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WORK PROGRAMMES FOR

EU REFERENCE LABORATORIES

FOR 2010 **********

Biological Risks **********

Milk and milk products

Salmonella Marine biotoxines

Bacteriological and viral contaminants of molluscs Transmissible Spongifiorm Encephalopathies

Listeria monocytogenes Coagulase positive Staphylococci

Escherichia coli, including Verotoxigenic E. coli (VTEC) Campylobacter

Parasites (in particular Trichinella, Echinococcus and Anisakis)

Antimicrobial resistance Animal proteins in feedingstuffs

EU REFERENCE LABORATORY FOR MILK AND MILK

PRODUCTS

WORK PROGRAMME 2010

EU REFERENCE LABORATORY FOR SALMONELLA

WORK PROGRAMME 2010

Work-programme CRL-S 2010 11-08-2009 Page 1 of 7

CRL-Salmonella work-programme 2010 Introduction The working programme of CRL-Salmonella consists of the following activities (the duration of the activities are indicated between brackets): 1. Organisation of interlaboratory comparison studies (yearly); 2. Organisation of a workshop with the NRLs-Salmonella (yearly); 3. Performance of research (depending on the subject: yearly or for a limited period); 4. Giving assistance to the Commission and ad hoc activities (yearly); 5. Communication (every 3 months and yearly); 6. Training (duration dependent on the subject). 7. Missions (duration dependent on the subject); 1. Interlaboratory comparison studies For 2010 it is planned to organise 3 interlaboratory comparison studies; • One study on bacteriological detection of Salmonella in a veterinary matrix; • One study on bacteriological detection of Salmonella in a food or feed matrix; • One study on typing of Salmonella. The exact timing of the studies in 2010 will be planned with the NRLs-Salmonella, but an indication of the probable time period is given below. The general set-up of the CRL-Salmonella interlaboratory comparison studies on the bacteriological detection of Salmonella has remained the same for many years. In these studies a Salmonella negative matrix is artificially contaminated with Salmonella reference materials containing Salmonella Typhimurium or Salmonella Enteritidis at different contamination levels. The differences over the years have been the choice of the matrix, the number of reference materials to be tested, the contamination level of these reference materials and the choice of the methods. Although it may be more realistic to test naturally contaminated samples, this has only occasionally been used as test matrix, because: a) It is complicated to obtain a sufficient amount of a naturally contaminated matrix; b) The Salmonella contamination in the naturally contaminated sample is in general not homogeneously distributed over the sample; c) The stability of the naturally contaminated samples may not be sufficient for the cause of the study; d) The contamination level and the Salmonella serovar present in the naturally contaminated sample can not be adapted; e) The results of different interlaboratory comparison studies are hard to compare to each other. Interlaboratory comparison study on bacteriological detection of Salmonella in veterinary samples Like for former studies, a Salmonella-free matrix will be selected which will be artificially contaminated with Salmonella reference materials. It will be discussed with statisticians whether the number of samples and contamination levels of the reference materials will be kept the same as for the study of 2009 or whether this set-up may need


some amendments. In the study of 2009, reference materials with low and high level of two Salmonella serovars (Salmonella Typhimurium and Salmonella Enteritidis) were used. The low level of the reference materials was chosen as close to the detection limit as possible. The high level samples were approximately 5-10 times higher than the low level materials. The following is planned for the 2010 veterinary ring trial: - Time period: February/March; - Matrix: animal faeces; - Number of samples and level of reference materials to be decided later; - Methods: Annex D of ISO 6579 (‘Detection of Salmonella in animal faeces and in

environmental samples from the primary production stage’), implying modified semi-solid Rappaport Vassiliadis (MSRV) agar as selective enrichment medium, and own method(s).

The results of each NRL will be evaluated and compared with the pre-set definition of ‘good performance’. In case of unexplainable ‘poor performance’, the follow-up will be discussed with the relevant NRL (e.g. sending extra samples which need to be tested according to a prescribed protocol, training at the CRL or visiting a NRL by a member of the CRL-Salmonella staff). Since 2008, also reference laboratories of two third countries participated, being: Tunisia and Israel. These countries participated on request of DG-Sanco. It is foreseen that these (two) third countries will again participate in the study of 2010. Additionally, two more third countries, Canada and the United States of America, may participate in the 2010 study (awaits for information from DG-Sanco). The results of the third countries will be analysed separately from the results of the NRLs of the European Member States. The justification for participation of these third countries was given in the work-programme of 2008 and is repeated below: Salmonella control programmes in live poultry are introduced in the European Member States by Regulation (EC) No 2160/2003. The control programmes in breeding hens include the monitoring of Salmonella by the testing of faecal materials in accordance with the provisions in Regulation (EC) No 1003/2005. Third countries, who want to remain or be added to the list of third countries from which Member States may import breeding hens or hatching eggs, should submit a control programme equivalent to the control programmes of the Member States. In order to evaluate the equivalence of testing in these third countries, they should participate in the ring trials organised by the CRL. Tunesia, Canada, Israel and the United States forwarded their control programme for breeding hens and should therefore be included in the ring trial. Interlaboratory comparison study on bacteriological detection of Salmonella in food or feed samples In 2008 the first interlaboratory comparison study on the detection of Salmonella in animal feed was organised. It will be discussed with DG-Sanco and with the NRLs for Salmonella, whether the 2010 study will again contain animal feed as matrix, or whether a food matrix should be the matrix of choice. In both cases, the set-up of the study may be similar to earlier studies. However, depending on the outcome of the discussion with statisticians on the set-up of the veterinary study, the set-up of the food/feed study may be amended as well. The prescribed method will be ISO 6579 (2002: Microbiology of food


and animal feeding stuffs – Horizontal method for the detection of Salmonella spp.), with selective enrichment in RVS and MKTTn. The requested method will be Annex D of ISO 6579, with selective enrichment on MSRV. The planning of the study is September/October 2010. Like for the veterinary study, the results of each NRL will be evaluated and compared with the pre-set definition of ‘good performance’. Actions will be taken in case of unexplainable ‘poor performance’ (see above). Interlaboratory comparison study on typing of Salmonella Up to 2008, EnterNet laboratories (analysing human isolates) had the possibility to participate in the typing studies through a collaboration between the CRL-Salmonella and the Health Protection Agency (HPA), Colindale (UK). In fall 2008, this collaboration was formalised in a contract with the ECDC. This collaboration is also profitable for the NRLs, as it still gives them the opportunity to test their phage typing capacities additional to the serotyping of Salmonella in the interlaboratory comparison study on typing. Like in former studies the CRL-Salmonella will select twenty Salmonella strains for serotyping, including serovars with public health significance, serovars with antigens similar to those of public health significant strains and serovars that had caused typing problems in previous studies. The strains will be blindly coded and send to the NRLs for serotyping. The HPA will select twenty Salmonella strains for phagetyping (10 Salmonella Enteritidis strains and 10 Salmonella Typhimurium strains). These latter strains will only be sent to the NRLs who have indicated to perform phage typing as well. The planning of the study is November/December 2010. In 2007 a definition on the evaluation of ‘good performance’ of the serotyping was agreed upon with the NRLs and for the first time applied on the results of the study of 2007. This definition was based on the fact that in EU legislation a distinction was made between the Salmonella ‘top 5’ and other Salmonella serovars. However, it is under discussion whether this distinction should be maintained or not in the EU legislation. Depending on the outcome of this discussion it may be necessary to amend the definition for good performance. If necessary this will be discussed with the NRLs at the CRL-Salmonella workshop in 2010. 2. Workshop At the annual workshop of the CRL-Salmonella in 2009, the NRLs indicated a preference for organisation of the annual workshop in conjunction with the international Salmonella symposium (i3s) in France (Saint-Malo) in June 2010. As this symposium last 3 days, the duration of the workshop may be shortened to 1-1,5 days instead of 1,5-2 days (as in earlier years) to facilitate the travelling of the participants of the NRLs. Extra costs are expected when the workshop is held in France, as the 3 CRL staff members as well as the participant of the Dutch NRL also need to travel and need daily allowances for their stay in France. The programme may contain the following items: - Introductory presentations (e.g., by EU representative and CRL-Salmonella); - Zoonoses in Europe (EFSA, DG-Sanco); - Results of research activities of CRL-Salmonella;


- Results of interlaboratory comparison studies of 2009 and 2010; - Experiences, problems, results in relation to monitoring surveys for Salmonella; - Plans and results of research activities of the NRLs-Salmonella; - Discussion on methods (e.g. typing, molecular, serological); - Activities in ISO and CEN; - Future working plan of CRL-Salmonella. 3. Research Activities concerning ISO and CEN The CRL-Salmonella (Kirsten Mooijman) is involved (as project leader or as member of working groups) in several activities of ISO and CEN. More specific in: - ISO/TC34/SC9: International Standardisation Organisation, Technical Committee 34

on Food products, Subcommittee 9 – Microbiology. - CEN/TC275/WG6: European Committee for Standardisation, Technical Committee

275 for Food analysis – Horizontal methods, Working Group 6 for Microbial contaminants.

Planned activities in which CRL-Salmonella will be involved (or is leading) in 2010: Enumeration of Salmonella (CRL-Salmonella project leader): The CRL-Salmonella

prepared in 2007 a draft ISO standard document on an MPN based method performed in microwell (12 wells) plates, called ‘mini-MSRV’ for the enumeration of Salmonella. Early 2009 a New Working Item Proposal (NWIP) for publication of the ‘mini-MPN’ document as an ISO Technical Specification (ISO/TS) was launched and accepted with some comments. The document has been amended, but it can only be finalised when a tool from the ISO working group on statistics for the calculation of the Most Probable Number (MPN) comes available. It is not known when this tool comes available and whether some final work on the document is necessary in 2010.

Revision of ISO 6579 (CRL-Salmonella project leader): At the ISO/TC34/SC9 meeting in Valencia (Spain) in 2009 it was agreed to revise ISO 6579 and that a new working group would be raised (WG9) with Kirsten Mooijman as convenor. Several items have been indicated which will need further review by consulting experts, literature research and or laboratory research. The working group will probably meet 1 to 2 times in 2010. One meeting may be held in conjunction with the plenary meeting of ISO/TC34/SC9 in 2010. Kirsten Mooijman will need to participate in this latter meeting to report the progress of the working group.

Guide or standard for serotyping Salmonella spp. (CRL-Salmonella project leader): At the annual ISO meeting in 2009 it was agreed to raise an ad hoc group which will deal with the document on serotyping and will be convened by Kirsten Mooijman. A first meeting of this ad hoc group is foreseen by the end of 2009. Depending on the outcome of this meeting further activities for the CRL-Salmonella may be needed for this subject (drafting a document, laboratory work). Furthermore it may be necessary that the working group will meet 1 to 2 times in 2010. One meeting may be held in conjunction with the plenary meeting of ISO/TC34/SC9 in 2010. Kirsten Mooijman


will need to participate in this latter meeting to report the progress of the ad hoc group.

CEN mandate on validation of microbiological methods: In 2007 the CRL-Salmonella was assigned as project leader for the validation of Annex D of ISO 6579. By then the CRL- Salmonella also submitted a work plan and budget forecast. In 2009 the first consolidated working plan (for all 15 methods) was given to the project leaders for comments. The work plan still needs to be submitted to the EC (by CEN). It is not clear when a contract for the performance of the study can be expected. As soon as this contract comes available, a further planning of the activities can be made, which will involve expertise of the CRL as well as of some NRLs for Salmonella.

Other working groups: Kirsten Mooijman is member of Working group (WG)3 (Validation of microbiological methods), of WG4 (Proficiency Testing in microbiology) and of the joint working group (WG)5 (quality assurance of culture media) of ISO/TC34/SC9. The items are of major relevance for the CRL-Salmonella as well as for the NRLs for Salmonella and it is therefore important to remain up to date with standardisation in these fields. The working groups meet approximately twice a year. Each member of a working group is expected to actively contribute to the documents which need to be prepared, either by writing a part of a document and/or by giving comments on draft documents.

