work programmes for community reference laboratories for 2008 · frame: the regulation 1664/2006...

WORK PROGRAMMES FOR COMMUNITY REFERENCE LABORATORIES

FOR 2008 **********

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

COMMUNITY REFERENCE LABORATORY FOR MILK AND MILK

PRODUCTS

WORK PROGRAMME 2008

CRL « Milk » 1/7 23/01/08 2008 Programme of Work

LABORATOIRE D’ETUDES ET DE RECHERCHES SUR L’HYGIENE ET LA QUALITE DES ALIMENTS

EU COMMUNITY REFERENCE LABORATORY FOR MILK AND

MILK PRODUCTS

2008 Programme of Work of the Community Reference Laboratory for Milk & Milk Products

Site de Maisons-Alfort


The AFSSA-LERQAP (Laboratory for Studies & Research on Quality of Foods & on Food Processes) foresees to undertake, as Community Reference Laboratory (CRL) for milk, the following works in 2008 according in particular to (a) the actions planned at the 10th Workshop of the National Reference Laboratories (NRLs) (28&29 June 2007), and (b) the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the CRL for the period 2006-2010. These actions are part of the new role of the CRL, restricted to the control of raw and heat-treated liquid milk (total flora, somatic cells count, phosphatase activity), as well as cheeses for phosphatase, in the frame of the Regulation 853/2004 laying down specific hygiene rules for food of animal origin. The Annex III, Section IX of Regulation 853/2004 is dedicated to raw milk and dairy products:

- Microbiological criteria on total flora at 30°C and on somatic cells count are fixed for: o raw cow’s milk and for raw milk from other species milk (Chapter I, III), o dairy products (Chapter II, III-criteria for the use of raw cow’s milk for further

processing). - Phosphatase activity:

o In Chapter I, I.3, a reference is made to a negative phosphatase test to characterize the heat-treatment applied to certain raw milks at the primary production stage (Chapter I, I.3).

o Chapter II(II) includes requirements on heat-treatment of raw milk or dairy products, applicable to food business operators. A cross reference is made to the general requirements of Regulation 852/2004, Annex II, Chapter XI.

The CRL foresees in particular to provide a support to the NRLs for the implementation of:

- the Regulation 853/2004; - the derived Regulation 1664/2006, recently published, defining amongst other the

testing methods for raw milk and heat-treated milk to be used by competent authorities and food business operators:

o to check compliance with the limits for total flora and somatic cells count laid down in Regulation 853/2004, Annex III/Section IX/Chapter I/Part III,

o to ensure appropriate application of a pasteurisation process to dairy products, as referred to in Regulation 853/2004, Annex III/Section IX/Chapter II/Part II.

NB: In brackets under each item, the scheduled duration of the action is indicated: either annual (limited to 2008) , either multi-annual (on-going programme on several years).


1. Inter-laboratory proficiency testing The inter-laboratory proficiency testing trials organised by the CRL for the NRLs aim at evaluating the ability of the NRLs to apply satisfactorily the methods for the analyses performed in the frame of official controls, prescribed by Regulation 1664/2006.

1.1 Somatic cells count (annual)

Frame : The Regulation 1664/2006 prescribes the reference method for somatic cells count, Standard ISO 13366-1 as well as conditions for the use of alternative methods.

The CRL (Unit HMPA) will organize an inter-laboratory trial on somatic cells count by the reference method, the Standard ISO 13366-1. Two CRL staffs will visit CECALAIT for establishing the experimental design of the samples for the trial, to be prepared by CECALAIT.

1.2 Determination of alkaline phosphatase activity (annual)

Frame: The Regulation 1664/2006 defines the reference method, the Standard ISO 11816-1, the legal limit for negativity of the test (350 mU/l for cow’s milk) and conditions to use alternative methods.

Criteria to evaluate the individual performance of NRLs At the last workshop of the NRLs Milk (7&8 June 2007), it was agreed that the CRL together with experts of some NRLs would define pertinent criteria to evaluate the individual performance of NRLs participating to PT trials organized by the CRL on AP activity. The outcome would be presented at the 2008 workshop dedicated to AP. It is intended to organize one meeting of the NRL working group in Brussels, in conjunction with one meeting of the DG AGRI chemical experts’ working group.


2 Analytical development These works are conducted in the CRL laboratory alone.

2.1 Determination of total flora at 30°C in raw milk (multi-annual)

Frame : The Regulation 1664/2006 prescribes the reference method for total flora at 30°C, Standard EN ISO 4833 as well as conditions for the use of alternative methods.

The reference method for evaluating this parameter is the Standard EN ISO 4833 (enumeration of bacteria on Petri dishes). However, this reference method is generally not used in routine analyses for the hygiene control of raw milk. Alternative methods are used instead, mainly instrumental ones based on flow cytometry (such as the Bactoscan or Bactocount apparatus). Their use is allowed in Regulation 1664/2006 under certain conditions.

a. Coordination of the NRLs Since the Standard ISO 21187 on the conversion factors between the routine method and the reference method has been recently published, the CRL will go on to supervise the NRLs on how they coordinate the implementation of the Standard by the network of laboratories in charge of routine control of raw milk. In particular, all conversion factors should be recalculated according to the Standard in each Member State and it is intended to have only one conversion factor per country. In 2008, the CRL will in particular update the enquiry on national practices by re-circulating the questionnaire to NRLs (update of NRL replies or additional replies). It will also prepare, in collaboration with the D and BE NRLs, a check-list for the NRLs to conduct the visits of laboratories in charge of establishing the conversion factors.

b. Study of the alternative methods The CRL (Unit HMPA) will go on the experimental studies using a flow cytometer (Bactocount), purchased in 2007, as an alternative method to the bacterial count, in order to investigate the questions linked to the correlation of this method to the reference method, especially the different factors influencing, for a same apparatus, the value of the conversion factor (variation in breeds, period of lactation, type of feeding,..). For that purpose, batches of raw milk delivered at regular intervals of time will be analysed in parallel by the reference method and by the Bactocount.

c. Scientific and technical support IDF/ISO intends to launch a revision of the Standard IDF 161 detailing the specificities of validation of a routine method against a reference method for the determination of total flora in raw milk. The CRL would follow this standardization work and would ensure a liaison with the works undertaken as CRL with the NRLs network.


2.2 Determination of total flora at 30°C in colostrums (multi-annual)

Frame: DG SANCO intends to add hygienic requirements for colostrums in Regulation 853/2004. But the CRL and NRLs consider that data on levels of total flora in colostrums are not readily available.

In 2008, the CRL (Unit HMPA) will launch a bibliographical study on the topic and will design the experimental study on determination of total flora at 30°C in colostrums, including the way to get appropriate samples, before conducting the study the year after.

2.3 Determination of somatic cell count in raw milk (multi-annual)

Frame : The Regulation 1664/2006 prescribes the reference method for somatic cell count in raw milk, Standard ISO 13366-1 as well as conditions for the use of alternative methods.

The reference method for evaluating this parameter is the Standard ISO 13366-1 (microscopic technique). However, this reference method is generally not used in routine analyses for the hygiene control of raw milk. Alternative methods are used instead, mainly instrumental ones based on flow cytometry (such as the Fossomatic or Bactocount apparatus). Their use is allowed in Regulation 1664/2006 under certain conditions. The CRL (Unit HMPA) will launch experimental studies using a flow cytometer (Bactocount), purchased in 2007, as an alternative method to the microscopic technique, in order to investigate the questions linked to the correlation of this method to the reference method, in parallel to the study of the use of the same apparatus for total flora (see 2.1.b).

2.4 Determination of alkaline phosphatase activity (multi-annual)

In 2008, the CRL (Unit CALAS) intends to conduct the following activities. It should be stressed that the following work program is scheduled on the basis of a normal yearly activity. If renovation of the CALAS laboratory is undertaken in 2008, the work may have to be re-scheduled to fit with the constrains of renovation. One CRL staff will follow a training course by the company manufacturing the Fluorophos instruments, as to be able to ensure itself part of the maintenance and revision of the Fluorophos instruments of the laboratory.

a. Determination of the phosphatase activity in other than cow’s milk The CRL will go on the pasteurisation studies on goat’s and ewe’s milk, whose purpose is to support EC advising an appropriate legal limit on alkaline phosphatase (AP) activity for ewe’s and goat’s pasteurised milk. Goat’s milk


For the 2008 NRLs workshop dedicated to AP, the CRL will finalise its proposal of a legal limit for correctly pasteurised goat's milk. The proposal has been postponed in order to study results of Greek goat's milk that might lead to a higher level than the one envisaged by the CRL and NRLs collaborating to this project (from Portugal, Spain, Norway, Romania and Switzerland). Ewe’s milk The CRL will study thermal inactivation of AP in ewe's milk, with input from collaborating NRLs. The CRL will establish the kinetics of AP inactivation in parallel with decrease of the microbial load of the milk samples in order to propose a legal limit for correctly pasteurised ewe's milk. Camel’s milk To characterize the heat-treatment of camel’s milk, the CRL intends to launch a study in collaboration with the Central Veterinary Research Laboratory (CVRL) of Dubai. The first phase of the study, to be launched in 2008, would aim at assessing the possibility to use AP activity as an pertinent indicator of pasteurisation of camel’s milk. The specific costs of the entire study is to be covered by a contract with CVRL.

b. Determination of alkaline phosphatase in cheeses As a first step to the relevant standardisation procedure, the CRL will prepare for IDF/ISO a revised draft Standard related to the determination of AP activity in cheese. The CRL will continue the study on the residual AP activity in soft cheese and will compile these results with those forwarded by other NRLs. The expected end-product is the fixation of a AP limit allowing for the distinction between cheeses made from pasteurised milk and cheeses made with non pasteurised milk.

c. Comparison of the chemiluminescent/fluorimetric methods The comparison performed in Spring 2007 between the chemiluminescent method (Novalum) and the current regulatory reference method, the fluorimetric method (Fluorophos) showed a bias between the two methods. Based on a modified protocol to be proposed by the manufacturer of Novalum, the CRL intends to conduct a new comparison study in 2008

3 Assistance to the NRLs

3.1 Training courses Upon requests of NRLs, the CRL may receive NRL staff for individual training on specific topics. 4 NRLs Workshop


The CRL will organise in 2008 the 11th NRLs Workshop, targeted on alkaline phosphatase activity. In particular, this workshop will enable: - to make an update of all the works undertaken by the CRL on this topic, especially since

the former workshop dedicated to this topic (2002); - to define limits for AP in goat’s and possibly in ewe’s milk; - to envisage the feasibility of a criteria approach for AP activity. 5 Technical and scientific assistance to the European Commission

5.1 Participation to ISO/IDF standardization works On behalf of DG SANCO (and official nomination as EC representative to CEN/ISO meetings), participation to - the IDF/ISO works on the analytical methods specific to the analysis of raw milk:

- somatic cells count: reference and alternative methods, - total flora: alternative methods, - phosphatase test: reference and alternative methods.

