received on the 17th March 2021. Analysis have been completed and results submitted, but trial results have not been confirmed yet.

4. Official control laboratory (OCL) engagement

There are currently no official control laboratories (OCL) or candidate OCLs in the UK for anisakis/parasite testing, so no activity was required during the reporting period. Some advice was provided to a Scottish laboratory (Aberdeen) in relation to their work on red vent syndrome in wild salmon, a pathological condition associated with high levels of nematode infection.

5. Maintenance of collection of reference material

A relatively small but varied collection of infected materials are in storage at Cefas. This comprises nematodes individually frozen in small vials extracted from various fish species during previous research cruises. As the nematodes are frozen, they are not viable (i.e. dead). Specific pathogen free material is available through our aquarium facility. Further details of what is in storage are as reported in the ‘Final technical report for the UK national reference laboratory for Anisakis April 2016 - March 2017’1. These materials will allow the production of larvae spiked fillet material for proficiency testing if needed, as detailed in protocol NRL007 (DOC 34 in Table 1).

1 https://www.cefas.co.uk/anisakis/annual-reports/anisakis-annual-report-16-17/

6. EURL Annual meeting (2020)

With agreement from FSA, Mr Cook attended the annual EURL meeting for foodborne parasites in December 2020. This took place during the EU transition period so approval for attendance at the meeting was sought and obtained from the Cabinet Office via Defra. Attendance was as an external expert observer only rather than as a direct representative of the UK NRL. The meeting was hosted by European Union Reference Laboratory for Parasites, Istituto Superiore di Sanità, Italy. As well as participants from EU member states, several individuals from outside the EU were also invited (Albania, Iceland, Republic of North Macedonia, Serbia and the UK). Most of the meeting was devoted to terrestrial parasites such as Trichinella, but there were some sections relating to nematodes in fish products. Dr Marco Lalle from the EURL presented the Anisakis proficiency testing results from 2019. Participants included one non-member. Of the 23 participants, 5 provided incorrect results for the enumeration of parasites in fish fillets and 18 laboratories passed. Most laboratories used the artificial digestion method as it requires no specialist equipment. Only 11 laboratories participated in the molecular identification element of the proficiency test, and of these 8 supplied correct results, 2 supplied incorrect results and one did not complete the analysis. Dr Patrizia Rossi from the EURL gave an update on the development of the ISO/CEN standards for examination of fish fillets by UV press and by artificial digestion (ISO 23036-1 and 23036-2). This process, which is being undertaken in parallel by CEN and ISO, is nearing its conclusion. Some minor editorial changes will be made to the documents then final votes will be held between January and March 2021. They should then be published as ISO and CEN standards in June 2021. Following this there was a series of talks on specific subjects by the various participants. Only two related to nematodes in fish products, and these were: • Proteomic profiling of Anisakidae nematodes (Maciej Kochanowski, NRL Poland) • Development and application of novel chemiluminescence immunoassays for highly sensitive detection of proteins in thermally processed seafood (Joanna Dąbrowska, NRL Poland)

Although of interest neither were of direct relevance to UK NRL function.

7. Representation on working groups

The NRL Director, Mr Cook, remains a member of the international Foodborne Parasites Working Group (ISO/TC 34/SC 9/WG 6 Committee). He is also a member of the parallel UK BSI AW009 microbiology working group. The ISO working group has developed new standards for the principal methods of Anisakis screening (UV press and digestion, ISO/DIS 23036-1 & ISO/DIS 23036-2) which have been subject to the various committee review processes and are now due to be formally issued in the summer of 2021.

8. Standard operating procedures

All standard operating procedures (SOPs) and the Code of Practice have been reviewed as required by our internal Quality Assurance processes and are listed in Table 2. Any changes have been recorded within the document management system. A new SOP covering the entire revised molecular identification process used successfully in 2018 and 2019 has been developed and added to the document control system and the NRL website. The EURL has confirmed they are content for these revised procedures to be applied and indicated that other NRLs have also deviated from the one described by the EURLP.