Methods Literature researches and experiments are foreseen in relation to the ISO/CEN activities in the field of Salmonella (see above). Especially for the revision of ISO 6579 (much) experimental work is expected. In 2007 some artificially contaminated samples (chicken faeces, pig faeces, minced meat) were tested for Salmonella using a Real-Time Polymerase Chain Reaction technique. All samples gave similar results with the RT-PCR and with the bacteriological detection method. In 2008 several naturally contaminated samples were obtained, with different contamination levels. These samples were frozen at -20 °C and still need to be tested with the PCR technique. Reference materials As indicated at 1. Interlaboratory comparison studies, it is planned to discuss with statisticians whether the studies can be further optimised in terms of number of reference materials and relevant contamination levels to be used. Depending on the outcome of this discussion it may be necessary to prepare and test new batches of reference materials before they can be used in interlaboratory comparison studies. Matrices Research activities may be necessary for testing relevant matrices for the interlaboratory comparison study on the detection of Salmonella in food or in animal feed. The matrix will be tested with and without the addition of Salmonella reference materials. Also the amount of background flora will be analysed.


4. Giving assistance to the Commission and ad hoc activities Each year the CRL-Salmonella is contacted by various parties, i.e. institutes in Member States, Candidate Member States or third countries, with requests for information or for participation in activities being organised. Also, requests for support from the European Commission with respect to certain issues (e.g., methods, participation in working groups, advices) are raised. In these cases the CRL-Salmonella will in principle always react positively and will try to include the ad hoc work required in the working plan although it is difficult to plan the time needed to answer the different questions. Participation in Working Groups of DG-Sanco and EFSA Staff members of the CRL-Salmonella participates in the working groups of DG-Sanco and of EFSA for (among others) discussion of results of and/or preparation of technical specifications of monitoring and control programmes in accordance with EU legislations. 5. Communication A staff member of the CRL-Salmonella has been trained to keep the CRL-Salmonella website up to date (www.rivm.nl/crlsalmonella). As it is foreseen that the RIVM website will be amended in 2010, it may be necessary to amend the CRL-Salmonella website as well. The newsletter of CRL-Salmonella is published every quarter with information from the CRL-Salmonella relevant for the NRLs-Salmonella or from NRLs-Salmonella relevant for the other NRLs-Salmonella. Also, a literature search is included in each newsletter covering the previous 3-month period. Results on the interlaboratory comparison studies, the workshop and relevant research will be published in RIVM reports. The reports will be distributed to the EC and to the NRLs and other interested bodies. Furthermore they will also become available at the CRL-Salmonella website. Summaries of several intercomparison studies and related research will be published (if possible) in the scientific literature. 6. Training On request of an NRL, the CRL can give a training for a specific need of an NRL. It is also possible that the CRL will advice an NRL to follow a training at the CRL, especially in case of (repeated) poor performance of the NRL in interlaboratory comparison studies. 7. Missions Summarising the information as given in the chapters above, the following missions may be needed in 2010. Per mission one (occasionally two) staff member of the CRL-Salmonella will participate. - Visit to a poor performing NRL (if necessary, see information under chapter

‘Interlaboratory comparison studies’), approximately 2 days, country unknown.


- Annual meetings of ISO/TC34/SC9 and CEN/TC275/WG6, probably in Argentina, date not yet set (see information under chapter ‘Research’). Total duration of the meetings approximately 5 days.

- Meetings of several working groups or ad hoc groups of ISO/TC34/SC9 (see information under chapter ‘Research’), approximately 2 meetings per working group, with 1 meeting, for most of the groups, in conjunction with the meeting of ISO/TC34/SC9. The meetings are not yet planned, but will be scheduled as soon as considered needed.

Mrs. drs. K.A. Mooijman Head CRL-Salmonella

EU REFERENCE LABORATORY FOR MARINE BIOTOXINS

WORK PROGRAMME 2010

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WORK PROGRAMME FOR THE COMMUNITY REFERENCE LABORATORY

FOR MARINE BIOTOXINS

January-December 2010

Community Reference Laboratory for Marine Biotoxins (CRLMB) Agencia Española de Seguridad Alimentaria y Nutrición (AESAN) Estación Marítima S/N. Muelle de Trasatlánticos 36200 Vigo, Spain E-mail: [email protected] http://www.aesan.msps.es/en/CRLMB/web/home.shtml

COMMUNITY REFERENCE LABORATORY FOR MARINE BIOTOXINS

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Work Programme for the Community Reference Laboratory for Marine Biotoxins Objectives for the period January-December 2010 LEGAL FUNCTIONS AND DUTIES The functions and duties of the Community Reference Laboratories for feed and food are described in Article 32(1) of Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules. 1. Coordination of activities of NRL network and

provision of technical assistance and training 1.1. Organization of the XIII Workshop of the European NRL network of marine

biotoxins Date: to be determined (expected to be in October-November) Place: to be agreed during the XII Workshop to be held in Madrid in October 2010. Duration: 1,5-2 days

The CRLMB will organise and coordinate the workshop and will edit the minutes that will be distributed to the participants and published on the CRLMB website.

1.2. Supply information to EU MS and third countries on analytical methods on marine biotoxins (e mail, website)

Duration: ongoing (as required) In addition to on-going assistance, the CRLMB website is available at: http://www.aesan.msps.es/en/CRLMB/web/home.shtml. There, it can be consulted about marine toxins legislation, methodologies, different meetings minutes and recommendations and all the activities related to CRLMB.

1.3. Assist NRL on the implementation, validation and accreditation of methods particularly by provision of advice and CRL SOPS.

Duration: ongoing The CRLMB will assist NRLs on the implementation and validation of official and alternative analytical methods by providing them with technical information, standards when available, contaminated materials, etc at their

COMMUNITY REFERENCE LABORATORY FOR MARINE BIOTOXINS

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request. Moreover, protocols for marine biotoxins determination are published on the CRLMB website.

1.4. Technical assistance to Third Countries on analytical methods. The CRLMB will assist third countries by providing them with samples from the CRLMB Proficiency Testing trials in order to have intercomparison results, useful for the validation and accreditation of methods for marine biotoxins detection. This provision will depend on samples availability.

1.5. Training activities for the staff of NRL MS and third countries (as required, maximum 4 countries).

1.6. Working group on LC-MS. The activities will be focused on lipophilic toxins.

See point 5.1.

1.7. Working group on EFSA Opinion on Marine Biotoxins. Activities focused on possibilities of the EU-NRLs network on the potential/practical implementation of the Opinion on marine biotoxins in shellfish provided by the EFSA Scientific Panel on Contaminants in the Food Chain (CONTAM). This Working Group will depend on discussions about the issue during the XII Workshop to be held in Madrid in October 2010.

NOTE: If it is required after the next XII Workshop of EU-NRLs (October 2009, Madrid) the ongoing working groups could be modified (eliminated, modified, or new ones created).

2. Scientific advice and support 2.1. Provide scientific assistance to DG-SANCO.

Duration: ongoing The CRLMB will assist DG-SANCO and FVO if it is required on issues related with the control of marine biotoxins in molluscs.

2.2. Provide technical assistance in cases where the results of analysis are contested between Member States and/or Member States and Third Countries.

Duration: ongoing, when requested. 3. Interlaboratory Proficiency Testing The interlaboratory proficiency testing trials organised by the CRLMB for the NRLs are aimed at evaluating the ability of the NRLs to apply satisfactorily the recognised testing methods for marine biotoxins for the purpose of Regulations (EC) Nos. 853/2004 and 854/2004 stated in Annex III of Commission Regulation (EC) Nº 2074/2005. At the same time, the equivalence of the different methods applied is studied. The CRLMB will organise one trial for each marine biotoxins group legislated in the EU:

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3.1. CRLMB 2010 Proficiency Testing for PSP toxins determination (CRLMB-2010-PT-PSP). 3.2. CRLMB 2010 Proficiency Testing for LIPOPHILIC toxins determination (CRLMB-2010-PT-LIPO). 3.3. CRLMB 2010 Proficiency Testing for ASP toxins determination (CRLMB-2010-PT-ASP). Schedule for the Proficiency Testing trials (participants registration, samples dispatch, results submission and PT report) will be agreed by the EU-NRLs network and published in the “EU CRLMB/NRLs timetable of activities for 2010”, which will be circulated to the network in the first quarter of 2010. NRLs will receive in advance the Programme and the “Application Form” to be registered at each trial together with information about the foreseen dates for samples dispatch and results submission. Samples for the trial will be accompanied with the identity code for each participant. At the same time, participants will receive by email the “Protocol” for the exercise, a “Sample Arrival Form” to be sent to the CRLMB just after samples reception and the “Reporting Sheet” for results submission. Preliminary results on CRLMB-2010-PT exercises will be presented by the CRLMB during the XIII Workshop of the European NRL network of marine biotoxins and discussed by the group. A final and detailed report (with all information on participants, aim of the exercise, schedule and instructions, materials preparation, methods used by participants, evaluation of results, proficiency results,..) will be elaborated and edited by the CRLMB for each PT, circulated to participants in the trial and published on the CRLMB website. A follow-up report on the management in cases of underperformance in the Proficiency Testing by NRLs will be submitted to DG SANCO together with the CRLMB technical report for 2010 (planned for March 2011). 4. Missions 4.1. Visit to NRLs when necessary, in order to solve problems with results after ring trials or validation studies (see points 3 and 5).

4.2. Participate in activities related to the involvement in the Working Group 5 ('Biotoxins') of CEN/TC 275 Technical Committee “Food analysis – Horizontal methods” (European Committee for Standardization). 4.3. Participate in activities related to the involvement in the AOAC Task Force for Marine Biotoxins.

4.4. On going participation in the Codex Alimentarius working groups and meetings related to marine biotoxins.

4.5. Assist third countries on technical issues on request.

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5. Development of analytical and detection methods 5.1. Development and validation of single toxins group methods, continuing with a method for AZA-group toxins, for LC-MS analysis of the lipophilic toxins legislated in the EU. 5.2. In view of the results got during the Single Laboratory Validation of the improved version of TOXILINE-DSP test, carried out in July-September 2009, continuation with the formal validation study of the method for the determination of Okadaic acid and dinophysistoxins in molluscs by a Fluorimetric Phosphatase Assay through the TOXILINE-DSP test. 5.3. Evaluation/Validation of other alternative phosphatase assays for the determination of Okadaic acid and dinophysistoxins in molluscs. 5.4. Validation and accreditation of a multitoxin method by LC-MS for the determination of Okadaic acid-group, Azaspiracid-group for the confirmative determination at the CRLMB of these toxins. 5.5. Pending on the reactivation of the activities related to phycotoxins through CEN/TC 275/WG5-Biotoxins, finalization of necessary requirements to get the validated method for “the determination of Domoic acid in shellfish and finfish by RP-HPLC using UV detection” as a CEN standard. 5.6. Pending on the reactivation of the activities related to phycotoxins through CEN/TC 275/WG5-Biotoxins, collaboration in the elaboration of a document about “Criteria of analytical methods for marine biotoxins”. 5.7. Participation in a collaborative study for the formal validation of a Rapid Postcolumn Methodology for Determination of Paralytic Shellfish Toxins in Shellfish Tissue. 5.8. Implementation of methodologies based on biological methods and LC-tandem mass spectrometry (LC/MS/MS) for the determination of ciguatoxins in fish. 5.9. Implementation of methodologies based on biological methods LC-tandem mass spectrometry (LC/MS/MS) for the determination of brevetoxins in shellfish. 5.10. Development of methodologies based on biological methods LC-tandem mass spectrometry (LC/MS/MS) for the determination of palytoxins in fish. 5.11. Identification of methodological options to changes in regulatory levels for marine toxins (see point 1.7).

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6. Implementation of DG SANCO “Protocol for the management of underperformance in comparative testing and/or lack of collaboration of National Reference Laboratories (NRLs) with CRLs activities”

6.1. Underperformance (i.e failure in proficiency test). See Point 3. The CRLMB will carry out a follow-up of those NRLs that fails in the CRLMB-2010-Proficiency Testing for PSP, lipophilic and ASP toxins determination in order to identify the origin of the bad results. 6.2. Lack of collaboration by the NRLs with the CRLMB. According to DG SANCO protocol, the CRLMB will contact those NRLs for which a lack of collaboration with the CRL is detected. This lack of collaboration can be due to lack of collaboration in working groups’ activities or to a lack of participation in the CRLMB proficiency testing exercises.