- the IDF/ISO Analytical Week and the meeting of the groups dealing with the topics mentioned above.

COMMUNITY REFERENCE LABORATORY FOR SALMONELLA

WORK PROGRAMME 2008

Work-programme CRL-S 2008 14-08-2007 Page 1 of 6

CRL-Salmonella work-programme 2008 Introduction The regular yearly working programme of CRL-Salmonella consists of the following activities: 1. Organisation of interlaboratory comparison studies; 2. Organisation of a workshop with the NRLs-Salmonella; 3. Performance of research; 4. Quality assurance of serotyping of isolates from (baseline) surveys 5. Performance of ad hoc activities; 6. Communication. The activities as specified for 2008 1. Interlaboratory comparison studies For 2008 it is planned to organise 3 interlaboratory comparison studies; • Two studies on bacteriological detection of Salmonella; • One study on typing of Salmonella. The exact timing of the studies in 2008 will be planned with the NRLs-Salmonella, but the general time table is indicated below. Interlaboratory comparison study on bacteriological detection of Salmonella in veterinary samples In 2007 no interlaboratory comparison study on veterinary samples (e.g. animal faeces) has been organised, it is therefore desirable to organise such a study at the beginning of 2008 (February/March). It is planned to use a similar set-up as in earlier studies, which implies the addition of Salmonella reference materials to a Salmonella negative matrix. The choice of matrix may be poultry faeces. It is planned to use reference materials with a lower number of Salmonella spp. than in earlier studies. The reason for the lower contamination levels is the fact that the last 2-3 years almost all NRLs score 100 % positives with all samples in the detection ring trials. Since the matrix is not longer mixed with glycerol the results of the interlaboratory comparison studies very much improved. To obtain more information on the performance of the laboratories and of the methods used, it is more interesting to test low level samples close to the detection limit, beside high level samples which are approximately 10 times above the detection limit. It is expected that when samples with a contamination level close to the detection limit are tested, circa 50 % of the tested samples will be negative. The choice of methods will be Annex D of ISO 6579, 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

Work-programme CRL-S 2008 14-08-2007 Page 6 of 6

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. 6. Communication In 2007 a fully amended version of the CRL-Salmonella website was launched. The address remained unchanged: www.rivm.nl/crlsalmonella. A staff member of the CRL-Salmonella will regularly update the website. 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 intercomparison 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 via the CRL-Salmonella website. Summaries of several intercomparison studies and related research will be published in the scientific literature. Missions Summarising from the information as given in the chapters above, the following missions may be needed in 2008. Per mission one staff member of the CRL-Salmonella will participate. - Visit to a poor performing NRL (if necessary, see information under chapter

‘Interlaboratory comparison studies’), ca 2 days, country unknown. - Meetings of ISO/TC34/SC9 and CEN/TC275/WG6, probably in Finland, date not yet

set (see information under chapter ‘Research’). Total duration of the meetings approximately 5 days.

- Meetings of two working groups of ISO/TC34/SC9 (2 meetings per working group): the working group on validation of methods (WG3) and the working group Proficiency Testing in microbiology (WG4). See information under chapter ‘Research’. Not all meetings are planned yet, except a (two days) meeting of the WG3 (method validation) in London, UK in January 2007.

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

COMMUNITY REFERENCE LABORATORY FOR MARINE BIOTOXINS

WORK PROGRAMME 2008

COMMUNITY REFERENCE LABORATORY FOR BACTERIOLOGICAL AND VIRAL CONTAMINANTS OF

MOLLUSCS

WORK PROGRAMME 2008

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

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

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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WORK PROGRAMME FOR THE CRL FOR BACTERIOLOGICAL AND VIRAL CONTAMINATION OF BIVALVE MOLLUSCS, 2008 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 2008 work programme year 27 Member States and 3 candidate countries (Croatia, Turkey and Former Yugoslav 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, 2008 Duration

1. Scientific advice and support

1.1. Assist DG Sanco in functioning and implementation of Community food hygiene legislation, e.g. drafting guidance documents, consideration of analytical tolerances, etc.

10 days

1.2. Participate in relevant EU and International scientific committees (ISO/CEN, WHO/FAO, ICMSS etc). In 2008 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.

• 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 (see resolution 33, 6th workshop of NRLs).

• 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.

45 days

1.3. Assist DG Sanco with specialist assistance in relation to food and veterinary inspections of Member States, Accession Countries and Third Countries and with other trade issues (e.g. equivalency negotiations, industry initiatives such as SUMO) as they arise.

15 days




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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 2008 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) and up to 3 missions in support of NRLs and DG Sanco activities.

35 days

1.6. Participation in relevant international scientific conferences 1.7. Participate as a member of the steering committee in the ICMSS’

international forum on harmonisation of approaches to bivalve shellfish sanitation, including standardisation of methodologies for indicator organisms, and human pathogenic viruses and bacteria.

5 days 3 days

1.8. Participation in the joint FAO/WHO/Codex Task Force undertaking risk profiling and formulating Food Risk Management Guidance on noroviruses in food, including bivalve shellfish.

5 days

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 seventh annual NRL workshop, produce resolutions and other workshop outputs (May 2008, CRL Weymouth). To include CRL administrative assistance.

45 days

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

Up to 153 days

2.4 Organise specialist, targeted, practical training for NRLs, MS competent authorities and the FVO on sanitary surveys - in accordance with the requirements of 854/2004 on official controls.

2.5 In collaboration with the US FDA organise and host a joint

workshop on implementation and approaches to sanitary surveys in the EU and US.

8 days 15 days

2.6 Supply specialist information and advice on bacteriological and 10 days




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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 and transfer of other technical information.

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.

5 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 5 of 6th workshop of NRLs). Analyse results, produce report, advice and recommendations (by May 08).

40 days

3.2 Organise Norovirus and hepatitis A ring trials (see resolution 26 of 6th workshop). Analyse results, produce report and recommendations (by May 08).

50 days

3.3 Undertake Vibrio parahaemolyticus ring trials appropriate for methods enabling enumeration of pathogenicity principles (thermostable direct and thermostable direct related haemolysins) (see resolution 32 of 6th workshop). Analyse results, produce report and recommendations (by May 08).

50 days

3.4 Undertake FRNA bacteriophage proficiency testing. Extend provision of EQA material, methods of analysis etc to national testing laboratories if required by NRLs (see resolution 4 of 6th workshop).

15 days

3.5 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 resolution 7 of 6th workshop) for NRLs, the scheme will be extended to selected Official Control Laboratories. Analyse results, produce report, advice and recommendations (by May 08).

30 days




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3.6 Prepare stable reference material using biological LENTICULE carriers for norovirus and Hepatitis A (see resolution 28 of 6th workshop). Perform homogeneity and stability analyses. Distribute data and LENTICULES to NRLs for use as control material on request.

30 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). To include maintenance of IEC ISO 17025 accreditation for detection of norovirus in bivalve shellfish and adoption of CEN method for viruses.

70 days

4.2 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.

70 days

4.3 Contribution to costs of the maintenance of CRL capability to perform analysis for marine vibrios in bivalve molluscs other than V. parahaemolyticus.

10 days

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

Included in above

5 Development of analytical methods (undertaken at CRL)

5.1 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).

Note. Validation of the TAG4 reference method for viruses in foods, including bivalve shellfish is a priority area. In the event of CEN mandate M/381 “Methods of Analysis of foodstuffs concerning food hygiene” (Mandate for standardisation M/381 addressed to CEN) not being fully funded, resources will be diverted from other areas within the work programme to enable validation of the virus method for bivalve shellfish.

5.2 With the likely introduction of a virus standard into the EU

legislative framework in the future, the importance of generating information on the public health significance of PCR results is paramount. In recognition of this and in support of resolution 31 of the 6th workshop of NRLs it is proposed that a PhD studentship should be established dedicated to researching norovirus infectivity

15 days 211 days sub-contracting costs €24,150




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and illness risk associated with PCR positive bivalve shellfish.

Note. A PhD studentship in collaboration with the School of Biomedical and Molecular Sciences, University of Surrey, UK represents the most cost effective and efficient means of resourcing this activity

5.3 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.

5.4 The existing E.coli enumeration reference method ISO TS 16649-3

specified in Commission Regulation (EC) No 2073/2005 is published as a technical specification with an expiry of December 2008. It is proposed that the CRL, in collaboration with the NRL network undertakes work to generate data enabling its adoption as a full horizontal standard. Thus enabling it to remain as an ISO recognised method for E. coli enumeration in live bivalve molluscs, tunicates, echinoderms and marine gastropods in Regulation.

20 days 50 days

Rachel Rangdale CRL Co-ordinator August 2007

COMMUNITY REFERENCE LABORATORY FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

WORK PROGRAMME 2008

CRL Work Plan 2008

2008 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 2008 (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 maintained. 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. Summary data will ultimately be accessible on the website (see section 3.3)

1.3

Collection and 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).

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.)

1.5 VLA was actively involved in the collection of BSE material for the exclusive use of European research, through project FAIR CT98-3651. This project has now finished, and the material (approximately 15,000 samples from 25 animals) remains stored at VLA. It is proposed that the CRL continues to maintain this collection on behalf of the Commission. Full details of this collection are on the Neuroprion website.

1.6 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 4 ARQ/ARQ sheep with bovine BSE in 2005, and a further 5 in 2006. This will provide material in the medium term against which other potential alternative supplies (such as the Tg mouse pool being created by IRMM) can be appropriately validated. Two of the original challenge sheep, and the five challenged in 2006, are still alive (as of August 2007). A further 10 sheep were challenged in 2007 to renew this initial resource. These 17 sheep will incur maintenance costs in 2008 if they do not succumb to disease within the next 6 months. It is proposed to challenge a further

CRL Work Plan 2008

2008 workplan.doc 31/05/11 2

5 sheep in 2008 .