Table 2: SOPs and Guidance Documents for Anisakis NRL

Name Version Title Status Detection of Anisakidae larvae in fish fillets using Live DOC26 1.0 ultraviolet transillumination (UVT) Summary of detection methods for Anisakidae larvae in Live DOC27 1.0 fish fillets DNA extraction and determination of species found within Live DOC36 2.0 the family Anisakidae United Kingdom Anisakis Reference Laboratory: Code of Live DOC32 3.0 Practice/Guidance Document DOC34 1.0 Production of Proficiency Testing (PT) material Live

9. Industry engagement

All testing for Anisakis in the UK is undertaken by the food production and preparation industry with no involvement from the NRL or OCLs. As part of our commitments as the UK NRL and at the request of FSA Delivery Team, Cefas is aiming to establish how controls are implemented by industry and whether these are effective.

The requirements for the hygiene of foodstuffs, including fish, are addressed under Regulation (EC) No. 853/2004 (EC, 2004), as amended by Regulation No. 1276/2011 (EC, 2011) and subsequently copied into UK law through Statutory Instrument 2019 No. 1247. The Regulation allows the marketing of farmed fish intended for raw consumption without freezing treatment and also allows national authorities to relax these requirements for wild fish populations where epidemiological evidence supports this. The exact requirements are listed in the Annexe.

In previous years we have contacted the industry directly to solicit information on the procedures and safeguards they have in place. The results were broadly reassuring in that all processors who replied were very much aware of the parasite, had screening in place, and would either clean or reject infected batches depending on the level of infection. However, screening was either via candling or visual inspection which

are not as effective as UV press or digestion, and only five processors responded despite wide circulation and reminders being sent out. An alternative source of unbiased information is from local authorities which inspect and approve food processing businesses. The FSA circulated a request for information to their network of local authority inspectors in June 2019. FSS and FSANI were also contacted shortly afterwards. Six individuals responded, and exploratory telephone interviews were conducted, covering the subjects listed in Box 1. Only two had been completed by the time the coronavirus lockdown commenced and only one other was successfully contacted since. The officers providing information were from Northern Ireland, Eastern Scotland and North East England.

Box 1. Subjects to be explored in semi-structured interviews with Local Authorities • How many businesses do you inspect which process wild marine fish? (brief description of each one, approx. volumes, species and type of processing) • How many of these produce material which they certify for use as sushi or for cold smoking? • For regular (non-sushi) material what parasite screening is undertaken (Is all incoming material screened? Is outgoing material screened? Candling or visual examination? How well are staff trained? How effective is the screening in your opinion? Are there differences in the way that different sizes/species of fish are screened?) • What do they do when they detect infected material? • For products certified for sushi/cold smoking do the operators demonstrably adhere to the legal requirements for freezing? • How do you check that all required controls are in place? • Are you aware of any significant issues with nematode infected material from these businesses? (rejection by caterers/retailers, supermarket contracts, consumer complaints, illness) • Were you aware that there is a National Reference Laboratory for nematodes in fish, and could it assist you in any way?

The officer from North East England inspects three factory/freezer trawlers at their point of landing. Two of these generally target demersal whitefish (cod, haddock etc) with one also targeting shrimp at times. The third targets pelagic species (e.g. mackerel, blue whiting). Quality control is undertaken on the vessel when the catch is processed by visual inspection of both incoming and processed material. Automated inspection systems are also used. The catch is frozen on board in all cases, so should not contain any viable worms. Freezing certification is inspected by the officer on landing. The operators indicate that they do not encounter much infected material. Any infected material may either be disposed of or rendered in the on- board fish meal plants. Quotas and catches for these vessels are large, and catches are mainly exported.