EU REFERENCE LABORATORY FOR BACTERIOLOGICAL AND VIRAL CONTAMINANTS OF BIVALVE MOLLUSCS

WORK PROGRAMME 2010

European Community Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs

CEFAS Laboratory, Weymouth, Dorset, DT4 8UB, UK

WORK PROGRAMME FOR THE CRL FOR BACTERIOLOGICAL AND VIRAL CONTAMINATION OF BIVALVE MOLLUSCS, 2010 LEGAL FUNCTIONS AND DUTIES The functions and duties of the CRL are specified in Article 32 of Regulation (EC) No 882/2004 (Official Journal of the European Communities No L 165 of 30.4.2004). In the 2009 work programme year 27 Member States and 3 candidate countries (Croatia, Turkey and Republic of Macedonia) are considered eligible for CRL assistance and invited to participate in CRL organised training programmes, ring trials/external quality assessments schemes etc. The full integration into the European Union of recent accession Member States continues to be a priority area, and is facilitated via the provision of additional advice, training and assistance. WORK PROGRAMME, 2010 1. Scientific advice and support

Duration

1.1. Assist DG Sanco in the application of the Community food hygiene legislation, e.g. drafting guidance documents, equivalency of CODEX standards for LBM, consideration of tolerances, alternative methods, and implementation of Water Framework Directive etc, and any other activities as required.

20 days

1.2. Participate in relevant EU initiatives and International scientific committees (ISO/CEN, WHO/FAO, expert working groups etc). In 2010 the CRL will:

Chair and co-ordinate the activities of the CEN/TC 275/WG6/TAG4

developing a CEN standard for detection of norovirus and hepatitis A in foodstuffs, including bivalve molluscs (see resolution 27, 8th workshop of NRLs). Two expert meetings are planned in 2010.

Progress plans for formal validation of the above virus standard in

preparation for work to deliver M/381 EU mandate to CEN for validation of methods in food microbiology.

Lead and co-ordinate the activities of CEN/TC 275/WG6/TAG3 in the

elaboration of molecular based enumeration methods for pathogenic marine vibrios in bivalve shellfish.

Chair and co-ordinate an expert working group to make recommendations

on fit-for-purpose methods for human pathogenic vibrio determination and their application in LBM.

Participate in ISO/TC34/SC9/WG3 working group on validation of methods

(revision of EN ISO 16140) to include the elaboration of ISO technical report on recommendations for establishing/revising reference methods.

Participate in working group CEN/TC 275/WG6/TAG6 on sampling methods

45 days

Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK Tel : +44 (0) 1305 206698, Fax : +44 (0) 1305 206601, e-mail : [email protected]

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(revision of ISO 6887) specifically ISO 6887 part 3 and make recommendations on sampling bivalve molluscan shellfish.

1.3. Provide DG Sanco with specialist assistance in relation to food and veterinary

inspections of Member States, Accession Countries and Third Countries and give advice and support on other technical issues related to trade as required.

4 days

1.4. Co-operate with, and assist DG Taiex in the provision of training and advice to Accession Counties.

4 days

1.5. Undertake CRL missions in support of the above activities.

During 2010 missions are foreseen in relation to the annual meetings of ISO and CEN (up to 2 missions); the CEN/TAG4 working group on viruses in food (2 missions); CEN/TAG3 working group on vibrios (2 missions); ISO/WG3 working group on validation of methods (2 missions), CEN/TAG6 working group on sampling and up to 6 missions in support of NRLs and DG Sanco activities.

Mission to the US associated with the second joint EU/US FDA workshop on implementation and approaches to sanitary surveys (item 2.4)

Up to 2 missions to JRC/IRMM Geel, Belgium (item 3.6)

55 days

1.6. Organise an annual review meeting between CRL representatives and designated DG Sanco representative in the area in Brussels or alternative mutually convenient location.

1.7. Complete a revision and publish (on the CRL website) the 4th issue of

Microbiological monitoring of bivalve shellfish harvesting areas, Guide to good practice: technical application.

5 days 6 days

1.8. Participate in relevant international scientific conferences, e.g. Food and Water borne microbiology symposium, Istambul, Turkey, 4th International calicivirus conference, location to be announced.

1.9. Participate as a member of the organising committee in “Vibrios in the

Environment”.

10 days Included in the above

2. Co-ordination of activities of NRL network and provision of technical

assistance and training

2.1. Participate in annual CRL Directors co-ordination meeting and other CRL co-ordination meetings/workshops as appropriate

5 days

2.2 Organise, host, and participate in the eighth annual NRL workshop, produce resolutions and other workshop outputs (May 2010 NRL Ancona, Italy). To include CRL administrative assistance.

40 days


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2.3 Undertake CRL activities and commitments agreed in resolutions at annual workshops (as posted on www.crlcefas.org).

Up to 100 days

2.4 Organise and participate in the second joint EU/US FDA workshop on

implementation and approaches to sanitary surveys to be held in US in 2010, as a follow up to the first workshop held in 2008 at the CRL (associated costs for travel for up to 6 staff members).

2.5 Produce a report on the implementation and approach to sanitary surveys of

LBM harvesting areas in EU member States.

25 days 5 days

2.6 Supply specialist information and advice on bacteriological and viral methods to NRLs (particularly new MS NRLs and accession countries), Official Control testing laboratories, and third county laboratories. To include assistance on implementation of methods, accreditation to IEC ISO17025, validation of alternative methods according to ISO16140, provision of CRL SOPs, protocols from TAG4 viruses in foods and transfer of other technical information.

5 days

2.7 Provide specialist training and/or training courses to NRLs, accession country NRLs and others in relation to analyses of E. coli, Salmonella spp., Vibrio spp., FRNA bacteriophage, norovirus, hepatitis A virus and other aspects of bivalve shellfish hygiene as required.

Up to 10 days

2.8 Continue to update and improve the CRL website (www.crlcefas.org) as a primary means of dissemination of information to NRLs and others.

7 days

3 Ring trials, comparative testing and quality assurance

3.1 Organise comparative (proficiency) testing for NRLs for E.coli and Salmonella spp. in bivalve molluscs via the CRL/HPA shellfish EQA scheme (see resolution 21, 8th workshop of NRLs). Analyse results, produce report, advice and recommendations (by May 10).

3.2 Produce a CRL guidance document on supervision of Official Control

laboratories with respect to assessment and follow-up activities in proficiency testing to encourage a harmonised approach across the network for dissemination through the CRL website.

3.3 To challenge aspects of the E. coli and Salmonella spp. methods not covered

by the standard shellfish EQA scheme organise a whole animal ring trial (see resolutions 22 and 23 , 8th workshop of NRLs) for NRLs, the scheme will be extended to selected Official Control Laboratories. Analyse results, produce report, advice and recommendations (by May 10).

30 days 5 days 40 days


3

http://www.crlcefas.org)/

http://www.crlcefas.co.uk/



3.4 Organise norovirus and hepatitis A ring trials (see resolution 24, 8th workshop of NRLs). Analyse results, produce report and recommendations (by May 10). Ring trial to be open to non-NRL laboratories on a cost recovery basis.

3.5 Organise norovirus and hepatitis A ring trials using matrix samples (i.e. whole

bioaccumulated bivalve shellfish) to test laboratory performance in application of the entire virus method. Ring trial to be open to non-NRL laboratories on a cost recovery basis.

3.6 In collaboration with JRC/IRMM Geel, Belgium undertake pilot studies on the

production of virus reference material using matrix samples, to include up to two meetings at Geel.

3.7 Prepare fully characterised stable reference material using biological carriers

for norovirus and Hepatitis A (see resolution 26, 8th workshop of NRLs). Perform homogeneity and stability analyses. Distribute to NRLs for use as control material on request.

40 days 60 days 25 days 5 days

3.8 Undertake Vibrio parahaemolyticus ring trials appropriate for methods enabling enumeration of pathogenicity principles (thermostable direct and thermostable direct related haemolysins) (see resolution 33, 8th workshop of NRLs). Analyse results, produce report and recommendations (by May 10).

35 days

3.9 Provision of reference material to support analysis of FRNA bacteriophage in LBM on request.

2 days

4 Confirmatory testing

4.1 Maintenance of CRL laboratory competence and expertise on analytical methods for monitoring virological contaminants of bivalve molluscs (norovirus and hepatitis A virus).

4.2 Maintenance of CRL laboratory competence and expertise on analytical

methods for molecular identification and characterisation of human pathogenic Vibrio spp. associated with LBM to include routine implementation of typing using Pulse Field Gel Electrophoresis.

20 days 25 days

4.3 Maintenance of CRL laboratory competence and expertise on analytical methods for monitoring bacteriological contaminants of bivalve molluscs (E.coli, Salmonella spp., FRNA bacteriophage, marine vibrios). To include maintenance of IEC ISO 17025 accreditation of enumeration of E. coli and FRNA bacteriophage and the detection of Salmonella spp. and Vibrio parahaemolyticus.

Up to 50 days

4.4 Performance of above tests on outbreak material or on occasion of disputed test results (on request of DG Sanco).

Included in the above


4



5 Development of analytical methods (undertaken at CRL)

5.1 Practical contribution as the project leader towards the validation of the TAG4 reference method for the detection of viruses in food (CEN/TC 275/WG6/TAG4).

5.2 Contribution as the project leader towards the elaboration and validation of the

TAG3 molecular based standard for the detection of potentially pathogenic vibrios in foodstuff, including bivalve shellfish using molecular methods - both nucleic acid hybridisation and real time PCR approaches.

5.3 Continue to contribute towards research to assist in the clarification of the

significance of quantitative PCR results for norovirus in bivalve molluscan shellfish in terms of public health risk is undertaken (see note below).

Note. The target date for a fully validated horizontal standard method for the detection of norovirus in bivalve shellfish is 2012. Thus previous restrictions on the implementation of norovirus testing within a European legislative framework, such as the absence of a standard reference method, will no longer apply. Norovirus cannot be routinely cultured in the laboratory and all existing assays in shellfish are based upon detection of a small fragment of viral genome. Application of these assays has shown that a high percentage of shellfish are contaminated with norovirus. Thus the implications of implementation of a European standard for norovirus in BMS may be very significant. It is however unclear whether detection of viral genome always correlates with presence of whole infectious virus and thus a health risk. studies on the use of analogous detection systems (i.e. RT-PCR) using the closely related but cultivable murine norovirus will assist in understanding the relationship between norovirus positive data generated by the proposed reference method and the presence of infectious virus in shellfish. It is proposed that a financial contribution of up to a maximum of €15,000 is provided to partially support a PhD studentship to develop this work area.

15 days 20 days 25 days

Rachel Rangdale CRL Co-ordinator August 2009


5

EU REFERENCE LABORATORY FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

WORK PROGRAMME 2010

CRL Work Plan 2010

2010 workplan.doc 31/05/11 1

Ref Function

1.0 Co-ordination and harmonisation of confirmatory methods and reference materials 1.1 With a view to establishing common practice, methodology and standards in

diagnosis of TSEs, the CRL will organise proficiency tests for the interpretation of histopathology and immunohistochemistry (IHC) in 2009 (see section 2 for details).

1.2 Data on the strengths and weaknesses of existing, as well as new/emerging antibodies and protocols used in confirmatory diagnostic tests will be assessed, and protocols reflecting best practice placed on the website. This, and other information relating to assessment of antibody performance will be made available through the technical IHC QA round (see section 2.2), and at the annual NRL meetings.

1.3

Storage of infected tissues from suspects in the UK will continue in order to maintain (as far as possible) a supply of reference materials for National Laboratories (or at least sufficient to enable appropriate characterisation of internal reference material). This collection of tissues is managed by the VLA TSE Archive, and all release of tissues from this collection to the CRL (or any other user) is subject to the approval of Defra’s Independent Archive Advisory Group (IAAG) and charges may be made (http://www.defra.gov.uk/corporate/vla/science/science-tse-arc-intro.htm). Standardised reference materials will also be prepared to facilitate batch testing activities (see section 6.1). Some reference materials, specifically for CRL use, will be generated through experimental challenge of animals (see sections 1.6, 1.7 and 1.8).