1.7 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. An experimental challenge of 5 animals with ‘atypical’ scrapie was started in 2006, with a further 5 challenged in 2007, to provide a bank of such material exclusively for EU QA purposes. (Detailed prediction of how long this challenge will take to produce disease is not possible at present, but the VLA has already begun such challenges for research purposes, and these sheep – mostly still healthy after almost three years –has resulted so far in two positive transmissions at 378 and 878 dpi.) Accurate prediction of timescale is still not possible. This is a long-term strategy, and material cannot be expected to be available for QA purposes until 2008 at the very earliest. These 10 animals are currently healthy and are predicted to incur maintenance costs into 2008. It is proposed to challenge a further 5 sheep in 2008.

1.8 The recent identification of BSE in a French goat has highlighted the importance of appropriate species-specific controls for the investigation of such cases. Given the extreme shortage of experimental caprine BSE, the CRL initiated goat challenges in 2005. These five animals remain healthy, and will incur maintenance costs in 2008 unless they develop disease within the next 6 months.

1.9 The identification and characterisation of ‘new’ forms of BSE (i.e. those provisionally classified as H and L type based on the molecular mass of the unglycosylated fragment on a Western blot) raises the need for reference material from such cases. Global supplies are currently very limited, and reserved forresearch. We have an offer from the German NRL to provide sufficient material to enable the intracerebral challenge of two cattle with H-type BSE and two with L-type BSE to generate a small bank of reference material for statutory testing purposes.

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 2008 and the second in October 2008.

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.

CRL Work Plan 2008

2008 workplan.doc 31/05/11 3

Information will also be gathered from the neuroprion cervid group IHC QA which is being coordinated by the CRL on behalf of Neuroprion.

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. It is not intended to make provision for caprine testing this year.

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. Initially this will cover classical scrapie only. Additionally, we would like to also include atypical scrapie in samples of cerebrum or cerebellum, but currently there are insufficient samples of this type available. However, if sufficient samples become available it would be our intention to extend the ovine rapid test QA round to cover atypical scrapie (Nor 98 like)-see 1.7. 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 be limited to classical scrapie until atypical samples become available.

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

those NRLs which are operating such methods.

• One proficiency test round for cervid rapid tests

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 be sent a copy of the results for each proficiency testing exercise. It is hoped that this will be superceded by the creation of a password-

CRL Work Plan 2008

2008 workplan.doc 31/05/11 4

protected area on the CRL website on which QA results will be posted .

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) will be possible across the whole of the EU27. Full instructions have been issued as part of the manual on Discriminatory Testing. However, the completeness and level of detail captured so far is far from ideal. 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.

3.2

The CRL will provide expert advice on the epidemiology and clinical manifestations of BSE and scrapie. The epidemiologists will also keep abreast of changes and developments in the pattern of TSEs throughout the world. 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. It is hoped that password protected areas will be available for the TSE CRL in late 2007/early 2008, which will enable effective but controlled sharing of confidential information, in addition to the dissemination of general information.

4.0 Provision of training

4.1 A workshop for National experts will be arranged in the first half of 2008. 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 2008 for three laboratory visits to assist with technical issues. Two NRLs have already been identified as requiring visits. An additional visit has been planned as a contingency.

CRL Work Plan 2008

2008 workplan.doc 31/05/11 5

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. We do monitor companies’ training methods and have already tested out one manufacturer's training system to assure NRLs that manufacturers train kit users.

5.0 Strain typing

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. [A self-financing representative from SEAC is also on this group, with the Commission’s approval]. This group plans to meet twice in 2008. Discussion will continue to focus on the validation of bioassay methodologies. Discussion of bioassay models will start within Neuroprion, and this group will then hold follow-up discussion, to include planning for a ‘ring-trial’ validation exercise. The group will also agree criteria for the classification of bovine isolates. 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. At present, only conventional mice are sufficiently evaluated and defined for this purpose, and interpretation is based on a full panel (i.e. RIII, VM and C57Bl6). [The choice of mouse strains is under active discussion (see section 5.1) including the use of Tg strains. However, none of these is sufficiently evaluated at present to be used for this purpose.] These estimates are based on the assumption that a maximum of 1 isolate will require further characterisation by bioassay.

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

CRL Work Plan 2008

2008 workplan.doc 31/05/11 6

cannot be readily predicted - if for example we have to undertake laboratory work to investigate a problem which arises in year. Additional costs may therefore arise in year which 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.

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 1.6 –1.9). There are also ongoing discussions with IRMM about the potential of using transgenic mouse material for this purpose. Deer/elk tissues and homogenates have been kindly supplied by North American collaborators, but these are finite. Homogenates have been prepared in 2007 for QA testing in 2008. There will be requirement to prepare further supplies of homogenate in 2008 for use in 2009.

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 Following on from discussions with the Commission in September 2006, the CRL plans to roll out EU wide batch testing and release of rapid test kits. The protocols have been agreed, NRLs nominated and a sample panel prepared. We will consider whether this should also include SR tests in 2008.

6.5 Following discussions with the Commission in May 2007, the CRL plans to roll out a programme of work to assess the analytical sensitivity (detection limit) of all TSE rapid tests. Initially BSE tests will be considered. The CRL will implement the protocol agreed with the Commission during 2007 (appended for ease of reference).

7.0 Discriminatory testing

7.1 A Handbook detailing precise methods for discriminatory blotting has been produced. This Handbook will be reviewed annually, revised and updated as necessary to include additional information of relevance to the surveillance of TSE,

CRL Work Plan 2008

2008 workplan.doc 31/05/11 7

and re-issued online. A protocol will be drafted to define the discriminatory criteria for classification of BSE isolates, based on the current data available for H- and L-type BSE. This protocol will be agreed at the STEG (see 5.1) and made available online.

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, and 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 .

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 (5 to date) of referrals to date, and assume that a maximum of 1 ring trial will be required. All ring trial results will be collated and reported to the Commission via the monthly update reports (see section 7.1).

CRL Work Plan 2008

2008 workplan.doc 31/05/11 8

Appendix 1

PROVISIONAL TIMETABLE FOR TSE CRL QA EXERCISES IN 2008

Intended Start

Date i QA activity

February 2008

Ovine genotyping

March 2008 Immuno-histochemical technique

April 2008 Histopathology and immunohistochemistry interpretation (round 1)

October 2008 Histopathology and immunohistochemistry interpretation (round 2)

October 2008 Bovine rapid testing incorporating confirmatory blotting if appropriate

November 2008 Ovine rapid testing, incorporating confirmatory blotting if appropriate

September 2008 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.

COMMUNITY REFERENCE LABORATORY FOR LISTERIA

MONOCYTOGENES

WORK PROGRAMME 2008

CRL « L. monocytogenes » 1/6 11/07/07 2008 Programme of Work


EU COMMUNITY REFERENCE LABORATORY FOR

LISTERIA MONOCYTOGENES

2008 Programme of Work of the Community Reference Laboratory for

Listeria monocytogenes



Introduction In May 2006, the laboratory LERQAP (Laboratory for Studies & Research on Quality of Foods & on Food Processes) of AFSSA (French Agency for Food Safety) has been nominated Community Reference Laboratory (CRL) for Listeria monocytogenes (see Regulation 776/2006). The CRL foresees to undertake the following actions in 2008, according to the actions planned at the 1st Workshop of the National Reference Laboratories (NRLs) (2&3 April 2007), as well as to the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the CRL for the period 2006-2010. Most of these activities aim at implementing, from an analytical point of view, the EC Regulation 2073/2005 on microbiological criteria for foodstuffs, which includes in particular 4 food safety criteria on L. monocytogenes (Annex I, Chapter 1):

- either qualitative criteria: absence of L. monocytogenes in 25 g, for o ready-to-eat foods intended for infants and for special medical purposes, o other ready-to-eat foods able to support the growth of L. monocytogenes, when

leaving the producer; - either quantitative criteria: a limit of 100 cfu/g, for

o ready-to-eat foods able to support the growth of L. monocytogenes, placed on the market during their shelf-life,

o ready-to-eat foods unable to support the growth of L. monocytogenes, placed on the market during their shelf-life.


1. Detection and enumeration of L. monocytogenes in food

1.1 Inter-laboratory proficiency testing for the NRLs (annual) The inter-laboratory proficiency testing (PT) trials organised by the CRL for the NRLs aim at evaluating the ability of the NRLs to apply satisfactorily the methods for the analyses performed in the frame of controls prescribed by Regulation 2073/2005.

a. Study of sample types for inter-laboratory trials The CRL (Unit HMPA) will conduct a study to develop the sample types to be used for the PT trial to be organized in 2008, using smoked salmon as matrix. In particular, the stability of this type of samples will be studied in different transportation conditions to the NRLs.

b. Detection of L. monocytogenes The CRL (Unit HMPA) will organize in 2008 a PT trial for the NRLs on the detection of L. monocytogenes by the reference method EN ISO 11290-1, using smoked salmon as matrix.