The officer from Northern Ireland inspects two businesses, both of which are small scale. Most whitefish landings to the port are shipped out, and only a small amount is processed locally, generally for the restaurant trade. None of this is certified for consumption raw. Species are a mix of higher quality demersal species, including cod, haddock, plaice, turbot, lemon sole, gurnard, and brill. Cod and gurnard are the species of greatest concern. There is anecdotal evidence that some fishing grounds are more affected than others. Only visual inspection is carried out, no candling tables are used as they are not considered necessary. Persons processing the fish are mostly extremely experienced (10 plus years). Any less experienced staff have to undertake an NVQ in fish processing, which includes formal training on parasitic worms. Where an infection is found, the belly flaps are removed and used as bait for potters. Flaps are left on if no infection is observed. Heavily infected material would be condemned, but this has never been observed. The officer has never heard of any complaints or rejections by customers. Inspections usually involve discussions with staff rather than a formal inspection of processes or final product.

The officer from Scotland only inspects one business which purchases white fish from fish markets, processes it and sells it on to retailers. Visual inspection is employed, and any worms found are picked out. Processing staff are all trained either via on-the-job training or sometimes via formal training such as NVQs in fish processing. Sometimes all material is screened, sometimes a subsample. This depends on species/area as this influences likelihood of being infected. They work to a standard of a maximum of about 1 worm per kg of flesh. They have never had any complaints from the fishmongers who purchase from them, who are instructed to report any parasite issues back to them.

No formal risk-based testing is carried out by the officers to independently evaluate the parasite burden of the material produced. All controls are undertaken by the processors themselves. Annex VI: F of Regulation 2019/627 (EC, 2019) indicates that ‘Risk-based testing shall take place to verify compliance’ with the parasite standards. Whilst this may potentially be satisfied by the checks the processors undertake themselves, an alternative interpretation may be that the officers should be undertaking periodic checks on the outgoing material from processors. Should this latter interpretation of the regulation be taken, it may be necessary to develop and implement an additional sampling programme for Anisakis.

10. Literature review, calendar year 2020

In order to maintain awareness of any new developments in this field, formal but brief annual reviews of the peer-reviewed literature are undertaken each year. The search term ‘anisakis OR pseudoterranova’ was entered into the Scopus search engine and all search results were extracted (including title and abstract) into a spreadsheet. The retrieved references were then reviewed and where relevant were divided into the following categories:

• Presence of anisakis in European fish stocks and fishery products. • European reports of illness due to anisakis. • Detection methods (for parasites within fishery products). • Any other papers of potential interest to the NRL.

Papers which were tagged as falling under one of these headings were reviewed in more detail, retrieving the full text as necessary and where possible. A synthesis of the findings of these various studies was then made. A total of 146 papers were retrieved by the search, of which 30 were deemed of potential relevance to the NRL. The most relevant papers for each heading are listed in Tables 3-6.

Table 3: Reported presence of anisakis in European stocks and fishery products

Species Area Description Reference Belgian 18% of belly flap portions carried the Mercken et Cod (Gadus morhua) wholesale infection al.,2021 81% of squid carried the infection. 37% had infection in the edible parts Cipriani et (Todarodes sagittatus) NE Atlantic (mantle) al.,2021 Gilthead bream (Sparus aurata) and European seabass Western No anisakis found in sample of 40 Guardone (Dicentrarchus labrax) Mediterranean wild bream and 47 wild bass et al.,2020 Octopus (Eledone cirrhosa and E. moschata) and longfin inshore Prevalence of 2.9% for squid and Guardone squid (Doryteuthis pealeii) Italy 6.7% in octopus et al.,2020 Infection levels were extremely high in both species, and although concentrated in the viscera Scabbardfish (Aphanopus carbo, Eastern nematodes were also present in the Hermida et A. intermedius) Atlantic flesh. al.,2020 Northern No viable larvae were found in air Bao et Cod (Gadus morhua) Norway dried cod fillets. al.,2020 200 cod livers were examined, and 194 were infected with third-stage nematode larvae (overall prevalence of infection 97%) with a mean West intensity of 10.3 (range between 1 Severin et Cod (Gadus morhua) Greenland and 44 parasites per fish). al.,2020 A total of 745 specimens were Western examined, and three Anisakis Menconi et Squid (Illex coindetii) Mediterranean nematodes were found al.,2020 In material collected from Spanish supermarkets, the overall prevalence and abundance of Anisakis type I larvae were 65.3 % and 23.2 helminths/host. 34.2 % of the total Horse mackerel (Trachurus Atlantic and fish sample was parasitized in the Debenedett trachurus) Mediterranean flesh. i et al.,2020