1.4

Positive and negative material necessary for annual QA purposes (see section 2 below) will be requested ‘en bloc’ from the TSE Archive, and reserved for CRL purposes. (This will require the continued maintenance of dedicated freezers. It was agreed in 2003 that the Commission would pay 1/5 depreciation costs each year.) We will request material in 2010, to prepare samples for EQA use in 2011.

1.5

As discriminatory tests become more widely used, it will be necessary for the CRL to maintain stocks of ovine BSE for the provision of QA and QC material. To obviate the need for repeat requests to the UK Archive Group (with no guarantee of continued successful application), we challenged four ARQ/ARQ sheep with bovine BSE in 2005, five in 2006, ten in 2007.and 5 in 2009 This provides material for test evaluation and sensitivity exercises, QA panels and species specific controls for STEG evaluations. The surviving 9 sheep will incur maintenance costs in 2009 if they do not succumb to disease within the next 6 months. It is proposed to challenge a further five sheep in 2010.

1.6

Atypical scrapie, its definition and detection is of increasing importance across the Community. Ideally the CRL proficiency testing regime should encompass such cases, but is unable to incorporate examples of such cases due to the relative rarity of this material, and the need to channel the limited amount of available material into research.

CRL Work Plan 2010

2010 workplan.doc 31/05/11 2

An experimental challenge of five animals with ‘atypical’ scrapie was started in 2006, with a further five challenged annually, to provide a bank of such material exclusively for EU QA purposes. To date, three animals have succumbed, but we have good evidence from parallel experiments now that the anticipated attack rate in this model will be 100%. The remaining twelve animals are currently healthy. Based on the incubation period of approximately 800 days observed in other research projects, it is predicted that up to five sheep may succumb within 2010, and that the remainder are predicted to incur maintenance costs into 2011. The amount of tissue required to include a sample in a QA round is approximately 65g (equivalent to two sheep). It is therefore proposed to challenge a further five sheep in 2010, to generate sufficient material to plan for the inclusion of atypical scrapie in the proposed batch testing panel (see 6.5).

1.7

The identification and characterisation of H and L type BSE raises the need for reference material from such cases. Global supplies are currently very limited, and reserved for research. We have challenged (Autumn 2008) two cattle with H-type BSE and two with L-type BSE to generate a small bank of reference material for statutory testing purposes. These animals are likely to incur maintenance charges in 2010, (based on a predicted minimum incubation period of 20-24 months).

2.0 Proficiency testing (see appended timetable)

2.1

The CRL will organise two proficiency tests for the interpretation of histopathology and immunohistochemistry (IHC) for BSE in bovines, and scrapie in sheep. The first of these will be in April 2010 and the second in October 2010. The format of these QA distributions will be based on a web-based QA system which will mean that we can use single existing slides to create electronic images, which can then be accessed by all readers at the same time. This will enable faster completion ofdistributions, and greater flexibility to include examples of unusual cases, challenging artefacts, different IHC protocols (drawn from the technical QA round –see below). This system will be administered through our QA Unit as before, but the web images will be hosted by ‘Slidepath’, an external company which specialises in web-based imaging.

2.2

An additional technical IHC test will take the form of a comparative test on unstained sections supplied by the CRL. Following staining and initial interpretation by the National Laboratories, the stained sections will be read by the CRL pathologists. Follow-up of any sub-optimal staining or inappropriate interpretation, if required, will be individually tailored to each participating laboratory. The previous rounds have raised a number of issues in relation to method optimisation for different species and tissues, so it is intended to keep the round at its current size.

2.3

A proficiency test panel of ovine blood samples will be provided for the QA of laboratories undertaking genotyping for statutory purposes. Information will be requested about the methods used in each country. Reporting on 4 codons (136, 141, 154 and 171) of the ovine PrP gene will be required from all labs.

CRL Work Plan 2010

2010 workplan.doc 31/05/11 3

QA Pilot Scheme for Caprine PrP Genotyping: It is increasingly recognized that susceptibility to scrapie in goats is regulated very similarly to sheep through the PrP gene and its variations (polymorphisms). While, similar to the ovine gene, the caprine PrP gene is highly polymorphic, the polymorphisms are usually at different positions (codons) compared to sheep. Recently, several publications associated various polymorphisms in the goat PrP gene to susceptibility or resistance to scrapie (e.g. Goldmann et al., J Gen Virol (1996) 77:2885-2891; Vaccari et al, J Gen Virol (2006) 87:1395-1402; Papasavva-Stylianou et al., Vet Journal (2007) 173:459-462; Colussi et al., J Gen Virol (2008) 89:3173-3176; Barillet et al., J Gen Virol (2009) 90:769-776) and it is probably only a question of time until one of these findings is considered significant enough to find its way into regulations. In the light of these developments, we suggest including a QA pilot scheme for the caprine PrP gene. This would include sourcing of blood with different polymorphisms from other countries in the EU, validation of the test for caprine polymorphisms at the CRL, and the distribution of the blood to 5 laboratories (including the CRL), who would voluntarily take part in this QA pilot scheme.

2.4

The CRL will organise: • One proficiency test for rapid diagnostic methods to assess PrP detection

in bovine brain tissue. Concurrently we will also issue proficiency test samples for confirmatory blotting methods to assess PrP detection in bovine brain tissue.

• One proficiency test for rapid diagnostic methods to assess PrP detection

in ovine brainstem tissue. In previous years this covered classical scrapie only. In 2009, it was extended to incorporate atypical scrapie samples of cerebrum or cerebellum as this is now possible following successful transmission in sheep in 2008 (see 1.6). Please note that it is not possible to use brainstem samples as there is insufficient tissue to supply all NRLs. However as rapid test EQA is based on homogenates the samples will not look different to other panel members and it is an important step to include atypical scrapie samples. At the same time we will also issue a proficiency test panel for confirmatory blotting methods to assess PrP detection in ovine brainstem tissue- this will also include an atypical sample as well as classical scrapie.

• One proficiency test round for BSE/scrapie discriminatory Western blots in

those NRLs which are operating such methods. Following successful transmission of ovine BSE (see 1.5), it is proposed that this round in 2010will include an ovine BSE sample in addition to the bovine BSE, to ensure the adequacy of the bovine BSE sample for routine use. However, please note that such ovine BSE samples are precious and will not necessarily be included in subsequent years, unless challenged animals continue to succumb.

Please note that such atypical and ovine BSE samples are precious and will not necessarily be included in subsequent years, unless challenged animals continue to succumb. The decision on whether to include this type of sample each year will depend partly on the performance of laboratories in receipt of the samples in the 2009 QA exercises, the outcome of which will not be known until near the end of

CRL Work Plan 2010

2010 workplan.doc 31/05/11 4

2009. The CRL will ask the COM to write to MS regarding their plans for the continuation of independent NRL status following the EFSA recommendations on changes to cattle testing. Until there is a clear indication of how many NRLs will continue to require full EQA panels, accurate predictions of future requirements cannot be made.

2.5

The CRL will monitor proficiency testing practices to ensure that they remain relevant, through discussion at the CRL meeting. We will attempt to maintain up-to-date data from NRLs, regarding the methods currently in use, the NRL purposes of such tests (e.g. confirmatory, discriminatory, research, etc.) national QC and QA approaches etc. to enable the effective provision of relevant and targeted advice. This will be done by provision to each NRL a summary of its situation, as understood by the CRL, with respect to tests, readers, contacts and addresses for the NRL and the testing labs of the member state. Feedback will be requested in the form of updating or confirmation of this base data. As the CRL has not so far undertaken inspections of NRLs (see 6.1), this is a good mechanism for gaining some understanding of current practices. It will also advise on any necessary changes to the CRL proficiency testing programme, monitoring of trend data from routine testing or general QA advice as the need is identified. Rapid test sample sets for each QA round will contain a range of diluted reactor homogenates for assessment of laboratory and test performance. The CRL will maintain an up-to-date database of all relevant NRL contacts and contact details.

2.6

The Commission will continue to have direct access to all QA results on the web-based systems.

3.0 Provision of diagnostic and confirmatory testing and advice

3.1

The demand for diagnostic testing will depend on individual countries. Most Member States have adequate arrangements and do not require significant help with routine diagnostic testing. However, confirmation of results may be an important task for CRL, which does not anticipate having to conduct large numbers of confirmatory tests but the service will be available on an ad hoc basis for difficult or perplexing cases (see also section 5). These tests will include HE sections, IHC sections and Western Blotting on unfixed material. The CRL will continue to attempt to collect data on cases which are in some way ‘unusual’, to enable comprehensive cross-referencing and collation of information on such cases for the Commission. For sheep, there is a standardised format for basic data collection and presentation on all positive cases (i.e. not pre-categorised by individual MS), so that more standardised interpretation of results as ‘usual’ or ‘unusual’ (to be undertaken by the CRL strain typing Expert Group) is possible across the whole of the EU27. Full instructions have been issued as part of the manual on Discriminatory Testing. The success of any parallel system for cattle data will be dependent on the willingness of MS to comply with such a request if our diagnostic opinion is not sought initially.

CRL Work Plan 2010

2010 workplan.doc 31/05/11 5

Additionally, as part of the approval for the heat treatment protocol for the IDEXX test, NRLs must send any samples which are initially reactive on the IDEXX tests, but where the signal is ablated by the heat treatment protocol, to the CRL.Depending upon the amount of such samples which arrive, investigations into the nature of these samples will be conducted by the CRL. No costs have been explicitly planned for this purpose on the assumption that the submission rate will be very low. However, if significant numbers of referrals are made, a separate plan of work will be proposed for this group of samples. No samples have been received to date (August 09)

3.2

The CRL will provide expert advice on the epidemiology and clinical manifestations of BSE and scrapie. The CRL will also provide scientific supervision of certain studies funded by the European Commission on request.

3.3

All relevant information will be published on the CRL website, when appropriate. Discussion fora are possible on the password protected TSE-LAB-NET system. The system provides links to QA, batch release data and workshop presentations, as well as closed discussion fora such as STEG.

4.0 Provision of training

4.1 A workshop for National experts will be arranged in the first half of 2010. This workshop will cover all aspects of NRL functions, with the exception of 4.2 below. Feedback will be provided and training needs identified following the outcome of the QA assessments outlined in 2.0.

4.2

Assistance and guidance will be provided to those laboratories experiencing difficulties. Specialist input to Commission fora on an ad hoc basis. Provision has been made in 2010 for three laboratory visits to assist with technical issues should the need arise.

4.3

Training in rapid diagnostic techniques will not be provided. All the evaluated tests are commercially available and it is assumed that the manufacturers will provide training/guidance on the use of the tests. Similarly, should problems be encountered then it is appropriate that the manufacturers address these directly with the test users. Feedback from the national laboratories will alert CRL to any problems and the CRL will liaise closely with the national laboratory and the test manufacturer. General advice and information will be posted (where relevant) on the website.

5.0 Strain typing

CRL Work Plan 2010

2010 workplan.doc 31/05/11 6

5.1

The CRL has established a working group of experts in the field of strain differentiation. It will continue to be responsible for the evaluation of any unusual results arising from TSE testing within Europe, and agree the criteria on which strains will be classified ‘BSE-like’ (and what that means). Advice will be provided on appropriate further investigation and interpretation, to enable the submitting NRL to appropriately and competently brief the relevant National authorities. The panel is drawn partly from experts within the CRL and NRLs, and partly from other sources. This group plans to meet twice in 2010. Discussion will continue to focus on the validation/interpretation of the increasing range of Tg bioassay methodologies. This group will coordinate the provision of material (see also 2.4 and 7.2) for the ring trial of any new potential discriminatory method not presented with sufficient supporting data to be approved by the group without further assessment.

5.2 MS undertake the initial and discriminatory testing of sheep isolates at their own cost. However, if an unusual isolate is referred to the CRL strain typing group for subsequent investigation by ring-trial, the CRL will be liable for any laboratory costs incurred (see section 7.4).