1.2 Analytical development (multi-annual)

Frame: The Standard method EN ISO 11290-parts 1&2 are cited as reference methods in the qualitative and quantitative criteria of EC Regulation 2073/2005 for L. monocytogenes.

a. Enumeration method using a membrane filtration

The Standard horizontal method EN ISO 11290-2 for enumeration of L. monocytogenes in food is characterized by a theoretical limit of enumeration of 10-100 cfu/g or ml. Meanwhile, it has been shown that the precision of this Standard method is quite poor, especially a low levels. Even if the Standard has been amended with a more precise method (an enumeration agar, ALOA, now more specific to L. monocytogenes), the method still lacks of enough sensitivity to control precisely a limit at 100 cfu/g or ml. The CRL (Unit HMPA) has developed and validated a more sensitive enumeration method, including a concentration step based on membrane filtration followed by transfer of the filter to a selective medium, for the enumeration of L. monocytogenes in cold-smoked fish. The CRL will continue in 2008 to test the applicability of this membrane filtration method to various foods, including cheeses, ready-to-eat foods, intermediate products (as grounded meat) and raw materials. b. Improvement of the enrichment step for the detection method The detection of L. monocytogenes in food at very low levels is required for a satisfactory implementation of the qualitative criteria on L. monocytogenes of Regulation 2073/2005 as well as for a correct prevalence’s estimation in the context of quantitative risk assessment. In that respect, the Standard reference method EN ISO 11290-1 can still be improved. The 2-phase selective enrichment is a key step: the growth of Listeria bacteria, sometimes stressed or associated with an abundant annex competing flora, can be hindered during the enrichment phase, thus leading to an under-estimation of the food contamination. The laboratory has already investigated the influence of the type of Listeria species/strains, of the annex flora and of the growth rate of each strain. The CRL (Unit HMPA) will go on in 2008 to study the influence of other factors, such as the bacterial stress and lag-time, the nutritional competition and the composition of enrichment media. In addition, the question of low numbers will be taken into account, investigating the growth of individual cells in broth, based on a model of predictive microbiology. c. Confirmation stage of the Standard methods Depending on the direction taken by the ISO/CEN working group for the modification of the confirmation stage of the Standards EN ISO 11290-1&2, the CRL (Unit HMPA) may have to conduct further trials to contribute to define the confirmation stage in the standard methods under revision. d. Environnemental sampling techniques After reviewing the ISO 18593 Standard, the CRL (Unit HMPA) intends to initiate in 2008 a bibliographic study in order to check whether the ISO Standard fully cover the case of


L. monocytogenes control in the environment of food production and food handling, or whether there would be a need to add to the ISO Standard specific guidance on sampling techniques for L. monocytogenes control.

1.3 Training of the NRLs (annual) The CRL will organize a training session for the NRLs on the estimation of measurement uncertainty, based on the Technical Specification ISO/TS 19036, as well as on the interpretation of results. This session will be organized in conjunction with the annual 2008 workshop.

2. Predictive microbiology

Frame: The EC Regulation 2073/2005 on microbiological criteria defines a quantitative limit for L. monocytogenes of 100 cfu/g, which is applicable to certain categories of products placed on the market during their shelf-life. The manufacturer needs to be able to demonstrate that the product will not exceed the limit of 100 cfu/g throughout the shelf-life. For that purpose, Annex II of the regulation lists the different types of data and studies that can be used.

In order to implement Annex II of Regulation 2073/2005, the CRL envisages to conduct the following works.

2.1 Guidelines to conduct challenge tests and durability studies (multi-annual)

A guide for (i) the implementation of durability studies and challenge tests and (ii) their interpretation as regards to L. monocytogenes criteria is developed by the CRL (Unit MQER) in collaboration with some NRLs, and is scheduled to be released by the end of 2007. In 2008, it may be necessary to prepare a 2nd version of the guide, detailing some aspects if necessary, such as the food classification as regards to the potential of L. monocytogenes growth. In addition, the CRL intends to assist the NRLs in supporting the national operators to implement the guide.

2.2 Expertise of challenge tests/durability studies (multi-annual)

If such a need would arise from NRLs or from DG SANCO, the CRL (Unit MQER) would give an opinion on challenge tests/durability studies conducted in MSs.

2.3 Training package (multi-annual)

The CRL (Unit MQER) intends to initiate the development of an English-language audio-visual training package on shelf-life of foods related to L. monocytogenes. This package would target food business operators and/or official control services. In 2008, a PowerPoint presentation will be at least prepared. The CRL intends to propose it to the NRLs for their own use, in particular during the training session scheduled in 2008.


2.4 Training of the NRLs (annual)

The CRL (Unit MQER) will organize for the NRLs a 3-day theoretical and practical training session on:

- durability studies; - challenge tests; - predictive microbiology softwares; - shelf-life of foods related to L. monocytogenes.

3. Characterization and typing of strains, epidemiosurveillance

Frame: In the DG SANCO support document to the call for the selection and designation of the new CRLs (SANCO/2214/2005), the Annex 1 describes the specific functions of the CRL L. monocytogenes, which includes to keep abreast of developments in Listeria epidemiology and to cooperate, as appropriate, with the Community structures involved into surveillance of Listeria.

3.1 Dispatch of strains

(multi-annual) Upon request of the NRLs, the CRL (Unit CEB) would send them L. monocytogenes field strains from its collection, as well as the control strain Salmonella Branderup H9812.

3.2 Training of the NRLs (multi-annual)

The CRL (Unit CEB) will organize in 2008 a 1st theoretical and practical training session of 5 days for the NRLs on L. monocytogenes subtyping by PFGE.

3.3 Investigation of recent molecular typing techniques (multi-annual)

The CRL (Unit CEB) will launch a study for the evaluation of recent molecular typing techniques, mainly Multi-Locus Sequence Typing (MLST), in comparison with PFGE. These new techniques may replace PFGE in the future as reference technique. The CRL will use MLST in addition to PFGE for some investigations.


4. Workshop of the NRLs (annual) The CRL will organise the 2nd Workshop of the NRLs in 2008, of general scope : - to make a progress report on works undertaken by the CRL since the 1st 2007 Workshop; - to envisage the work programme for 2009 and further.

5. Technical and scientific assistance to the European Commission

5.1 DG SANCO activities (multi-annual)

Upon request of the services of DG SANCO in charge of food hygiene: - If needed, participation of the CRL coordinator, for the analytical aspects, to the update of

Regulation 2073/2005 on microbiological criteria related to L. monocytogenes; - and any new question which may arise during the year.

5.2 Participation to CEN/ISO standardization and Codex activities (multi-annual)

On behalf of the CRL and as EC representative: - Participation to the activities and to the joint plenary meeting of ISO/TC 34/SC 91 &

CEN/TC 275/WG 62 for aspects related to the standardization of reference methods for L. monocytogenes;

- Participation to the ad’hoc group of ISO/TC 34/SC 9-CEN/TC 275/WG 6 on L. monocytogenes, in charge of the revision of the Standard reference methods EN ISO 11290-1&2 (1 meeting);

- Participation to the WG Listeria of the Codex Committee on Food Hygiene (1 meeting).

1 Sub-Committee 9 « Microbiology » of Technical Committee 34 « Food products » 2 Working Group 6 « Microbial Contaminants » of Technical Committee 275 « Food analysis – Horizontal methods »

COMMUNITY REFERENCE LABORATORY FOR COAGULASE POSITIVE STAPHYLOCOCCI

WORK PROGRAMME 2008

CRL “CPS” 1/5 12/07/07 2008 Programme of Work


EU COMMUNITY REFERENCE LABORATORY FOR

COAGULASE POSITIVE STAPHYLOCOCCI

2008 Programme of Work of the Community Reference Laboratory for

Coagulase Positive Staphylococci (including Staphylococcus aureus and their toxins)



Introduction In May 2006, the laboratory LERQAP (Laboratory for Studies & Research on Quality of Foods & on Food Processes) of AFSSA (French Agency for Food Safety) has been nominated Community Reference Laboratory (CRL) for Coagulase Positive Staphylococci (CPS), including Staphylococcus aureus and their toxins (see Regulation 776/2006). The CRL foresees to undertake the following actions in 2008, according to the actions planned at the 1st Workshop of the National Reference Laboratories (NRLs) (7&8 June 2007), as well as to the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the CRL for the period 2006-2010. Most of these activities aim at implementing, from an analytical point of view, the new EC Regulation 2073/2005 on microbiological criteria for foodstuffs, which includes in particular:

• 5 process hygiene criteria on CPS, defining a quantitative limit in: o cheeses made from raw milk or from heat-treated milk, ripened cheeses, and

unripened soft cheeses, o milk/whey powder, o cooked crustaceans and molluscan shellfish.

• 1 food safety criterion on staphylococcal enterotoxins (SETs), requiring absence in 25 g in cheeses, milk/whey powder, to be tested when CPS enumeration is higher than 105 cfu/g when testing the above mentioned criteria on CPS.


1. Detection/enumeration of coagulase positive staphylococci in food

Frame: The Standard methods EN ISO 6888-1 or 2 are cited as reference methods in the quantitative criteria of EC Regulation 2073/2005 for CPS.

1.1 Study of sample types used for inter-laboratory trials

(annual) As to be able to organize a PT trial on another matrix than liquid milk, the CRL (Unit HMPA) will conduct in 2008 an investigation study as to find a way, such as the addition of a chemical stabilizer, to stabilize sufficiently the CPS contamination of a cheese matrix, in order to prepare and dispatch the samples used for quantitative determinations. It is intended to select a formula adapted to CPS; formula which would allow the bacteria to grow on plates after the dilution steps.

1.2 Improvement of the reference method (confirmation step) EN ISO 6888-1 (multi-annual) As to improve the reference method of EN ISO 6888-Part 1, using BP medium, the CRL (Unit HMPA) will complete its investigation launched in 2007 of an alternative confirmation procedure: a stabbing test on RPF agar. This could be used as an optional confirmation procedure, in alternative to the coagulase test in tubes of the current EN ISO 6888-1, that would be easier, quicker and less expensive to perform than the coagulase test.


In case of a positive outcome of the study, the CRL would propose to ISO/TC 34/SC 9 to introduce this optional confirmation in the Standard EN ISO 6888-1.

2. Characterization and typing of strains, epidemiosurveillance

Frame: In the DG SANCO support document to the call for the selection and designation of the new CRLs (SANCO/2214/2005), the Annex 1 describes the specific functions of the CRL CPS, which includes to keep abreast of developments in CPS epidemiology and to cooperate, as appropriate, with the Community structures involved into surveillance of CPS.

2.1 Dispatch of strains

(multi-annual) Upon request of the NRLs, the CRL would send them CPS field strains from its collection.

2.2 Optimisation of molecular typing by PFGE (multi-annual) The CRL (Unit CEB) will optimise the method currently used for PFGE sub-typing of CPS strains. Once optimised, the CRL will dispatch the method to the NRLs.

2.3 Development of SE genes detection by multiplex PCR (multi-annual) The CRL (Unit CEB) intends to launch the development of a multiplex PCR technique to detect the main SET coding genes in the strains isolated.