Fillets in oil (90 samples) from EU Anchovies (engraulis retailers were examined, and 30 encrasicolus) and sardines Atlantic and non-viable anisakis larvae were Smaldone (sardina pilchardus) Mediterranean found et al.,2020 A prevalence of 84.5% of infected fish was found, and the mean intensity and mean abundance were Horse mackerel (Trachurus Eastern 32.1 and 27.1 parasites per fish, Lopes et trachurus) Atlantic respectively. al.,2020 A total of 415 fish samples, belonging to 36 different fish species, were collected from a Belgian whole-sale company. An overall prevalence of 53% in the viscera and 27% in the muscles was observed. No larvae were detected in 13 fish species, while a high Belgian prevalence (>78%) was observed in Mercken et Various species markets pollack, halibut, and gurnard. al.,2020 A review of published information was undertaken. Over a 37-year period from 1978 to 2015 the authors reported a 283-fold increase in Anisakis spp. abundance but no change in the abundance of Fiorenza et Various Various Pseudoterranova spp. al.,2020 Anisakis simplex (s.l.) was the most prevalent (100%) nematode in wild salmon with the highest mean intensity (259.9 ± 197.3). The mean intensity of A. simplex (s.l.) represents a four-fold increase compared to published data (63.6 ± 31.9) from salmon captured in Kent et Atlantic salmon (Salmo salar) Scotland Scotland in 2009. al.,2020 Compared with the period 1991- 1996, in 2017-18 there was a strong increase of prevalence, abundance and extension of ulcerative lesions in most cetacean species, based on Pons- post mortem examination of Bordas et Cetaceans NE Atlantic stranded in NW Spain. al.,2020 No nematodes were found in farmed fish destined for human Rainbow trout (Oncorhynchus consumption. Occasional infections Roiha et mykiss) Norway were found in discarded runts. al.,2020

These articles indicate the continuing presence of parasitic nematodes in a variety of fish species and fishery products. This year they also contain some reports of the parasite in European cephalopods. Some fish species (horse mackerel, scabbard fish, wild salmon) typically carry quite heavy parasite burdens, and viable parasites were recovered from retail samples. There were no reports of live worms in ready to eat products.

Two of these were review articles which suggested increasing levels of infection in fish stocks and cetaceans in the North Atlantic, and another article identifies an increase in intensity of infection in wild Scottish salmon over the last decade.