5.3 Any isolate still considered BSE-like following ring trial (see section 7.4) will be forwarded for bioassay in mice. Historically, only conventional mice were sufficiently evaluated and defined for this purpose, and interpretation was based on a full panel (i.e. RIII, VM and C57Bl6). However, the choice of mouse strains has been under active discussion (see section 5.1) including the use of Tg strains. Although TSE strains have traditionally been characterised using wild-type mice, recently a number of transgenic lines have been used extensively in several European laboratories, and a large amount of data regarding their usefulness in identifying different TSE strains is now available, including unpublished data which is available to STEG. Indeed, some have already been adopted by STEG for the bioassay of demanding referred samples. A major advantage of these Tg lines over the wild-type lines is their enhanced susceptibility to certain TSE isolates e.g. transgenic mouse lines are susceptible to atypical scrapie when conventional lines are not. Consequently we propose to change the STEG referral bioassay panel from 3x20 mice of three conventional lines to 10x Tg338 (VRQ ovine), Tg110 (bovine) and TgShpXI (ARQ ovine). The use of TgShpXI line is advisable because it is susceptible to certain classical scrapie isolates where Tg338 mice show prolonged incubation periods (e.g. Suffolk and Italian scrapie isolates). The cost estimates are based on the assumption that a maximum of 2 isolates will require further characterisation by bioassay.

5.4 A spiked pool discriminatory testing proposal is currently under separate consideration by the Commission for inclusion in this workplan–see annex. At the time of submitting this workplan, no decision has been reached with regard to thesuitability of the spiked pool proposal for inclusion in this workplan. For costing purposes, the maximum costs in 2010 have been calculated based on:

CRL Work Plan 2010

2010 workplan.doc 31/05/11 7

All of the in vitro discriminatory testing phase, 60% of bioassay phase 1, together with the start of some or all of the bioassay phase 2. (This depends on the scale of phase 2, which cannot be determined until the completion of the in vitro phase.)The costs here are based on the assumption that a maximum of 14 isolates could be started for phase 2 on a rolling programme (due to breeding capacity) so cost for starting and maintaining 14 isolates for 6 months have been included. Remaining maintenance costs and all analysis would fall into 2011/12.

6.0 Rapid diagnostic methods

6.1 The CRL will contribute actively (on an ad hoc basis) to the continual assessment of existing rapid tests by contribution to relevant discussion fora, laboratory visits and comparative trials. The workload and costs for this component of CRL work cannot be readily predicted - if for example we have to undertake laboratory work to investigate a problem that arises in year. Additional costs may therefore arise in-year that would require additional funding, or a reassessment of CRL commitments to enable delivery within the agreed annual budget. The CRL has an ongoing commitment to assess changes to approved rapid test kits or sampling methods, which are proposed by manufacturers. This involves discussion with companies, input into protocol design, assessment of evaluation data and consideration of the impact of proposed changes. The proposals are then either accepted, further work requested or they are rejected. If proposals are accepted the company is required to update kit inserts or SOPs as appropriate. If changes are made to kit instructions, NRLs and the Commission are notified. If changes to production are necessary as a part of kit changes, Quality Control data may need to be provided by the manufacturer and assessed by the CRL to confirm adherence to the manufacturer’s Quality System. An annual statement will be sent to the COM confirming which manufacturers continue to comply with these requirements, so that the listing in the regulation is kept up to date.

6.2

The CRL will continue investigations into a sustainable standardised source of positive control material. Currently, this is based upon a macerate or homogenate of bovine brain material to provide all NRLs with the same material, which could be used to compare test performance. However, attempts will also be made to assess more sustainable source materials for the long term. (This issue is partially addressed under sections 1.5 –1.7). Homogenates have been prepared in 2009 for QA testing in 2010. There will be requirement to prepare further supplies of homogenate in 2010 for use in 2011.

6.3 In 2004, a document was produced by a ‘virtual working group’ defining what ‘minor test changes’ are, and how such changes should be assessed. This information is now on the CRL website. The document will be subject to annual review.

6.4

Batch testing of approved rapid tests for the detection of BSE in bovine samples was introduced in 2008. Nominated NRLs are responsible for testing to an agreed protocol and the CRL approves batches for release and communicates this information to NRLs for cascade to testing labs throughout the EU. Costs are

CRL Work Plan 2010

2010 workplan.doc 31/05/11 8

based on current test usage, and an estimate of 40 batch releases per year.

6.5 A workshop held at the CRL meeting in June 2009 discussed extending batch testing to kits approved for scrapie diagnostics. We agreed that the approval may be based on an NRL/CRL review of test data generated by manufacturers’. This will be instigated as part of the current system and will have negligible cost. However, the group also felt that the value of the assessment would be enhanced if the test panels included atypical scrapie material. This is technically demanding because the material is relatively unstable when prepared as homogenates and also difficult because it is currently quite scarce. This task will require infection and maintenance of 5 sheep to produce sufficient material (costed in section 1.6), assessment of protocols and data from various National Reference laboratories to select the most promising protocols. Next year it will also be necessary to include time to trial this method at the CRL.

7.0 Discriminatory testing

7.1 The Discriminatory Testing Handbook detailing precise methods for discriminatory blotting was produced in 2005, updated in 2007 and again in 2009. This Handbook will be reviewed annually, revised and updated as necessary to include additional information of relevance to the surveillance of TSE, and re-issued online (.http://www.defra.gov.uk/vla/science/docs/sci_tse_rl_handbookv2mar07.pdf) A protocol , as presented at the STEG meeting in April 2008, for the discrimination of H and L type BSE from C type will be made available on line. It should be notedthat the material does not yet exist (see section 1.7) to perform adequate - or indeed any - QA or QC and any testing performed in NRLs using this protocol will, by definition, be ‘out of control’. (A referral system will be recommended, but cannot be enforced).

7.2 There will be an annual QA round for discriminatory blotting (see section 2.4).

7.3 The CRL will continue the evaluation of cervinised mice (developed by Glenn Telling, University of Kentucky) for susceptibility to BSE relative to experimental cervid passaged BSE, to establish the suitability of this model for investigation of strain should any European cervid be identified through the surveillance programme. The experimental challenge of these mice was started mid –2008. To date (June 2009) the cervid-passaged BSE has resulted in disease, but the bovine BSE source has yet to result in disease in these mice. This would be anticipated from first principles given the presence of a species barrier). Maintenance of the Tg line at the CRL - either by minimal breeding, or the cryopreservation of the line, should further challenges not be required in the short to medium term – will incur some cost.

7.4 Any positive isolate which presents a discriminatory blot result which is BSE-like should be sent to the CRL. Such cases will be referred to the STEG (see section 5.1) and material distributed around ring-trial laboratories. Cost estimates have been based on the very low level (12 so far) of referrals to date, and assume that a maximum of 1 ring trial will be required. (see 5.3)

CRL Work Plan 2010

2010 workplan.doc 31/05/11 9

CRL Work Plan 2010

2010 workplan.doc 31/05/11 10

Appendix 1

PROVISIONAL TIMETABLE FOR TSE CRL QA EXERCISES IN 2009

Intended Start

Date i QA activity

February 2010

Ovine genotyping

March 2010 Immuno-histochemical technique

April 2010 Histopathology and immunohistochemistry interpretation (round 1)

October 2010 Histopathology and immunohistochemistry interpretation (round 2)

October 2010 Bovine rapid testing incorporating confirmatory blotting if appropriate

November 2010 Ovine rapid testing, incorporating confirmatory blotting if appropriate

September 2010 Ovine discriminatory Western blotting i Some QA exercises (such as the technical and slide interpretation) take several weeks or months to complete. Any follow-up activities will also lengthen the duration. It is not therefore possible to accurately predict completion dates for these activities.

EU REFERENCE LABORATORY FOR LISTERIA

MONOCYTOGENES

WORK PROGRAMME 2010

EU REFERENCE LABORATORY FOR COAGULASE POSITIVE STAPHYLOCOCCI

WORK PROGRAMME 2010

EU REFERENCE LABORATORY FOR ESCHERICHIA COLI, INCLUDING VEROTOXIGENIC E. COLI

(VTEC)

WORK PROGRAMME 2010

of 9

Community Reference Laboratory for E.coli Department of Veterinary Public Health and Food Safety

Unit of Foodborne Zoonoses and Veterinary Epidemiology Istituto Superiore di Sanità

Community Reference laboratory (CRL)

for Escherichia coli,

including Verotoxigenic E. coli (VTEC)

Work Programme

1st January - 31st December, 2010

of 9

Introduction

The work programme of CRL for VTEC (CRL-VTEC) for the year 2010 will consist of

the following activities:

1. Consolidating the CRL structures

1.1. Staff 1.2. Accreditation of the laboratory 1.3. MLVA-typing of VTEC O157

2. Coordination of the NRLs network and provision of technical assistance and

training 2.1. Annual workshop 2.2. Assistance to NRLs 2.3. Training

2.4. Characterization of VTEC strains isolated by the NRLs 3. Implementation of the CRL-VTEC web site 4. Co-operation with other European Community structures

4.1. The European Food Safety Authority (EFSA)

4.2. The European Committee for Standardization (CEN) 4.3. The European Centre for Disease Control (ECDC) 4.4. MED-VET-NET, the Zoonoses Network of Excellence 4.5. The Pathogenic E. coli Network (PEN)

5. Inter-laboratory comparison studies

5.1. PCR typing of vtx genes variants 5.2. Detection of VTEC non-O157 in food samples 5.3. Validation study of the method EN/ISO 16654 for E. coli O157

6. Research on VTEC accessory virulence factors

7. Missions

The duration of each action is indicated, and will be either limited to 2010 or multi-

annual (ongoing program).

of 9

1. Consolidating the CRL structures

1.1. Staff The permanent staff of ISS will continue to devote significant working time to the

CRL’s activities. Four persons, hired with CRL funds, will work full time at the CRL-

related activities with the status of “temporary staff employees”.

(Duration: ongoing)

1.2. Accreditation of the laboratory The CRL-VTEC will maintain the accreditation UNI EN/ISO 17025:2005 (obtained

from the Italian body for accreditation, SINAL, accreditation N. 0779) covering the

methods for detection and typing of VTEC related with CRL’s tasks and activities. The

possibility to submit additional methods for accreditation will be evaluated.

(Duration: ongoing)

1.3. MLVA-typing of VTEC O157

Most bacterial genomes contain tandem duplications of short DNA sequences,

termed "variable-number tandem repeats" (VNTR). A subtyping method targeting

these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool

for characterization of VTEC O157, as a complement or even as an alternative to

pulsed-field gel electrophoresis (PFGE).

The CRL-VTEC will introduce this technique into its array of routine typing methods,

and it will be applied, in parallel to PFGE and phage-typing, to representative subsets

of the VTEC O157 strains isolated by the NRLs in their countries from different

sources. The CRL-VTEC will evaluate the applicability of MLVA as a routine tool for

VTEC O157 typing that could replace the more expensive and cumbersome PFGE.

This could allow a larger number of NRL to perform molecular subtyping of VTEC

O157. The shipment of the strains from each NRLs could be made periodically to

contain the cost of the shipments. The methods will be harmonized with those used

by the ECDC Food- and Waterborne Diseases (FWD) network for human infections.

Therefore, such a project could allow the comparison of the characteristics of VTEC

O157 isolated from human infections and from non-human sources throughout

Europe (see also point 2.4).

Duration: ongoing

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2. Coordination of the NRLs network and provision of technical assistance and

training 2.1. Annual workshop with NRLs The 5th annual workshop will be held in the second half of 2010 in Rome. In

alternative, upon agreement of DG SANCO, one of the NRLs could host the

workshop at its own Institute. The results of the 2010 inter-laboratory studies will be

presented and discussed. The training program for the benefit of NRLs will be

discussed as well and plans for the following year will be established according to the

NRLs needs.

2.2. Assistance to NRLs The CRL-VTEC will continue to assist NRLs in the field of VTEC detection and typing,

providing methods via the web site, standard operating procedures, reference strains.

If needed, the CRL-VTEC will visit NRLs to help in solving problems. Drafts of other

standard operating procedures for detection of other pathogenic Escherichia coli in

animals, food, and in other relevant matrices and for typing of the isolated strains will

be developed and discussed with the NRLs.