2.4 Investigation of recent molecular sub-typing techniques (multi-annual)

The CRL (Unit CEB) will launch a study for the evaluation of recent molecular typing techniques, mainly Multi-Locus Sequence Typing (MLST) and spa typing, in comparison with PFGE sub-typing. These new techniques may replace PFGE in the future as reference technique. The CRL will use MLST and spa typing in addition to PFGE for some investigations.


3. Detection of staphylococcal enterotoxins in food

3.1 Inter-laboratory PT trial for the NRLs (annual) The inter-laboratory PT trials organised by the CRL for the NRLs aim at evaluating the ability of the NRLs to apply satisfactorily the methods for the analyses performed in the frame of controls prescribed by Regulation 2073/2005. The CRL (Team TOP-BAC) will organize in 2008 an inter-laboratory trial on SET detection in cheese, using the European screening method (under revision, see below). For the samples, the same protocol (freeze-drying of SET spiked cheese matrices) than the one used to fully validate the Vidas SET2 detection kit will be used as the homogeneity and stability studies gave satisfactory results.

3.2 Improvement of the official screening method (multi-annual)

Frame: The European screening method of the CRL Milk/CPS is cited as reference method in the criterion for SETs by the EC Regulation 2073/2005.

The screening method, used for the own checks made by the food producers and for the official controls, has been developed by the laboratory and adopted as European screening method (ESM) in the frame of the former mandate of the CRL “Milk & Milk Products”. At the 1st Workshop of the NRLs “CPS” (7&8 June 2007), it has been decided to modify in 2007 the ESM version 3 of the CRL “Milk” into the ESM version 1 of the CRL “CPS”, including a paragraph dealing with interferences. Given the serious analytical difficulties specifically linked to the SET detection, this method still needs to be improved. After the past two past years dedicated to the improvement of the detection step, it is necessary to put the main emphasis on the improvement of the extraction step, at first for dairy products. In 2008, the CRL (Team TOP-BAC) will finalise the investigation of an alternative to dialysis-concentration, the use of trichloacetic acid, for the extraction of staphylococcal enterotoxins in milk and milk products.

3.3 Evaluation of commercially available antibodies against SEs (multi-annual)

In order to be able to transfer to the NRLs a confirmatory method, the CRL (Team TOP-BAC) will launch the evaluation of commercially available antibodies to perform a quantitative and specific ELISA test to detect SEA to SEH in milk-based products. Mice monoclonal antibodies will be used as capture antibodies and rabbit polyclonal antibodies coupled to horseradish peroxidase as probing antibodies. Once the characterization study conducted, the CRL will provide the NRLs with the instructions for use.


3.4 Training of the NRLs (annual) The CRL (Team TOP-BAC) will organize one (or two, depending on the needs) training session(s) on the detection of SETs, especially for the new NRLs CPS (not formerly NRLs Milk).

4. Workshop of the NRLs (annual) The CRL will organise the 2nd Workshop of the NRLs in 2008, of general scope : - to make a progress report on works undertaken by the CRL since the 1st 2007 Workshop; - to envisage the work programme for 2009 and further.

5. Technical and scientific assistance to the European Commission

5.1 DG SANCO activities (multi-annual)

Upon request of the services of DG SANCO in charge of food hygiene: - If needed, participation of the CRL coordinator, for the analytical aspects, to the update of

Regulation 2073/2005 on microbiological criteria related to CPS and SETs; - and any new question which may arise during the year.

5.2 Participation to CEN/ISO standardization activities (multi-annual)

On behalf of the CRL and as EC representative, follow-up by the CRL coordinator of the activities of ISO/TC 34/SC 91 & CEN/TC 275/WG 62 for aspects related to the standardization of reference methods for CPS and SETs (1 jointed plenary meeting –budget CRL L. monocytogenes). In particular, participation to the works of: - 2 working groups of ISO/TC 34/SC 9 of specific interest for the CRL activities and for

DG SANCO: WG 3 “Method Validation” (2 meetings) and WG 4 “Proficiency Testing” (1 meeting);

- 1 working group of CEN/TC 275/WG 6, in charge of the standardization of a reference method for the detection of SETs in food (group to be convened by Jacques-Antoine HENNEKINNE, Team Leader TOP-BAC) (1 meeting).

1 Sub-Committee 9 « Microbiology » of Technical Committee 34 « Food products » 2 Working Group 6 « Microbial Contaminants » of Technical Committee 275 « Food analysis – Horizontal methods »

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

(VTEC)

WORK PROGRAMME 2008

of 8

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

Unit of Foodborne Zoonoses and Veterinary Epidemiology Istituto Superiore di Sanità

Community Reference laboratory (CRL)

for Escherichia coli,

including Verotoxigenic E. coli (VTEC)

Work Program

1st January - 31st December, 2008

of 8

Introduction

The work programme of CRL for VTEC (CRL-VTEC) for the year 2008 will consist of

the following activities:

1. Consolidating the CRL structures

1.1. Accreditation of the quality assurance system 1.2. Phage-typing of VTEC O157

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

2.1. Development of a consensus PCR method for vtx gene subtyping 2.2. Annual workshop 2.3. Assistance to NRLs 2.4. Training

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

4.1 Enter-net, the Community network for human VTEC infections

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

5. Inter-laboratory comparison studies 5.1. 5.1. 2nd inter-laboratory study with the NRLs

5.2. 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 2008 or multi-

annual (ongoing program).

1. Consolidating the CRL structures 1.1. Accreditation of the quality assurance system

At present, the CRL-VTEC has presented a formal request to the Italian body for

accreditation (SINAL) to obtain the accreditation (UNI EN/ISO 17025) of its quality

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assurance system. The visit by SINAL should occur within the end of the year 2007

and the UNI EN/ISO 17025 accreditation should therefore be obtained.

Accreditation will include the following methods:

• Horizontal method for microbiological detection of VTEC O157 (UNI EN/ISO

16654).

• PCR detection of VTEC virulence genes (internal method). • Determination of the serogroup of VTEC strains, with respect to the serotypes

mainly involved in severe human infections (internal method).

During 2008, the CRL-VTEC will consolidate its quality assurance system and will

submit additional methods for accreditation.

Duration: ongoing

1.2. Phage-typing of VTEC O157

The inventory of the activities of the E.coli NRLs in EU countries performed in 2007

has shown that at present there are no laboratories performing phage-typing of

VTEC O157 in the field of food safety and veterinary public health. Considering the

epidemiologic value of phage-typing, the CRL-VTEC is planning to introduce this

technique into its array of typing methods. The phages will be purchased from the

Laboratory of Enteric Pathogens of the Health Protection Agency (HPA), Centre for

Infections, Colindale, London, which represents the reference laboratory for both

salmonella and VTEC O157 phage-typing. A person from ISS will spend a 3-weeks

visit at the HPA Centre for Infections, to learn how to perform the technique, with ISS

covering the related expenses. This person will then be included in the CRL-VTEC

organization chart as a part-time temporary employee to establish the technique at

the CRL. Once phage-typing will be established, the NRLs could send

representative subsets of the VTEC O157 strains isolated in their countries from

different sources for typing. The shipment of the strains from each NRLs could be

made periodically to contain the cost of the shipments.

Such a project could allow the comparison of the phage-types of VTEC O157

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

throughout Europe, as determined by a fully harmonized method.

Duration: ongoing

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

training 2.1. Development of a standard PCR method for vtx gene subtyping There are two major types of VTs, encoded by vtx1 and vtx2 genes. Several variants

or alleles of VT2 and, in a lesser extent, VT1 have been described. Epidemiological

studies have revealed that strains associated with severe human disease, like bloody

diarrhoea and HUS, produce VT2 more frequently than VT1. Moreover, the different

vtx2 genotypes seem to be associated with different clinical manifestations:

• vtx2 and vtx2c are the most frequently found genotypes in human infections and

are usually associated with HUS;

• vtx2d is also isolated from human infections, but has been mainly found in cases

of non bloody diarrhoea and asymptomatic carriers, while association with HUS

has been reported in a few cases.

• vtx2e vtx2f, typically associated with strains from pigs and birds, respectively,

have been rarely found in human infections

A number of primer sets able to detect vtx genes has been developed during the

years and there is a need for consensus protocols for vtx genes typing. The CRL

VTEC will develop a standard PCR method for vtx gene sub-typing, in collaboration

with the WHO International Escherichia and Klebsiella Centre at the Statens Serum

Institut in Copenhagen, which act as reference laboratory for Enter-net, the network

for surveillance of VTEC infections in humans. Such a project could allow the

comparison of the vtx gene types of VTEC strains isolated from human infections and

from non-human sources throughout Europe, as determined by a fully harmonized

method. Moreover, vtx gene typing could be a tool for predicting the virulence of

VTEC strains isolated from food and therefore could represent a parameter to be

considered in risk analysis.

Duration: ongoing

2.2 Annual workshop with NRLs The 3rd annual workshop will be held in the second half of 2008 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 second inter-laboratory study on

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VTEC identification and typing performed in the first half of the year will be presented

and discussed, together with the draft standard PCR method for vtx gene subtyping.

The training programme for the benefit of NRLs will be discussed as well and plans

for the following year will be established according to the NRLs needs.

2.3. Assistance to NRLs

CRL-VTEC will continue to assist NRLs by provision of methods via the web site,

standard operating procedures, reference strains. In particular, the draft standard

PCR method for vtx gene subtyping will be prepared and distributed (see point 2.1).

Drafts of other standard operating procedures for detection of 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.

CRL-VTEC will perform typing of selected strains of E.coli upon request from NRLs

and, if needed, it may visit NRLs to help in solving problems.

Duration: ongoing (as required) 2.4. 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 (as required) 2.5. Maintaining the European database for VTEC strains isolated by NRLs

The European database for VTEC strains isolated from food and from animals will be

agreed during the 2nd workshop in December 2007 and data collection should start by

January 2008. This database will collect on a regular basis the data on the strains

isolated by NRLs and will be harmonized with the Enter-net network database for

human isolates, maintained by the ECDC, to allow an easy comparison of the data.

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.

A catalogue of the consolidated diagnostic methods as well as of the innovative

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molecular techniques available at CRL-VTEC for detection and typing of E.coli

isolates will be published on the CRL web-site.