10.1 Reports of illness in Europe Table 4: Studies reporting illness associated with parasitic nematodes in Europe

Country Description Reference A retrospective survey was carried out in the years 2010 to 2014 among all medical parasitology laboratories from university hospitals in France. Thirty- seven cases of anisakidosis have been reported, including 18 cases of allergic Dupouy- anisakidosis. Six additional cases of severe Anisakidae allergy were reported to Camet et France the National Allergovigilance Network over the same period. al.,2020 Report of the first case of gastric anisakiasis in Poland, in a 59-year-old female patient, after eating raw Atlantic salmon (Salmo salar). The larva was still alive and causing pain until it was removed, which occurred more than 5 weeks after Kołodziejczy Poland infection. k et al.,2020 A 59-year-old female who had returned from Japan 2 months previously was Iacomino et Spain found to have a larva embedded in her oral mucosa al.,2020 A 33-year-old patient suffering from rhinitis for 1.5 years recovered a worm larva from the nose. Diagnosis was performed by morphological and molecular Denmar characterization, showing the causative agent to be a third-stage larva of Nordholm k Pseudoterranova decipiens (sensu stricto). et al.,2020 The observed anti-Anisakis seroprevalence inferred by indirect ELISA was significantly higher in fish processing workers (1.8%, 95% CI 0.9–3.3%) Jerončić et Croatia compared to the control population (0%, 0–0.8%). al.,2020 A 58-year-old woman was diagnosed with gastro-allergic anisakiasis, in which the patient developed an acute food-induced IgE-mediated hypersensitivity Decruyenae reaction as well as concurrent gastro-intestinal manifestations after re et Belgium consumption of raw fish. al.,2020 Describes three cases of anisakiosis in patients from Barcelona who had consumed undercooked hake. All patients described epigastric pain and several larval nematodes were removed endoscopically from their stomachs. Hake Roca- consumption stands out as a risk factor for anisakiosis, since this fish species Geronès et Spain can be highly parasitized. al.,2020

There continue to be reports of both infections and allergic reactions in Europe. There were no reports of any illness in the UK.

10.2 Detection methods Table 5: Studies on methods for detection of nematodes in fish products

Method type Description Reference Describes the development and validation of high-sensitivity chemiluminescent (CL) immunoassays for the detection of A. simplex Kochanowsk Immunoassay proteins in processed seafood i et al, 2020

A Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in Cammilleri LAMP assay processed fish products. et al., 2020

Both of these methods are for the detection of Anisakis remains in processed products to detect potential allergens, so neither of these studies are of particular relevance to NRL activities.

Table 6: Other peer reviewed studies of potential interest

Description Reference Investigated larval viability and antigen release following freezing. High freezing rates, Sánchez- preferred in order to maintain optimum fish quality for eating, are able to kill Anisakis Alonso et larvae faster, but may cause a higher release of antigens to the surrounding medium. al., 2020a Ángeles- Reviewed the geographic distribution of Anisakidae. These species are located mainly in Hernández the European continent, Asia and South America, as well as in North and Central America et al., and Australia. 2020 Reviewed the use of substituted (mis-labelled) fish in the context of Anisakis risks. Substituted fish included species vulnerable to infection with a zoonotic parasite not Williams commonly found in the fish which it substituted for. The study focussed on species of et al., relevance to Australian markets. 2020 Investigated viability and antigen release under simulated oral and gastric conditions following freezing. Anisakis able to survive freezing showed lower resistance to gastric fluid. Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in gastric fluids. In treatments rendering complete larvae mortality, Sánchez- the highest loss of larvae integrity was found upon fast freezing, and loss of integrity was Alonso et associated with increased antigen levels. al., 2020b

Two of these studies presented some evidence of increased antigen release from fast-frozen material. The mislabelling of fish species is known to occur in the UK, and this could lead to an increased risk of anisakis in some cases.

11. References

Ángeles-Hernández J.C., Gómez-De Anda F.R., Reyes-Rodríguez N.E., Vega-Sánchez V., García-Reyna P.B., Campos-Montiel R.G., Calderón-Apodaca N.L., Salgado-Miranda C., Zepeda-Velázquez A.P. (2020). Genera and species of the anisakidae family and their geographical distribution. Animals 10, 23741-23.

Bao M., Cipriani P., Giulietti L., Roiha I.S., Paoletti M., Palomba M., Levsen A. (2020). Air-dried stockfish of Northeast Arctic cod do not carry viable anisakid nematodes. Food Control 116, 107322.