Duration: ongoing

2.3. Training Upon request from NRLs within EU or from governmental institutions of third

countries, CRL-VTEC will be available to receive short visits of staff for individual

training on specific topics related with detection and typing methods.

Duration: ongoing

2.4. Characterization of VTEC strains isolated by the NRLs After the introduction of phage-typing and MLVA in the panel of typing methods

available at CRL-VTEC, the NRLs will be asked to send representative subsets of the

VTEC O157 strains isolated in their countries from different sources for

characterization by these techniques, as well as pulse-field gel electrophoresis

(PFGE). The shipment of the strains from each NRLs will be made periodically to

contain the delivery costs. The data obtained will be collected in a European

database for VTEC strains isolated from food and from animals, harmonized with the

Enter-net database for human isolates, maintained by the ECDC in the framework of

the FWD Network. Such a project could allow the comparison of the characteristics of

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VTEC O157 isolated from non-human sources with those of the strains causing

human infections throughout Europe.

Duration: ongoing

3. Maintaining and implementing the CRL-VTEC web site

The web site of the CRL-VTEC will be maintained, and updated on a regular basis. The European database for VTEC isolated by NRLs will be included in the “private”

section, to allow NRLs to directly upload their data.

Duration: ongoing

4. Co-operation with other European Community structures The CRL-VTEC will continue the cooperation with EC structures working in the field of

human and animal health and food safety. The following liaisons will be maintained

and implemented:

4.1. The European Food Safety Authority (EFSA) The CRL-VTEC will contribute as reference laboratory (elaboration of methods,

pahge-typing of VTEC O157, etc.) to any program on VTEC Monitoring that will be

implemented by the EFSA Task Force on Zoonoses Data Collection, according to the

“Guidance on the technical specifications for the monitoring and reporting of VTEC in

animals and food” developed in 2009.

4.2 The European Committee for Standardization (CEN), Technical Committee 275 – Food analysis – Horizontal methods, WG 6 – Microbial contamination.

4.2.1. Validation study of the method EN/ISO 16654 for E.coli O157

The CRL-VTEC, as project leader, will coordinate the revision study of the method

EN/ISO 16654 for E.coli O157 in foodstuffs. The study, which will involve 13 NRLs for

E.coli, had been approved by CEN and will begin as soon as the financial support

from CEN will be available.

4.2.2. Real-Time PCR-based method for the detection of VTEC in food

The CRL-VTEC, as leader of the “STEC ad hoc Group” of the CEN/TC275 WG6 has

edited a draft Technical Specification (TS) “Horizontal method for the detection of

Shiga toxin-producing Escherichia coli (STEC) belonging to O157, O111, O26, O103

and O145 serogroups - Qualitative Real-time polymerase chain reaction (PCR)-based

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Method”. The document has been approved by ISO/TC 34/SC9 on February the 24th,

2009 as a New Work Item Proposal. The final form of the draft ISO Technical

Specification will be prepared considering the outcome of the discussion that will take

place at the last meeting of the STEC ad hoc group organized by the CRL-VTEC in

Rome on October the 29th, 2009. Such a meeting has been organized upon request

of the CEN/TC275 WG6 members at the plenary meeting held in Valencia in may

2009. The plenum asked the STEC ad hoc group to clarify whether the primers and

probes indicated in the draft norm should be set as normative or should be included in

an informative annex (see Res. 200 in Annex YY). The final form of the ISO/TS will be

presented by the CRL-VTEC at the next CEN/TC275 WG6 plenary meeting in 2010.

CRL-VTEC and the Institute for Health and Consumers Protection of the Joint

Research Centre (JRC) have reached a collaboration agreement with the aim of

designing and performing a collaborative study for the validation of the ISO/TS.

The technical annex of the agreement, including both the scientific and financial

aspects of the validation project, will be discussed during a meeting to be held on

September the 24th at JRC. The study will benefit from the collaboration between the

two European Reference Laboratories, each contributing with their specific

expertises.

4.3. The European Centre for Disease Control (ECDC) Food- and Waterborne Diseases (FWD) Network (formerly Enter-net) The CRL-VTEC will continue to take part into the Coordination Group of the ECDC

FWD Network, with the aim of ensuring connection and activity harmonization

between this network and the network of Reference Laboratories in the veterinary and

food safety fields. The CRL-VTEC will also continue the liaison with the ECDC

reference laboratory for VTEC infections (the WHO International Escherichia and

Klebsiella Centre of the Statens Serum Institut, Copenhagen), which is in charge of

the external quality assurance activities for the network. This will allow the

harmonization of the identification and typing schemes and making the respective

monitoring programs and databases compatible for comparison of human and non-

human data (see also point 5.1). In particular, the collaboration will be aimed at the

refinement of the methods for the characterization of the different vtx genes subtypes,

as defined by the nomenclature scheme proposed in 2009. In particular, such

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methods will be tested on a shared collection of VTEC reference strains for the

different vtx variants and strains from HUS by a group of laboratories specialized in

VTEC diagnostics, including the CDC of Atlanta, GA, US.

4.4. MED-VET-NET, the European Network of Excellence for research on the prevention and control of zoonoses.

The MED-VET-NET Work-Package no. 26 of which Dr. Morabito is the deputy

coordinator (http://www.medvetnet.org/cms/templates/doc.php?id=64), closed on

2009. However, MED-VET-NET partners are establishing an association that will

continue the scientific networking activities. CRL-VTEC is exploring the possibility to

have new Work-Packages on VTEC, funded.

4.5. The Pathogenic E. coli Network (PEN). The activities of the PEN network will end on January 2010. The last technical booklet

“Control and management of pathogenic Eschericha coli” will be edited and

published, either as hard copy or via PEN-project website, within that date.

5. Inter-laboratory comparison studies Two studies are planned for 2010. A third one could be carried out upon availability of

CEN funding (see points 5.3 and 4.2.1)

5.1. PCR typing of vtx genes variants This study will be focused on the PCR typing of vtx genes variants. It will be agreed

with the ECDC reference laboratory for VTEC infections (the WHO International

Escherichia and Klebsiella Centre of the Statens Serum Institut, Copenhagen), which

is in charge of the external quality assurance activities for the network. A same panel

of VTEC strains harboring different vtx gene subtypes will be sent to the NRLs of both

the medical and veterinary/food Networks. The NRLs will be provided with a

conventional PCR protocol to perform the typing and a set of reference strains for the

vtx subtypes. This study will facilitate the harmonization of the identification and

typing schemes and the compatibility of the respective monitoring programs and

databases for comparison of human and non-human data (see also point 4.3).

5.2. Detection of VTEC non-O157 in food samples

After the 2009 study had been dedicated to the detection of VTEC in animal samples,

the 2010 detection study will involve food samples. It will be organized according to

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the draft EFSA “Guidance of on the technical specifications for the monitoring and

reporting of VTEC on animals and food”, which proposes, as secondary objective of

the monitoring, to investigate on the presence of VTEC selected serogroups in food

items considered as likely sources of infection. The study will consist on the

examination of food samples containing VTEC non-O157 strains belonging to

serogroups involved in severe human infections (e.g. O26, O103, O111 and O145),

together with background microbial flora. According to the EFSA Guidance, the

detection method will be the Real-time PCR-based horizontal Technical Specification

developed in the framework of CEN TC275/WG6. Basically, this method has already

been experienced by those NRLs who performed the facultative parts of the 2008

study on strain identification and typing, and of the 2009 study on the detection of

VTEC non-O157 in carcass swabs. 5.3. Validation and revision study of the standard method EN/ISO 16654 for

E.coli O157 in food and feeding stuffs The CRL-VTEC is project leader of the validation and revision study of the method

EN/ISO 16654 for E.coli O157 in foodstuffs (see point 4.2.1). The study is still in

standby and will begin as soon as the financial support from CEN will be available.

The CEN-approved proposal involves 13 NRLs selected on the basis of a call for

expressions of interest. The validation study will also function as external quality

assurance test for these 13 NRLs, with respect to the EN/ISO 16654 method.

6. Research on VTEC accessory virulence factors

The studies on the identification of potential virulence-related Mobile Genetic

Elements in VTEC still remain the main research topic of the CRL-VTEC. The

completion of the virulence genes asset of this pathogroup is of primary importance to

elucidate the molecular mechanisms underlying the VTEC pathogenicity and can lead

to the discovery of virulence predictors that can facilitate the development of less

complex detection methods to identify the pathogenic VTEC strains in the food

matrices regardless their serogroup.

Duration: ongoing

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7. Missions

The following missions may be needed in 2010:

• Participation of a CRL representative in 2 meetings of the Coordination Group of

the ECDC Food- and Waterborne Diseases and Zoonoses (FWD) Surveillance

Network, presumably in Stockholm.

• Participation of a scientist at the 2010 CEN/TC275 WG6 annual plenary

meeting, presumably in Buenos Aires. • Participation of a scientist at two CRL-VTEC/JRC meetings for defining and

managing the ISO/TS validation study.

• A visit to one NRL can be planned for 2009, upon agreement with the EC and

the interested country.

August 20th, 2009

Dr. Alfredo Caprioli Director, CRL for Escherichia coli Reparto Zoonosi Trasmesse da Alimenti ed Epidemiologia Veterinaria Dipartimento di Sanità Pubblica veterinaria e Sicurezza Alimentare Istituto Superiore di Sanità Viale Regina Elena 299, 00161 Rome Italy

EU REFERENCE LABORATORY FOR CAMPYLOBACTER

WORK PROGRAMME 2010

CRL- CAMPYLOBACTER WORK PROGRAMME FOR 1st OF JANUARY 2010 TO 31st OF DECEMBER 2010 Introduction The activities in the working programme for 2010 for the Community Reference Laboratory (CRL) for Campylobacter will follow EU legislation on CRLs functions, duties and designation (Regulation (EC) No 882/2004 and Commission Regulation (EC) 776/2006). The work programme for 2010 will consist of the following key activities, repeated annually:

1. Ring tests (interlaboratory comparison studies) 2. Workshop 3. Research 4. Scientific and technical assistance to the European Commission and to the NRLs-

Campylobacter, including ad hoc activities 5. Missions 6. Communication 1. Organisation of ring tests in 2010 In May 2009, a ring test for detection and enumeration of Campylobacter in broiler meat (breast meat) was organised for the NRLs- Campylobacter. Basically, the protocol for analysis followed the standardised protocols of ISO 10272 Part 1 and Part 2: 2006 “Microbiology of food and animal feeding stuffs – Horizontal method for detection and enumeration of Campylobacter spp”. The majority of NRLs performed very well with the analyses. 1.1. Detection and enumeration of Campylobacter in different matrices The planned ring test in 2010 will consist of detection and enumeration of Campylobacter in different matrices, using the abovementioned ISO standards, but including different enrichment broths and applying different times and temperatures for incubation. The use of freeze dried cultures as reference material was very successful in 2009 and will be used also for the ring test in 2010. A revision of the ISO 10272 standards has been initiated. The use of alternative enrichment broths and incubation times and temperatures has been discussed and there is a need for collaborative tests in order to find optimal conditions for the analyses. The ring test is planned to be distributed by courier in the spring of 2010. 1.2. Identification of Campylobacter species in swab samples Another ring test is planned in 2010,“Identification of strains”, which will consist of pure cultures to be identified by the NRLs. Some isolates submitted by the NRLs to the CRL from the EU baseline survey of the prevalence of Campylobacter in broiler flocks and broiler carcasses in 2008 (Commission Decision of 19 July 2007, 2007/516/EC) will be included, especially isolates that appeared difficult to species identify. Strains of

Campylobacter- related organisms will also be included. The NRLs will be allowed to use any method for analysis. The purpose will be to evaluate the NRLs ability to correctly identify different thermophilic Campylobacter species and distinguish between true Campylobacter spp and Campylobacter- related or Campylobacter- look-a-like organisms. 2. Organisation of a workshop The workshop will be organised in the fall (probably in October) in 2010. Representatives from the Member States´ NRLs for Campylobacter will be invited. The EU Candidate Countries and Iceland, Norway and Switzerland will also be invited to participate at own costs. Candidate Countries can apply for financial assistance by TAIEX. As in previous years, experts from DG- SANCO, the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) will be invited. The agenda will include presentations and discussions on:

- Campylobacter activities in the EU at Community level. Results of zoonosis monitoring, surveys and control of Campylobacter in animals, food stuffs and humans (where applicable).