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. The following liaisons will be implemented:

4.1 Enter-net, the Community network for epidemiological surveillance and control of salmonella and VTEC infections.

The CRL-VTEC will participate in the 2008 Enter-net workshop in order to harmonize

the respective future proficiency tests on strain typing and strain databases.

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

The coordination of the validation study of the method EN/ISO 16654 for E.coli O157

in foodstuffs that has been assigned to the CRL-VTEC and will be carried out, upon

fund availability from CEN.

The CRL-VTEC, as co-leader of the project involving the VTEC/STEC ad hoc group

charged with the drafting of the “Standardized PCR-based horizontal method for the

detection of Shiga toxin producing E.coli other than E.coli O157 in food and animal

feeding stuffs”, will continue to manage the discussion and will present a draft

proposal at the next WG6 plenary meeting.

4.3 MED-VET-NET, the European Network of Excellence for research on the

prevention and control of zoonoses. The collaboration with several institutes on the virulotyping of VTEC will continue

within the framework of the MED-VET-NET Work-Package no. 26

(http://www.medvetnet.org/cms/templates/doc.php?id=64), of which Dr. Morabito is

the deputy coordinator.

4.4 The Pathogenic E. coli Network (PEN). The collaboration with this EU coordination action will continue. In particular, the CRL-

VTEC will host the 3rd PEN meeting on VTEC pathogenesis and virulence, which will

take place in Rome on 6-8 March.

Duration: ongoing

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5. Inter-laboratory comparison studies

5.1. 2nd inter-laboratory study with the NRLs The 2nd inter-laboratory study will presumably still be focused on VTEC identification

and typing and will be calibrated on the basis of the results of the 1st study that is still

ongoing. It will be performed in the first half of the year and the results will be

presented and discussed at the annual workshop. The program of the study will be

discussed and harmonized with the Enter-net surveillance network.

5.2. Validation and revision study of the standard method EN/ISO 16654 for E.coli O157 in food and feeding stuffs

The project for the validation and revision study of the standard horizontal method for

the detection of Escherichia coli O157 in food and feeding stuffs (EN/ISO 16654) has

been approved by CEN (see point 4.2). Upon availability of funds by CEN, the study

will be carried out within 2008, and several NRLs will be involved in the study (see

point 4.2).

Duration: ongoing

6. Research on VTEC accessory virulence factors

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

Elements in VTEC will continue. The aim of these investigations is the complete

definition of the virulence genes that make a VTEC strain fully pathogenic to humans

and that could represent suitable targets for diagnostic assays aimed at detecting the

most virulent VTEC clones, beside E.coli O157, in foodstuffs and live animals.

The findings obtained so far by comparing VTEC O157 strains by microarray

analyses will be completed by investigating the polymorphic region identified on the

VT2-converting phages.

Long-PCR tools will be deployed to investigate the distribution of such

polymorphisms among VTEC O157 strains belonging to different phage-types. The

same approach will be applied to VTEC O157 strains isolated from patients

presenting different levels of disease severity, chosen among those present in the

CRL-VTEC culture collections. The study will include strains from cases of hemolytic

uremic syndrome, hemorrhagic colitis, and uncomplicated diarrhea.

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Afterward, the study will be extended to other VTEC isolates belonging to the sero-

pathotypes from A through E, as described by Karmali et. al (J Clin Microbiol 2003,

41:4930-40). With this respect, a protocol to perform these long PCR will be prepared

and used for typing a large number of strains. Finally, the polymorphic regions

identified, when meaningful, will be sequenced and investigated at the nucleotide

level to identify possible markers to be used as tools for early detection of VTEC with

significant impact on human health.

Duration: ongoing

7. Missions The following missions may be needed in 2008:

• Participation of a scientist at the 2008 CEN/TC275 WG6 annual plenary

meeting.

• Participation of a scientist at the WG6 TAG3 general meeting, which is planned

to be held in Sweden in January 2008. • Two visits to NRLs can be planned for 2007, upon agreement with the EC and

the interested countries.

August 24th, 2007

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

COMMUNITY REFERENCE LABORATORY FOR CAMPYLOBACTER

WORK PROGRAMME 2008

CRL- CAMPYLOBACTER WORK PROGRAMME FOR 1st OF JANUARY 2008 TO 31st OF DECEMBER 2008 Introduction The activities in the working programme for 2008 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 2008 will consist of the following key activities which will be repeated annually:

1. Organisation of a ring test (interlaboratory comparison study) for the National Reference Laboratories for Campylobacter in the EU Member States.

2. Organisation of a work shop for the NRLs for Campylobacter in the MS 3. Perform research within the Campylobacter area 4. a) Assist the European Commission with scientific and technical advice on questions

concerning Campylobacter b) Assist the EC and the NRLs for Campylobacter with Ad hoc issues

5. Communication 1. Organisation of a ring test in 2008 In 2007, two ring tests were organised. The first one was voluntary, it was not included in the work programme for 2007, and could be regarded as a test of practicalities involved with sending samples and communication between the NRLs and the CRL. The second ring test was included in the 2007 work programme. The purpose was to detect and identify Campylobacter in broiler caecal samples inoculated or naturally contaminated with Campylobacter. The reason for choosing caecal samples as matrix in the ring test, was the planned EC baseline study of the prevalence of Campylobacter in broiler flocks and broiler carcasses (Commission Decision of 19 July 2007, 2007/516/EC). There is no standardised reference method for detection of Campylobacter in broiler faeces material, but basically the ISO 10272-1: 2006 method for detection of Campylobacter in food and animal feeding stuffs should be applied for the baseline study. For the 2007 ring test on caecal samples, a similar protocol was applied to evaluate the performance of the NRLs using this type of matrix and protocol. Also the ring test in 2008 will relate to the abovementioned baseline survey, in this case to perform enumeration of Campylobacter in broiler carcasses. In the baseline study, neck skin and skin of the carcasses shall be analysed for presence and numbers of Campylobacter using ISO 10272-2: 2006 “Microbiology of food and animal feeding stuffs – Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique”. The planned ring test will consist of broiler neck skins inoculated with two or three different concentrations of Campylobacter and the ISO 10272-2:2006 shall be applied for

the analysis. The CRL will have a training course for the NRLs on enumeration of Campylobacter in broiler carcasses in November 2007, using the same ISO protocol. The baseline study will start in January 2008, and the ring test in 2008 will therefore be a good way to evaluate the performance of the NRLs for doing the enumeration analysis. The ring test is planned to be distributed in the fall 2008. More information about the reference materials for the ring tests is given under point 3 (Research). 2. Organisation of a workshop The workshop in 2008 will be organised in the fall. Representatives from the Member States´ NRLs for Campylobacter will be invited. The EU Candidate Countries (financed by TAIEX) and Norway and Switzerland (on their own budgets) will also be invited to participate. Further, experts from EC, 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. Results of zoonosis monitoring (EFSA), baseline study in broilers (EC) and monitoring and control of Campylobacter in humans (ECDC).

- Campylobacter activities in MS will be presented, including monitoring and research studies

- Results of ring tests will be presented and discussed - Information about ring tests/ comparative tests to come - Information from meetings with ISO/TC34/SC9 and CEN/TC 275/WG6 - Future CRL- NRL collaboration and activities will be discussed

3. Research 3.1. Studies related to ring tests. Bacterial and matrix reference materials. 3.1.1. For the first ring test (2007), lenticule discs containing ~103 cfu of Campylobacter/disc were purchased and tested. However, the level of Campylobacter was lower than indicated and the discs were not found suitable for the ring test. Alternative methods, i.e. to prepare chicken isolates to be used as bacterial reference material, will therefore be investigated for the ring test in 2008. The possibility to make stable and freeze- dried preparations of known concentrations of animal isolates of Campylobacter will be investigated. 3.1.2. Stability of matrices. Chicken matrices inoculated with different concentrations of Campylobacter will be tested for stability/Campylobacter survival at +4 ºC and –20 ºC. Before the ring test in 2008, homogenates of broiler neck skins, both naturally contaminated neck skins and neck skins inoculated with Campylobacter will be tested. 3.2. Other 3.2.1. Quantification of Campylobacter in chicken caeca. It has been suggested that a high level of Campylobacter in chicken caeca constitutes a high risk for heavy contamination of broiler carcasses at slaughter. This, in turn, implies a higher risk for the consumer to become infected with Campylobacter. Today there is no standardised, validated method available for quantification of Campylobacter in chicken

caeca. However some NRLs are testing protocols for estimating levels of Campylobacter in chicken caeca at flock level and at individual bird level, respectively. The CRL will review and test methods and protocols in use and plan an interlaboratory comparison study with a limited numbers of NRLs using methods that seem most appropriate. The intention would be to organise such a study in 2009. 3.2.2. DNA-based, and other methods for detection, species identification and strain characterization of Campylobacter. Many NRLs are performing PCR-based methods, including realtime PCRs for detection and identification of Campylobacter. The CRL will continue to review methods and in collaboration with the NRLs plan a voluntary comparative study of PCR-based methods. Rapid and automated methods for detection and characterization of Campylobacter are of interest to investigate. In 2007, the CRL has been involved in a pilot study, using mass spectrometry for identification and strain characterization of Campylobacter. The study will continue in 2008. For subtyping of strains, the CRL will continue to perform pulsed- field gel electrophoresis (PFGE) following restriction with SmaI using the Campynet protocol (http://campynet.vetinst.dk/PFGE.html). The method is used for routine subtyping of isolates and proved valuable in several studies, for example of Campylobacter epidemiology in broiler production. A Swedish surveillance programme for Campylobacter in broilers has been carried out since 1991. The National Veterinary Institute (SVA) as a NRL- Campylobacter, performs the analyses of samples within the programme. This means that the CRL in collaboration with the NRL has access to a great number of Campylobacter isolates from broilers and broiler environment for investigation in studies on validation and comparison between analytical methods. 3.2.3. Participation in international research networks etc. In 2008, CRL staff members will participate in:

- EU funded network Med-Vet-Net, WP 24 “Development of a European consensus frame work on risk assessment of Campylobacter in broiler meat”, WP 28 ”Methods of attributing human Salmonella and Campylobacter infections with different food and food animals”, WP 30 “Towards a combined microbiological and epidemiological approach for investigation of host-microbe interactions of C. jejuni” (Campynet III), and WP 33 “Early host responses to Salmonella and Campylobacter” (EOE and IH)

- Safefoodera project “Foodborne zoonoses – Campylobacter and E. coli – a

network project” (CampEc-NET). WP 1 “Molecular epidemiology of Campylobacter: Comparing European regions” and WP 3 “Role of specific immunity in risk reduction of human infections during exposure to Campylobacter” (EOE).