Cammilleri G., Ferrantelli V., Pulvirenti A., Drago C., Stampone G., Del Rocio Quintero Macias G., Drago S., Arcoleo G., Costa A., Geraci F., Di Bella C. (2020). Validation of a commercial loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Anisakis spp. DNA in processed fish products. Foods 9, 92. Cipriani P., Palomba M., Giulietti L., Bao M., Mattiucci S., Levsen A. (2021). Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) hidden in the mantle of European flying squid Todarodes sagittatus (Cephalopoda: Ommastrephidae) in NE Atlantic Ocean: Food safety implications. International Journal of Food Microbiology 339, 109021.

Debenedetti Á.L., Codes F., Laza S., Hernández S., Madrid E., Trelis M., Fuentes M.V. (2020). Ascaridoid nematodes in horse mackerel, Trachurus trachurus, sold in Spanish supermarkets—Factors able to diminish consumer risk. Fisheries Research 230, 105669.

Decruyenaere P., Van de Maele B., Hulstaert E., Van Vlierberghe H., Decruyenaere J., Lapeere H. (2020). IgE- mediated gastroallergic anisakiasis with eosinophilic oesophagitis: a case report. Acta Clinica Belgica: International Journal of Clinical and Laboratory Medicine, in press.

Dupouy-Camet J., Gay M., Houin R. (2020). New eating habits, new parasitic risks: The example of fish [De nouvelles habitudes alimentaires, de nouveaux risques parasitaires : l'exemple du poisson]. Bulletin de l'Academie Nationale de Medecine 204, 1010-1016.

Fiorenza E.A., Wendt C.A., Dobkowski K.A., King T.L., Pappaionou M., Rabinowitz P., Samhouri J.F., Wood C.L. (2020). It’s a wormy world: Meta-analysis reveals several decades of change in the global abundance of the parasitic nematodes Anisakis spp. and Pseudoterranova spp. in marine fishes and invertebrates. Global Change Biology 26, 2854-2866. Guardone L., Bilska-Zając E., Giusti A., Malandra R., Cencek T., Armani A. (2020). Larval ascaridoid nematodes in horned and musky octopus (Eledone cirrhosa and E. moschata) and longfin inshore squid (Doryteuthis pealeii): Safety and quality implications for cephalopod products sold as fresh on the Italian market. International Journal of Food Microbiology 333, 108812.

Guardone L., Susini F., Castiglione D., Ricci E., Corradini C., Guidi A., Armani A. (2020). Ascaridoid nematode larvae in wild gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) caught in the Tyrrhenian Sea (Western Mediterranean Sea): a contribute towards the parasitological risk assessment on two commercially important fish species. Food Control 118, 107377.

Hermida M., Simon C., Amorim A., Delgado J. (2020). Anisakid infection in scabbardfishes, Aphanopus carbo and A. intermedius (Perciformes: Trichiuridae), from the Eastern Atlantic. Regional Studies in Marine Science 40, 101538.

Iacomino E., Sinatti G., Pasqua M., Tucci C., Picchi G., Cipolloni G., Marco G.-P.D. (2020). Anisakis in oral cavity: A rare case of an emerging disease. Oral and Maxillofacial Surgery Cases 6, 100136.

Jerončić A., Nonković D., Vrbatović A., Hrabar J., Bušelić I., Martínez-Sernández V., Lojo Rocamonde S.A., Ubeira F.M., Jaman S., Jeličić E.Č., Amati M., Morales M.A.G., Lukšić B., Mladineo I. (2020). Anisakis sensitization in the Croatian fish processing workers: Behavioural instead of occupational risk factors? PLoS Neglected Tropical Diseases 14, e00080381-21.

Kent A.J., Pert C.C., Briers R.A., Diele K., Rueckert S. (2020). Increasing intensities of Anisakis simplex third- stage larvae (L3) in Atlantic salmon of coastal waters of Scotland. Parasites and Vectors13, 62.

Kochanowski M., Różycki M., Dąbrowska J., Karamon J., Sroka J., Antolak E., Bełcik A., Cencek T. (2020). Development and application of novel chemiluminescence immunoassays for highly sensitive detection of anisakis simplex proteins in thermally processed seafood. Pathogens 9, 7771-18.