- Results of ring tests - Updates on analytical methods, including molecular methods for identification

and characterization of Campylobacter - Campylobacter activities in MS, including national monitoring and research

studies - Information about ring tests/ comparative tests to come - Information from meetings with ISO/TC34/SC9 and CEN/TC 275/WG6 - Information about revision of ISO standards - Future CRL- NRL collaboration and activities, depending on recent and urgent

matters of common interest

3. Research 3.1. Studies on bacterial and matrix reference materials for the ring tests. 3.1.1. As for the ring test in 2009, the CRL plans to purchase vials with freeze dried reference material consisting of Campylobacter and other bacterial species of varying and specified concentrations. The CRL will test batches of the freeze dried reference material with selected matrices and different conditions (enrichment broths, incubation times and temperatures) to ascertain the stability of the test procedure. The ring test will be delivered by courier and should be delivered within 48 hours. However, the aim is to have samples that are stable for at least 5 days. 3.1.2. For the other ring test (“Identification of strains”), approximately 10 selected isolates will be tested for stability to be transported as swab samples in Amies transport medium containing charcoal. 3.2. Validation study. ISO 10272 Part 1 and Part 2:2006 have not been validated in collaborative tests. The ring test that will be organized in 2010 will serve as a pre-study for validation. Validation

could be initiated after revision of the current methods. However, this may take some time and the CRL will keep updated on the progress of revision in order to be prepared for a validation study later on. 3.3. DNA-based (molecular) methods for detection, species identification and strain characterization of Campylobacter. The traditional methods for detection and identification of Campylobacter include culture in selective media, followed by phenotypic methods for genus and species identification. Although molecular or DNA based methods are considered more reliable, there are no ‘golden standard’ methods for detection, species identification or strain characterization (subtyping/fingerprinting). 3.3.1. PCRs for identification. The voluntary PCR ring test in 2008, when three traditional PCR assays were tested showed that the assay by Denis et al. (Lett. Appl. Microbiol., 1999, 29: 406-410) was most successful for identification of Campylobacter jejuni and C. coli. Since these two Campylobacter species are by far the most commonly isolated species from broilers (and humans), the assay can be recommended for identification of Campylobacter colonies in routine diagnostics. For “negative” results by the PCR by Denis et al., other PCRs eg the assay by Wang et al. (J. Clin. Microbiol., 2002, 40: 4744-4747) and/or phenotypic methods could be applied. Real-time PCR assays are becoming commonly used and constitute a more rapid technique with less risk for contamination. However, the equipment for real-time PCR is still quite expensive and not available in all NRLs. The CRL has reviewed published real-time PCR assays and plans to perform or initiate a validation study in 2010 of an assay that appears adequate and robust for identification of C. jejuni and C. coli.

3.3.2. Subtyping/fingerprinting In 2009, the CRL sent out a short questionnaire to the NRLs- Campylobacter, in order to collect information about molecular methods used for subtyping at the NRLs and needs for assistance or training. Seven of 29 responding NRLs responded that they intended to set up PFGE in the future and that they would need a training course. Earlier in 2009, EFSA reported the results of a questionnaire survey carried out in EU MSs and non-MSs on the availability of molecular typing methods for the main food-borne pathogens in animals, food and feedingstuffs (The EFSA Journal (2009) 272, 1-52). For Campylobacter, PFGE was the most commonly used method followed by MLST. Other techniques were used by some MSs, ie short variable region (SVR) sequencing of the flaA gene, amplified fragment length polymorphism (AFLP), ribotyping etc. For the monitoring of zoonoses and zoonotic agents (Zoonoses Directive 2003/99/EC) a request of molecular (subtyping) data may be considered in the future. In order to assist the NRLs, the CRL will in 2010 continue to establish routines for molecular typing methods. The CRL has for many years routinely performed PFGE using the standardised Campynet protocol (http://campynet.vetinst.dk/PFGE.html) and has also some experience with the protocol developed by PulseNet (USA- PulseNet) (http://www.cdc.gov/PULSENET/protocols.htm). The PulseNet protocol differs somewhat from the Campynet protocol but will also be established as a routine method in the laboratory.

The CRL will continue the discussions with IRMM about collaboration in order to provide reference materials for PFGE standards and possibly a feasibility study. The CRL will start the process to establish MLST as an in-house typing method, basically according to the protocol of Dingle et al. (J. Clin. Microbiology, 2001, 39: 14-23).

3.4. Database and analyses of Campylobacter isolates from the EU baseline survey From the EU baseline survey of Campylobacter in broilers in 2008, 434 isolates submitted by the MSs have been analysed by the CRL. The CRL has documented all information about the strains (“database”) and stored them in – 70 ºC. Subtyping of the isolates have been initiated in 2009 and will be continued 2010. Subtyping by PFGE and MLST will have priority and will provide information about what type of strains are prevalent and degree of strain diversity among Campylobacter in broilers in Europe. 3.5. Participation in international research networks, research projects and international meetings In 2010, CRL staff members will participate in: MedVetNet Association. The European Network of Excellence MedVetNet ends in 2009 and will be followed by an Association with the same name. The CRL will participate in relevant activities of the new Association. ISO/CEN standardization activities:

- Working group CEN/TC 275/WG 6/TAG 5, considering documents

regarding Campylobacter in Primary Production (EN‐ISO 10272 part 4) and Sampling techniques ‐ Primary Production Stage.

- - Revision of ISO 10272 Part 1 and Part 2. The work started in 2009 and will

continue in 2010

- Other relevant national and international seminars and research meetings in order to assure competence and knowledge on recent advancement within the Campylobacter area, for example in the international conference Food Micro 2010 in Copenhagen (August 30-September 3, 2010)

- As Member of the Advisory Board to a new EU FP7 financed project

“Campylobacter control – novel approaches in primary poultry production” (acronym: CamCon). Coordinator of project: Merete Hofshagen, National Veterinary Institute, Norway

4. Assistance to the European Commission and the NRLs including ad hoc activities

The CRL will provide scientific and technical assistance to the Commission and the NRLs on issues regarding Campylobacter.

Results from the baseline study for Campylobacter spp. in broiler flocks and Campylobacter spp. and Salmonella spp. in broiler carcasses (performed in 2008) will be published in 2010 and it is anticipated that there will be questions in relation to the release of this information. A plan for a baseline survey on Campylobacter and Salmonella in poultry meat and meat products at retail was prepared by EFSA in 2008, but the study was postponed by the Commission. However, the need of this survey might be re-considered after the results of the survey in 2008 have been presented in 2010. CRL staff participates in an EFSA Task Force on Zoonoses Data Collection Working Group on the analysis of the baseline survey on the prevalence of Campylobacter in broiler flocks and on Campylobacter and Salmonella in broiler carcasses in the EU, 2008 Depending on the interest and need for training courses, the CRL will make plans to organise training for molecular typing by PFGE for a limited number of participants. Study visits will be organized on an ad hoc basis Request from the NRLs and the Commission for support and advice will be handled by the CRL scientific staff as soon as possible. Assistance to the Commission and EFSA services will have priority. 5. Missions

The following missions are planned to be made (or may be necessary) in 2010:

- The 29th meeting of ISO/TC34/SC9 and the 17th meeting of

CEN/TC275/WG6, which will be in Buenos Aires, Argentina in 2010. Total duration of the two meetings will be 5 days.

- 1-2 meetings with working group CEN/TC275/WG6 TAG 5 “Primary

production stage”, date not set yet. Duration is probably 2 days per meeting. - For the revision of ISO 10272 standards, it is expected that 1- 2 meetings

will be organised. Location and dates have not been determined yet.

- If requested, the CRL will visit NRLs- Campylobacter for assistance with issues related to Campylobacter analyses

- Plan to participate in two meetings of relevant Commission working groups

under the Standing Committee on the Food Chain and Animal Health (SCFCAH), section biological safety of the food chain.

- Participation in the FoodMicro 2010 meeting in Copenhagen, DK (30

August – 3 September 2010).

6. Communication

The CRL- Campylobacter webpage will be updated with information about and presentations from the workshops, ring test results, NRL- Campylobacter contact details, and other relevant issues. The CRL will cooperate with EU Commission Services and other organisations and authorities working in the field of human and animal health.

EU REFERENCE LABORATORY FOR PARASITES (IN PARTICULAR TRICHINELLA,

ECHINOCOCCUS AND ANISAKIS)

WORK PROGRAMME 2010

EU REFERENCE LABORATORY FOR ANTIMICROBIAL RESISTANCE

WORK PROGRAMME 2010

Work plan for 2010 The main purpose of the Community Reference Laboratory on Antimicrobial Resistance (CRL-AR) is to ensure the quality of antimicrobial susceptibility testing in the Member States and to harmonise the procedures and methodologies used. Thus, most of the activities aim at implementing, from an analytical point of view, the provisions of monitoring of antimicrobial resistance set down in Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents. In addition the CRL-AR provides assistance to the member States and the Commission on other relevant aspects of antimicrobial resistance. Furthermore, the CRL-AR is requested to work in an international context and ensure that EU influences and follows global standards and guidelines. Scientific advice and support to the Commission During 2010 the CRL will provide advice as stated under the general terms. The CRL will participate in workshops and working groups on antimicrobial resistance initiated by EFSA, Codex, FAO/WHO/OIE and if relevant other organisations. In addition, the CRL-AMR will participate ad hoc in meetings and work shops arranged by EMEA. The WHO has recently established an Advisory Group in Surveillance of Antimicrobial Resistance (AGISAR), which has as the aim to develop global standards for monitoring. The CRL-AMR has and will actively support this initiative that also involves EFSA. Co-ordination of National Reference Laboratories and provision of technical support Workshops In 2010, the CRL will host a workshop held in April at the Bundesinstitut für Risikobewertung (BfR), Berlin with the following tentative agenda:

- Introduction and presentation - Update of the functions of the CRL-AMR - Update on other CRL’s - Update from EFSA, the commission and other parties - Updates on ongoing projects (streptomycin, ESC, qnr, beta-lactamases) - Break points and cut-off values - MRSA isolation, detection and epidemiology - Results of the ring trials performed in 2009 - Presentation and discussions on national programmes on susceptibility testing, control

of antimicrobial usage, criteria for categorising isolates as multiple resistant, criteria for when results should be verified or isolates send to other laboratories for verification and criteria for when the CRL or other NRL’s should be notified.

- Presentation of activities at the NRL’s - Experiences from other CRL’s

Meetings on standardization of monitoring of antimicrobial resistance The most important problem in relation to ring trails and monitoring of resistance is the lack of common interpretive criteria. This is a global problem and not only related to EU. The CRL will in 2010 continue the work with WHO (AGISAR) and other important stakeholders such as CLSI in order to promote a common international standard. This is also essential for the work planned in Codex Alimentarius.

Maintaining the network of NRL’s The CRL will during 2010 maintain and continuously update a full list of contact persons from all NRL’s. In addition, the CRL will attempt to identify expected members from applicant countries to include in the network. This list will also be maintained during the following years. Dissemination of knowledge and information The CRL will maintain the CRL web page (www.crl-ar.eu) where relevant information is posted. In addition, the CRL will distribute if any; updates, highlights or other relevant information through newsletters to the NRL’s as pdf-files. Specifically for 2010 the CRL will provide updates on the current situation in Europe and suggested methods used for isolation and identification of extended spectrum beta-lactamase producing and quinolone resistant bacteria, as well as methicillin-resistant Staphylococcus aureus and other issues considered critical important. The CRL will provide updated lists of suggested break points based on the work done by EUCAST (www.eucast.org) and other international standardization committees. Specially the CRL will host and participate in meetings with the aim to get standardised break points and cut-off values globally as mentioned above. In addition, knowledge on which antibiotics to test for and ranges to use as well as other problems encountered will also be disseminated between the NRL’s. Collection of information on activities at the NRL’s The CRL will through the NRL’s continue to gather information on the activities in relation to antimicrobial resistance from the different member countries. This will include information on:

- Antimicrobial agents tested and ranges and methodologies used - Ongoing monitoring programmes - National programmes or policies for control of antimicrobial resistance - Procedures for maintaining strain collections - Initiatives for taking action when observing special or unwanted antimicrobial

resistance (re-testing, notification, rapid alerts, verification at other laboratory) - Knowledge on standardized antimicrobial susceptibility testing - Needs for training, protocols or methodologies.