- ISO /TC working group on validation and revision of EN/ISO 10272- 1 and 10272-2 and in a working group on ISO 17604- Microbiology of food – carcass sampling (IH/EOE)

- As Consultant (IH) on Defra project OZ0610 “Survival and persistence of

campylobacters in poultry farm environments and suggested control measures”.

- In national and international seminars and research meetings on zoonotic

issues including Campylobacter in order to keep updated on new methods and developments in the area and on Campylobacter epidemiology.

4. Assistance to the European Commission and the NRLs including ad hoc activities The CRL will provide scientific and technical advice to the Commission and the NRLs on issues regarding Campylobacter. It is anticipated that there will be many questions in relation to the baseline study on Campylobacter in broilers and broiler carcasses (2007/516/EC) that will take place in 2008 in the MS. For quality assurance, a maximum of 16 Campylobacter spp isolates per MS from the baseline study shall be sent to the CRL- Campylobacter for confirmation and speciation. A maximum of 8 caecal and 8 carcass isolates per MS shall be submitted. The CRL has prepared a routine for this activity, including analysis by phenotyping and molecular identification, and reporting and storage of strains. Request from the NRLs and the Commission for support and advice will be handled by the CRL scientific staff as efficiently as possible. Assistance to the Commission and EFSA services will have priority. 5. Communication. The CRL- Campylobacter webpage will be continuously updated in order to give relevant information about the CRL and activities such as work shops, work programmes, ring tests and reports. A list of NRLs- Campylobacter with contact details will be provided. The CRL will cooperate with EU Commission Services and other organisations and authorities working in the field of human and animal health.

COMMUNITY REFERENCE LABORATORY FOR PARASITES (IN PARTICULAR TRICHINELLA,

ECHINOCOCCUS AND ANISAKIS)

WORK PROGRAMME 2008

Community Reference Laboratory for Parasites

Istituto Superiore di Sanità

page 1 of 8

COMMUNITY REFERENCE LABORATORY

FOR

PARASITES

WORK PROGRAMME

2008



page 2 of 8

CRL for Parasites work programme 2008

Introduction The 2008 working programme of CRL for Parasites (CRLP) consists of the following activities: 1. Ad hoc activities 1.1 Trichinella

o To increase and maintain the serum bank of Trichinella-infested pigs (multi-years)

o To establish a Trichinella-positive standard pig serum

o To increase and maintain the serum bank of Trichinella-infested humans (multi-years)

o To produce reference Trichinella antigens for serology (multi-years)

o Maintenance of reference strains of Trichinella in vivo (multi-years)

o Diagnostic activity with both accredited and non-accredited tests (multi-years)

o Collection of serum and/or meat juice samples from wild boars and foxes (multi-years)

1.2 Anisakidae worms

o To increase and maintain the collection of Anisakidae worms and/or their DNAs (multi-years)

o To prepare plasmids containing ITS regions from Anisakis spp. and ITS2 region from Pseudoterranova spp.

1.3 Echinococcus

o To establish a genetic bank of cestodes of the genus Echinococcus (multi-years)

2. Research 2.1 Barcoding of zoonotic and non zoonotic helminths and protozoa parasitizing domestic

animals and foodstuffs (multi-years)

2.2 Identification of proteins specific at the oocyst stage of Toxoplasma gondii (multi-years).

2.3 Identification and development of analytical methods for the speciation of parasites of the family Anisakidae (one year)

2.4 Identification of intraspecific genetic variability of Trichinella spiralis and T. britovi (multi-years)

2.5 Identification and development of analytical methods for the speciation of parasites of the genus Echinococcus (multi-years)



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2.6 Collection of epidemiological data on the prevalence of the trematode worm Alaria alata in wild boar populations of MS

3 An interlaboratory comparison study

4 Workshop

5 Visit to NRLs

6 Training for Personnel of NRLs and from developing countries 7 Further development and up to date of the web site of the CRL for parasites



page 4 of 8

1. Ad hoc activities 1.1 Trichinella 1.1.1 To increase and maintain a serum bank of Trichinella-infested pigs (multi-years)

Serum samples will be collected from Trichinella-infested pigs, pigs infested and/or infected with other parasites and from domestic pigs known to be Trichinella-free. All samples will be tested by the validated ELISA, distributed in aliquots, lyophilised and stored at +4°C. The database of the serum bank will be up to dated. Pig serum samples from different world regions and pig races (infested and not infested with Trichinella) will be collected, in order to get control sera and to up to date the most appropriate cut-off which will be useful for serological studies carried out on different swine races. If the CRL will receive a high request of reference serum samples, SPF pigs will be experimentally infested with Trichinella spiralis larvae. Before the infestation, sera will be collected from each pig. After the infestation, the kinetics of anti-Trichinella antibodies will be screened and, when the serum conversion will be detected (approximately 20-25 days p.i.), pigs will be sacrificed and sera will be collected, tested, distributed in aliquots and lyophilised.

1.1.2 To establish a Trichinella-positive standard pig serum

A freeze-dried preparation of pooled swine sera containing immunoglobulin G (IgG) antibodies against Trichinella spp, will be prepared. The material will be proposed as an International Standard containing a number of arbitrary units of anti-Trichinella IgG for use in quantitative assays to determine the anti-Trichinella IgG in swine. A panel of sera from Trichinella-positive and negative swine will be tested with different tests in different laboratories using the pooled swine sera as a reference standard. In addition to EU laboratories, three laboratories from Canada, USA and Switzerland will be invited to joint this project to evaluate the standard serum.

1.1.3 To increase and maintain the serum bank of Trichinella-infested humans

Serum samples will be collected from infected people during trichinellosis outbreaks occurring in different European countries or outside of Europe. Serum samples from people with a confirmed diagnosis of trichinellosis will be tested by ELISA, distributed in aliquots, lyophilised and stored at +4°C. The database of this serum bank will be up to dated.

1.1.4 To produce reference Trichinella antigens for serology

Excretory/secretory (E/S) antigens will be produced from Trichinella larvae in order to supply to each laboratory within EU the reference antigen for diagnostic purposes.

1.1.5 Maintenance of reference strains of Trichinella in vivo

Reference strains for each species or genotype of Trichinella identified so far, will be maintained in laboratory animals. Fresh mouse carcasses infested with Trichinella species/genotypes will be provided to laboratories for training of



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personnel and for typing of wild isolates. Trichinella larvae from reference strains will be stored in ethyl alcohol and forwarded to laboratories as reference material for typing of wild isolates. To increase the quality and the reliability of the maintenance of these reference strains in vivo, the identity of each infected mouse will be monitored by a microchip which will be inserted under the skin.

1.1.6 Diagnostic activity Diagnostic samples provided from NRLs will be tested with the most appropriate tests:

a. Accredited tests: i. Identification of parasites of the genus Trichinella by a multiplex-PCR

analysis ii. Identification of anti-Trichinella antibodies in swine sera

b. Non-accredited test: i. A number of other diagnostic tests are available at the CRLP for the

diagnosis of other foodborne parasitic infections in food and animals 1.1.7 Collection of serum and/or meat juice samples from wild boars and foxes

Samples will be collected from wild boars and foxes of MS with the aim to establish serum banks which will be used to evaluate the usefulness of serology to monitor the Trichinella sp. infection in wildlife.

1.2 Anisakidae worms

1.2.1 To increase and maintain the collection of Anisakidae worms and/or their DNAs Reference larvae and/or DNA will be directly collected from naturally infected fish or will be request to European and extra-European laboratories skilful on this matter.

1.2.2 To prepare plasmids containing ITS regions from Anisakis spp. and ITS2 region from Pseudoterranova spp.

The protocol is based on selective PCR amplification of the entire ITS region for Anisakis spp followed by endonuclease digestion with either HinfI or HhaI restriction enzymes. In order to have a collection of reference ITS region from the different Anisakis species, the ITS fragments obtained by PCR from the DNA of reference larvae will by cloned in plasmid vector, introduced in bacteria, verified by sequencing and the transformed bacteria as well as the purified plasmid will be stored at -80°C. The plasmid material could be used as positive control in PCR assay and shared with NRLs for the setting of the PCR-RLFP identification methods

1.3 Echinococcus 1.3.1 To establish a genetic bank of cestodes of the genus Echinococcus.

Most species and genotypes belonging to the genus Echinococcus (Cestoda) show different hosts and transmission patterns which play an important role in their



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epidemiology. The main species circulating in Europe are E. multilocularis, E. ortleppi, E. granulosus and several related genotypes. There is the need to develop a molecular epidemiology for the evaluation of the transmission patterns and the risk assessment in the member states. On this purpose, DNA from adult, larval and egg stages will be collected to establish a genetic bank of cestodes of the genus Echinococcus which will be useful for the development of molecular identification tests.

2 Research 2.1 Barcoding of zoonotic and non zoonotic helminths and protozoa parasitizing

domestic animals and foodstuffs. Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. The prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon ‘barcodes’. DNA barcoding - the recently proposed DNA-based project for species identification, has attracted much attention. A short DNA fragment can be used to diagnose taxa, increasing the speed, objectivity, and efficiency of species identification. Initial tests of genetic barcoding using mitochondrial markers on animals reported near-100% accuracy, indicating that the method can be highly accurate under certain conditions. Accurate species identification—assignment of an unknown to a known—requires a comprehensive comparative molecular database against which unknowns can be compared. DNA Barcoding has two essential goals: (1) differentiate the species (diagnostic role); (2) discover new species (investigative role).