Kołodziejczyk L., Szostakowska B., Sobecka E., Szczucki K., Stankiewicz K. (2020). First case of human anisakiasis in Poland. Parasitology International 76, 102073.

Lopes P., Vilares A., Caçador T., Martins S., Ferreira I., Carvalho L.M., Gargaté M.J. (2020). Occurrence of larval anisakids in horse mackerel (Trachurus trachurus) caught in Portuguese waters. Parasitology Research 119, 2799-2811.

Menconi V., Pastorino P., Burioli E.A.V., Righetti M., Scanzio T., Favaro L., Bona M.C., Pavoletti E., Dondo A., Prearo M. (2020). Occurrence of ascaridoid nematodes in Illex coindetii, a commercially relevant cephalopod species from the Ligurian Sea (Northwest Mediterranean Sea). Food Control 116, 107311.

Mercken E., Van Damme I., Šoba B., Vangeenberghe S., Serradell A., Lumain J.P.L., De Sterck T., Lalle M., Gabriël S. (2021). High occurrence of Anisakidae at retail level in cod (Gadus morhua) belly flaps and the impact of extensive candling. Food and Waterborne Parasitology 22, e00108.

Mercken E., Van Damme I., Vangeenberghe S., Serradell A., De Sterck T., Lumain J.P.L., Gabriël S. (2020). Ascaridoids in commercial fish: Occurrence, intensity and localization in whole fish and fillets destined for the Belgian market. International Journal of Food Microbiology327, 108657.

Nordholm A., Kurtzhals J.A.L., Karami A.M., Kania P.W., Buchmann K. (2020). Nasal localization of a Pseudoterranova decipiens larva in a Danish patient with suspected allergic rhinitis. Journal of Helminthology, e187.

Pons-Bordas C., Hazenberg A., Hernandez-Gonzalez A., Pool R.V., Covelo P., Sánchez-Hermosin P., López A., Saavedra C., Fraija-Fernández N., Fernández M., Aznar F.J. (2020). Recent increase of ulcerative lesions caused by Anisakis spp. In cetaceans from the north-east Atlantic. Journal of Helminthology, e127.

Roca-Geronès X., Alcover M.M., Godínez-González C., González-Moreno O., Masachs M., Fisa R., Montoliu I. (2020). First molecular diagnosis of clinical cases of gastric anisakiosis in Spain. Genes 11, 452.

Roiha I.S., Maage A., Levsen A. (2020). Farmed rainbow trout (Oncorhynchus mykiss) in Norway are at low risk of carrying anisakid nematodes. Journal of Applied Aquaculture, 1-12.

Sánchez-Alonso I., Carballeda-Sangiao N., González-Muñoz M., Navas A., Arcos S.C., Mendizábal A., Cuesta F., Careche M. (2020)a. Freezing kinetic parameters influence allergenic and infective potential of Anisakis simplex L3 present in fish muscle. Food Control 118, 107373.

Sánchez-Alonso I., Carballeda-Sangiao N., Rodríguez S., Tejada M., Navas A., Arcos S.C., González-Muñoz M., Careche M. (2020)b. Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release. Journal of the Science of Food and Agriculture, in press.

Severin N.L., Yurchenko M., Sørensen J.S., Zuo S., Karami A.M., Kania P.W., Buchmann K. (2020). Anisakid nematode larvae in the liver of Atlantic cod Gadus morhua L. from West Greenland. Parasitology Research 119, 3233-3241.

Smaldone G., Ambrosio R.L., Marrone R., Ceruso M., Anastasio A. (2020). Anisakis spp. Larvae in deboned, in- oil fillets made of anchovies (Engraulis encrasicolus) and sardines (Sardina pilchardus) sold in eu retailers. Animals 10, 18071-10.