Specific assistance to individual laboratories, site visits or individual training courses Some NRL’s might have a need for special assistance. The CRL will to the extent possible within the financial limits provide specific assistance to individual laboratories based on individual needs. E-learning The continuous changing of staff at the different NRL’s makes it difficult to ensure sufficient training though individual and larger training courses. The CRL-AMR will during 2010 test the possibilities to provide e-learning on the web-site and thereby ensure a real-time visual manual for the most basic principles. Ring trials, comparative testing and quality assurance External quality control is an important part of ensuring and maintaining the analytic quality of laboratory tests performed. The CRL will in the spring and autumn 2010 organize the following ring trials on antimicrobial susceptibility testing for the NRL’s:

1. Salmonella 2. Campylobacter 3. Escherichia coli 4. Enterococci 5. Staphylococci 6. Detection of MRSA (together with CRL Staphylococci) 7. Genotypic characterization of one MDR Salmonella isolate (optional)

The organization and evaluation of the results are given under the general terms. Confirmatory testing The CRL will provide confirmatory testing for NRL’s on bacterial isolates of particular relevance or on request by the Commission. Specifically, the CRL will in 2010 provide reference testing of putative Salmonella and E. coli isolates producing beta-lactamases with extended spectrum, fluoroquinolone resistant Salmonella and isolates with transferable fluoroquinolone resistance and confirmation of MRSA. Evaluation and development of analytic methods Reference strains Reference strains for use in quality control or other analyses are an important part of the internal quality control and validation of on-going analyses. The CRL will extent it’s already available strain collection and make the strains available for NRL’s on request. Interpretative criteria The CRL will if needed perform studies on the susceptibility of food borne pathogens to various antimicrobial agents in order to provide data for the establishment of interpretative criteria for categorizing isolates as susceptible or resistant. MRSA detection The emergence of MRSA in food animal production is a matter of increasing concern. The CRL will together with other institutions continue to evaluate different methods for the optimal detection of MRSA from animal sources and if possible food of animal origin. Detection of streptomycin resistance The routine detection of streptomycin resistance poses a special problem. During 2009 the CRL conducts a study together with the different NRL’s with the aim of selecting an optimal break point for resistance. This project will be finalised in 2010. Transferable quinolone resistance Transferable quinolone resistance has recently emerged. The CRL will continue the 2009 project into 2010. Extended spectrum beta-lactamases The emergence of ESC producing isolates poses a major problem for human health. The CRL will together with the NRL’s collect information on the occurrences of ESC resistance in Europe and if necessary initiate studies into this.

EU REFERENCE LABORATORY FOR ANIMAL PROTEINS IN FEEDINGSTUFFS

WORK PROGRAMME 2010

CRL-Animal Proteins

CRA-W

Work programme 2010

CRL-AP Workprogramme 2010 ver 01 final

1

PROPOSED 2010 WORK PROGRAMME FOR THE COMMUNITY REFERENCE

LABORATORY “Detection of animal proteins in feedingstuffs”

CRL-AP

Walloon Agricultural Research Centre – CRA-W (Belgium)

1 Scientific advice and support to the European Commission (41 p/m)

1.1 Provide scientific and technical assistance to the European Commission in

relation to the development of EC feed legislation. (6 p/m)

1.2 Upon the request of the European Commission or in order to fulfil his role as

Community reference laboratory, participate to international fora/committees

relating to the detection of animal proteins in feedstuffs (EFSA, WHO/FAO,

JRC, etc) with eventual presentations to prepare for it. As up to 2 European or

international missions/year are foreseen in support to DG Sanco and/or CRL

activities. (1 p/m)

1.3 Upon the request of the European Commission or in order to fulfil his role as

Community reference laboratory, participate in meetings for the standardisation

of analytical methods relating to the detection of animal proteins in feedstuffs

and their implementation (CEMA, ISO/CEN, OIE, IAG, etc). Up to 3 European

missions/year are foreseen in support to DG Sanco and/or CRL activities. (1

p/m)

Among the possible activities in 2010 there might be a CEN working group

(within CEN TC327) defining guidelines for PCR in detection of PAP in feed.

1.4 To actively participate in technical and scientific support of the European

Commission in the context of incidents or crises linked to incorrect use of

animal proteins. (3 p/m)

1.5 To keep at CRL the highest standard possible of technical skill, scientific

awareness and quality management under accreditation (ISO17025) on

CRL-Animal Proteins

CRA-W

Work programme 2010


2

analytical methods for detection, quantification and identification of animal

proteins in feed ingredients and in feedingstuffs. To maintain and extend the

accreditation scope of the CRL lab. (16 p/m)

1.5.1 To maintain scientific awareness in general about techniques that

might be helpful in relation to topics of interest of the CRL-AP

1.5.2 To maintain the accreditation scope

1.5.3 Maintenance of the competences of the CRL-AP team and formation of

the new recruits

1.5.4 Extend the accreditation scope of the CRL-AP by including additional

animal targets DNA in the scope for PCR analyses

1.5.5 Preparation of the dossier for the accreditation of the organisation of

interlaboratory studies

1.6 On the request of DG SANCO or the NRLs, to perform analyses on samples

with disputed results. (12 p/m)

1.7 Organisation in collaboration with TAIEX of a training/workshop for the

candidate and potential countries (Turkey, Croatia, Iceland, FYR Macedonia,

Albania, Bosnia and Herzegovina, Montenegro, Serbia) (2 p/m)

1.7.1 Request of financial support to TAIEX

1.7.2 Preparation and organisation of the workshop

1.7.3 Redaction of the proceeding

CRL-Animal Proteins

CRA-W

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2 Coordination of activities of NRL network (17 p/m)

2.1 Maintenance and update of CRL website (internet/intranet) to disseminate and

share information with NRLs and others stake holders. (6 p/m)

2.1.1 Information collection and validation

2.1.2 Maintenance of the website

2.1.3 Development of additional management tools of the website

2.1.4 Test of the information system and validation

2.2 Prepare and send a six-months newsletter for NRLs. (1 p/m)

2.3 Organisation of the annual CRL-AP meeting/workshop (3 p/m)

2.3.1 Organisation of the 4rd annual CRL-AP workshop

2.3.2 Preparation of the agenda

2.3.3 Invitation of the attendees

2.3.4 Realisation of the workshop

2.3.5 Minutes of the annual workshop

Note : For 2010 it is proposed to organise the annual workshop in Italy (Turino). The

Italian NRL agrees to host this meeting.

2.4 Supply information, scientific advices and protocols to NRLs, testing

laboratories, detection, quantification and identification of animal proteins in

feed ingredients and feedingstuffs. (5 p/m)

2.5 Participate in the annual CRL Directors co-ordination meeting.

2.6 Prepare the six months and annual reports of activities according to the report

guidelines transmitted by DG SANCO. (2 p/m)

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3 Interlaboratory studies and quality assurance (28 p/m)

3.1 Coordinate the preparation, reception, storage, maintenance and distribution to

national reference laboratories (NRL) of samples containing animal proteins

derived from different species and in particular from fish, poultry, pigs and

ruminants to be used as reference materials or to carry out comparative testing.

This task includes the preparation of the samples for the interlaboratory studies

(12 p/m)

3.1.1 Definition of the needs

3.1.2 Collection of the raw materials to use in the preparation of the

samples

3.1.3 Control of the raw materials

3.1.4 Production of the samples

3.1.5 Test of the homogeneity of the samples produced

3.1.6 Report on the produced samples

3.1.7 Distribution of the samples

3.2 Organize interlaboratory study for the determination of PAPs in feed using

classical microscopy. (9 p/m)

3.2.1 Redaction of the report of the CRL-AP interlaboratory study of

Autumn 2009

3.2.2 Definition (with the collaboration of the DG-Sanco) of the objectives

of the ring trial to perform at the end of 2010

3.2.3 Preparation of the interlaboratory study.

3.2.4 Invitation of the NRLs to participate.

3.2.5 Packaging and sending of the samples (cf. task 3.1)

3.2.6 Collection of the results.

3.3 Audit NRLs, coordinate training on methods of analysis and assist staff from

NRLs if comparative testing reveals limited experience. Up to 3 European

missions/year are foreseen in support to DG SANCO and/or CRL activities

(1 p/m)

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3.4 To help to develop, extend and keep in the NRLs the highest standard of

technical skill and quality management under accreditation on analytical

methods for detection, quantification and identification of animal proteins in

feed ingredients and in feedingstuffs. (6 p/m)

3.4.1 Definition of the needs of the NRLs

3.4.2 Provide the requested help to the NRLs

3.4.3 Preparation of a syllabus including all the information needed for an

appropriate detection of processed animal proteins in feedingstuffs

3.4.4 Preparation of permanent slides set at the disposal of the NRL

4 Development of analytical methods and tools (34 p/m)

4.1 Contribute to the development of new methods of analysis and improvement of

existing methods of analysis. (4 p/m)

4.1.1 Establishment/maintain of contact with the laboratory in charge of the

development in order to be frequently informed about the progress of their

development

4.1.2 Definition of the potential support of the CRL to these initiatives

4.1.3 Establishment of the needs in the development of methods

4.2 Contribute to the development of complementary analytical methods necessary

to assure the correct implementation of official methods and explorative or

alternative methods. (6 p/m)

4.2.1 In this context the capacity of DGGE technique (and eventually even

the TGGE technique) to determine the origin of fish meals based on bacterial

profiles will be tried out.

4.3 Coordination of evaluation studies on alternative methods. As soon as they

become available, methods specifically detecting ruminant, pig or poultry

proteins should be evaluated. (12 p/m)

4.3.1 Organisation of interlaboratory studies based on alternative tests

(PCR, immunology,...) developed by NRLs or by companies

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4.3.2 On the basis of the former interlaboratories regarding PCR methods,

define the strategy for the optimum implementation in the NRL

4.3.3 Preparation of CRL-AP protocol at the destination of the NRL for the

implementation of the PCR methods

4.3.4 Organisation of the transfer of validated PCR methods to the NRLs

network – training courses and manuals will be prepared during 2010

4.4 Performing CRL available methods or adapting them on outbreak material to

make them available for the NRLs network. (2 p/m)

4.5 Extension of the samples bank with a special focus on specific animal material

(e.g. sea mammals, rodents). Test, packaging and storage of the new samples as

well as production of microscopic image representative of the particles making

up the samples collected and selected to be included in the CRL samples bank.

(10 p/m)

4.5.1 Establishment of the specification for the CRL samples bank

4.5.2 List of the priority needs regarding the materials to include in the

samples bank

4.5.3 Maintenance of informatics tools for the appropriate management of

the samples

4.5.4 Collection/production of samples of animal origin

4.5.5 Test of the samples collected

4.5.6 Storing of the samples

4.5.7 Maintenance of the samples bank

5 Workshops/trainings (5 p/m)

5.1 Provide specific workshop for the benefit of NRLs for the correct application of

the method described in the Annex VI of the 152/2009/EC Commission

regulation to detect animal proteins in feed (Classical microscopy) and any new

development or regulation related to the detection, identification and

quantification of animal proteins in feed. (1 p/m)

5.1.1 On the basis of the former interlaboratories regarding PCR methods,

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decide of the opportunity to organise a specific workshop in 2011 for the

implementation of the PCR method

5.2 Provide specific workshop of experts from candidate Member States for the

correct application of the 152/2009/EC directive to detect animal proteins in

feed (Classical microscopy) and any new directive linked to the detection,

identification and quantification of animal proteins in feed. (1 p/m)

See section 1.7., workshop for candidate and potential countries

5.3 Provide training through dissemination tools like CD’s or DVD’s. Development

of analytical support and libraries for the training and the maintenance of the

skill of laboratories performing classical microscopy or other validated method

(3 p/m).

5.3.1. Presentation to the NRL of ARIES 2 (Safeed-pap)

5.3.2. Didactical support for the correct implementation of PCR method

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