Williams M., Hernandez-Jover M., Shamsi S. (2020). Fish substitutions which may increase human health risks from zoonotic seafood borne parasites: A review. Food Control 118, 107429.

12. Annexe: Legislative standards for parasites in fishery products

The requirements for the hygiene of foodstuffs, including fish, are addressed under Regulation (EC) No. 853/2004 (EC, 2004), as amended by Regulation No. 1276/2011 (EC, 2011) and subsequently copied into UK law through Statutory Instrument 2019 No. 1247. The sections relating to parasites in fishery products are listed below.

‘Laying Down Specific Hygiene Rules on the Hygiene of Foodstuffs’ Annex III, Section VIII, Chapter III: Requirements for Establishments, Including Vessels, Handling Fishery Products. Section D: Requirements Concerning Parasites 1. Food business operators placing on the market the following fishery products derived from finfish or cephalopod molluscs: (a) fishery products intended to be consumed raw; or (b) marinated, salted and any other treated fishery products, if the treatment is insufficient to kill the viable parasite; must ensure that the raw material or finished product undergo a freezing treatment in order to kill viable parasites that may be a risk to the health of the consumer. 2. For parasites other than trematodes the freezing treatment must consist of lowering the temperature in all parts of the product to at least: (a) – 20 °C for not less than 24 hours; or (b) – 35 °C for not less than 15 hours. 3. Food business operators need not carry out the freezing treatment set out in point 1 for fishery products: (a) that have undergone or are intended to undergo before consumption a heat treatment that kills the viable parasite. In the case of parasites other than trematodes the product is heated to a core temperature of 60 °C or more for at least one minute; (b) that have been preserved as frozen fishery products for a sufficiently long period to kill the viable parasites; (c) from wild catches, provided that: (i) there are epidemiological data available indicating that the fishing grounds of origin do not present a health hazard with regard to the presence of parasites; and (ii) the competent authority so authorises; (d) derived from fish farming, cultured from embryos and have been fed exclusively on a diet that cannot contain viable parasites that present a health hazard, and one of the following requirements is complied with: (i) have been exclusively reared in an environment that is free from viable parasites; or (ii) the food business operator verifies through procedures, approved by the competent authority, that the fishery products do not represent a health hazard with regard to the presence of viable parasites.

4. (a) When placing on the market, except when supplied to the final consumer, fishery products referred to in point 1 must be accompanied by a document issued by the food business operator performing the freezing treatment, stating the type of freezing treatment that the products have undergone. (b) Before placing on the market fishery products referred to in points 3(c) and (d) which have not undergone the freezing treatment or which are not intended to undergo before consumption a treatment that kills viable parasites that present a health hazard, a food business operator must ensure that the fishery products originate from a fishing ground or fish farming which complies with the specific conditions referred to in one of those points. This provision may be met by information in the commercial document or by any other information accompanying the fishery products.

Chapter V: Health and Safety Standards for Fisheries Products Section D. Parasites Food business operators must ensure that fishery products have been subjected to a visual examination for the purpose of detecting visible parasites before being placed on the market. They must not place fishery products that are obviously contaminated with parasites on the market for human consumption.

Regulation 2019/627 (EC, 2019) indicates that: Article 70 Official controls of fishery products shall include at least the practical arrangements laid down in Annex VI as regards parasites;

Article 71 which requires the competent authorities to declare fishery products unfit for human consumption if: (a) official controls carried out in accordance with Article 70 reveal they are not in compliance with organoleptic, chemical, physical or microbiological requirements or requirements for parasites as established in Section VII of Annex III of Regulation (EC) No 853/2004 and/or Regulation (EC) No 2073/2005;

Annex VI: F. Parasites Risk-based testing shall take place to verify compliance with Part D of Chapter III of Section VIII of Annex III to Regulation (EC) No 853/2004 and Section I of Annex II to Regulation (EC) No 2074/2005.