2.2 Identification of proteins specific at the oocyst stage of Toxoplasma gondii. The

increasing awareness of the risk of toxoplasmosis in both food animals and humans, along with the difficulty of detecting T. gondii oocysts in cat faecal samples and in environmental samples, calls for the production of specific reagents for the development of an efficient and standardized procedure. To this aim, we plan to produce monoclonal antibodies (Mabs) recognizing T. gondii oocyst wall proteins (TgOWPs), a class of molecules which has not been characterized yet. These Mabs could be used to develop an immuno-magnetic capture method for oocyst detection and concentration. The experimental approach will include: i) cloning of genes, identified in the ToxoDB database, encoding putative TgOWPs; ii) production in bacteria of recombinant fragments of distinct TgOWPs; iii) production of polyclonal antisera for the validation of putative TgOWPs as true oocyst wall components; iv) production of Mabs against selected TgOWPs. The proposed study would have an important additional implication. Indeed, the availability of recombinant oocyst wall proteins might be exploited to carry out a serologic survey on human and food animal sera, aimed at assessing the potential of these antigens in the identification of oocyst-driven, versus tissue cyst-driven, T. gondii infections. The possibility of discriminating between the two sources of infection would have a great impact on the understanding of the epidemiology of T. gondii infection in food animals and humans.

2.3 Identification and development of analytical methods for the speciation of parasites

of the family Anisakidae. Of the large amount of Anisakidae worms parasitizing fish,



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only a few of them are zoonotic. Consequently, it is of great importance to identify those which can present a risk to human health from those which have only veterinary importance. Literature data and sequence information available in the Genebanks, will be used to prepare specific primer sets to develop a standard protocol for the identification of single larvae belonging to the Anisakidae family (e.g. Anisakis, Pseudoterranova, Phocanema, Contracaecum) at the species level by PCR, PCR-RFLP and sequencing.

2.4 Identification of intraspecific genetic variability of Trichinella britovi. To trace back

the source of infection “from fork to farm”, a large number of isolates will be collected from Europe and the presence of loci with multiple alleles will be investigated. Putative polymorphic loci will be detected by the screening of the parasite genome for low complexity regions. Low complexity regions are sequences of DNA where the base composition is extremely uniform and for this reason they are the source of mutations (i.e. new alleles) during the cell division. Selected DNA regions will be amplified by PCR and the products from different isolates compared by electrophoresis and sequencing to identify the presence of alternative alleles. If the segregation of specific alleles in different geographic areas will be proven, these alleles will be useful to investigate dispersion of isolates inside the EU countries.

2.5 Identification and development of analytical methods for the speciation of parasites

of the genus Echinococcus. Literature data and sequence information available in the Genebanks, will be used to prepare specific primer sets to develop a standard protocol for the identification of the different parasite stages (eggs in faecal samples of definitive hosts; protoscolices in cysts of intermediate hosts) at the species or genotype level by PCR, PCR-RFLP and sequencing.

2.6 Collection of epidemiological data on the prevalence of the trematode worm Alaria alata in wild boar populations of MS. In the course of the workshop, which was held in Rome from 14 to 15 June, 2007, several participants expressed their concern on the increased detection of Alaria alata infestations in wild boars. Alaria alata is an intestinal trematode of carnivores (e.g. dogs, cats, foxes, wolves) which can reach other mammalian hosts such as the wild boar, which acts as paratenic host. This trematode can infect the human being, even if the number of documented infestations in humans is very limited. Data on the prevalence of Alaria alata infestations in wild boars will be collected in the MS to have a base line information which will be useful to evaluate the human risk.

3 Interlaboratory comparison study

A ring trial will be organised among NRLs to evaluate the sensitivity of the magnetic stirred method as reported in the EU legislation 2075/2005 on trichinellosis. Test samples will be prepared with nurse cells (capsules) containing T. spiralis larvae obtained from experimentally infected mice by partial digestion of tissue and subsequent repeated washings. Capsules will be added in known numbers to 100gr meatballs made with diaphragm tissue from pigs. Each laboratory in the trial will receive 9 samples of 100 grams containing a different number of Trichinella capsules. The number of added capsules will be of 0 larva/100g, 3-5 larvae/100 g, 6-10 larvae/100



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g, and 11-20 larvae/100 g. Samples will be packed and sent as bio-hazardous material in cool freeze containers to ensure a stable temperature. Every participating partner in the proficiency test will be coded (lab code) and notified in advance about the timetable and when to receive the test panels along with the protocol. The test results from each laboratory will be evaluated, and possible critical points will be identified and corrected.

4 Workshop

In the first half of 2008, a two day-workshop will be held at the Istituto Superiore di Sanità of Rome or in another Italian place to present and discuss the results of the ring trial on the digestion technique, and other issues including epidemiological problems related to foodborne parasitic zoonoses occurring in the MS. Some experts in the field of foodborne parasitic zoonoses will be invited to present the most recent knowledge on the epidemiology, diagnosis and control of some zoonoses.

5 Visit to NRLs

Qualified personnel of the CRLP will visit two NRLs to assist them as required by circumstances. The selection of the NRLs will be done with an agreement among NRL, CRLP and the Commission. The outcome of the visits will be reported to the Commission.

6 Training for Personnel of NRLs and from developing countries

On request from NRLs within EU or from governmental institutions of developing countries, personnel will be hosted at the CRLP to be trained on different detection methods of foodborne parasites and quality control systems.

7 Further development and up to date of the web site of the CRL for parasites

The web site of the CRLP established on the server of the Istituto Superiore di Sanità, according to the work programme 2008, will be up dated with epidemiological information (accessible to the NRLs and the public).

Rome, 31st July, 2007 The Director of CRL for Parasites

Dr. Edoardo Pozio

COMMUNITY REFERENCE LABORATORY FOR ANTIMICROBIAL RESISTANCE

WORK PROGRAMME 2008

COMMUNITY REFERENCE LABORATORY FOR ANIMAL PROTEINS IN FEEDINGSTUFFS

WORK PROGRAMME 2008

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CRL 2008 work programme ver 00 email 140807.doc 1

2008 WORK PROGRAMME FOR COMMUNITY REFERENCE LABORATORY

“Detection of animal proteins in feedingstuffs”

CRL-AP

1 Scientific advice and support to the European Commission (35.5 p/m)

1.1 Provide scientific and technical assistance to the European Commission in

relation to the development of EC feed legislation. (3 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, this means one for the six months period of 2008. (1 p/m)

1.2.1 Preparation and participation to international meeting/fora

1.2.2 Report/minutes following completion of the mission

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

Community reference laboratory, participate to 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, this

means one (as it is a starting year) for the six months period of 2007. (1 p/m)

1.3.1 Participation to the CEMA, CEN and IAG meetings

1.3.2 Reports/minutes of the meetings

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

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

animal proteins. (12 p/m)

1.4.1 Provide technical and scientific support

1.4.2 Submission of the report on the technical and scientific support

provided

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

awareness and quality management under accreditation (ISO17025, later on

maybe even ISO9001) on 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. (12 p/m)

1.5.1 Maintain of the accreditation scope

1.5.2 Extend of the accreditation scope

1.5.3 Preparation of the file for the organisation of interlaboratory studies

1.6 On the request of DG SANCO, to perform analyses on samples with disputed

results. (6 p/m)

1.6.1 Perform the requested analyses

1.6.2 Report on the analyses performed

1.7 Assist TAIEX with targeted assistance towards specific training/workshop for

new member states or/and candidate countries (0.5 p/m)

1.7.1 Administration and participation of candidate countries (Croatia,

Turkey) to the annual NRLs network workshop.

1.7.2 Request of financial support for candidate countries (Croatia, Turkey)

to TAIEX

2 Coordination of activities of NRL network (15 p/m)

2.1 Construction, development and maintenance 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 Development of the website (internet and intranet)

2.1.3 Development of the management tools of the website

2.1.4 Test of the information system and validation

2.2 Prepare and send a four-months newsletter for NRLs. (2 p/m)

2.2.1 Preparation and sending of three newsletters in 2008

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2.3 Organise and host the annual NRL meeting/workshop and produce minutes of

the meeting. (3 p/m)

2.3.1 Organisation of the 2nd 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

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

laboratories, detection, quantification and identification of animal proteins in

feed ingredients and feedingstuffs. (3 p/m)

On the request of the NRLs, supply of information and scientific advice

2.5 Participate to annual CRL Directors co-ordination meeting.

2.5.1 Participation to the 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. (1 p/m)

2.6.1 Prepare and submit the 6-months report (January 2008 – June 2008)

and annual report (January 2008 – December 2008)

3 Interlaboratory studies and quality assurance (19 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.

(3 p/m)

3.1.1 Definition of the needs

3.1.2 Set up of a planning of the samples to produce in the 2008-2009

period

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

samples

3.1.4 Control of the raw materials

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3.1.5 Production of the samples

3.1.6 Test of the homogeneity of the samples produced

3.1.7 Report on the produced samples

3.1.8 Distribution of the samples

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

classical microscopy. (12 p/m)

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

November 2007

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

of the ring trial to perform at the end of 2008.

3.2.3 Preparation of the interlaboratory study.

3.2.4 Invitation of the NRLs to participate.

3.2.5 Preparation and homogeneity test of the samples (cf. task 3.1)

3.2.6 Sending of the samples (Link with task 3.1.)

3.2.7 Collection of the data.

3.3 Au dit 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

(2 p/m)

3.3.1 On the basis of the results of the interlaboratory studies, organisation

and planification of the audit

3.3.2 Report of the audits

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. (2 p/m)

3.4.1 Definition of the needs of the NRLs

3.4.2 Preparation of the programme, in consultation with the European

Commission, including actions to be undertaken

3.4.3 Provide the requested help to the NRLs

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4 Development of analytical methods and tools (31 p/m)

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

existing methods of analysis. (1 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. (2 p/m)

4.2.1 Specification of the needs

4.2.2 Report of the results of the tests

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. (8 p/m)

4.3.1 Transfer of validated PCR methods to the CRL

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

make them available for the NRLs network. (6 p/m)

4.4.1 Preparation of the framework for the transmission of methods to NRLs

network

4.4.2 On the basis of the results of the validation of the PCR and

immunological dipstick methods (see tasks 1.5.3.2. and 1.5.3.3.) preparation

of CRL protocol to apply these methods in the NRLs labs

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4.5 Construction and extension of the samples bank with a special focus on the

animal meals of one single species origin (e.g. fish, poultry, pig, bovine, sheep)

from different processes. 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.

(14 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 Production and validation of informatics tools for the appropriate

management of the samples

4.5.4 Collection/production of samples of animal meals of one single species

origin (e.g. fish, poultry, pig, bovine, sheep)

4.5.5 Collection/production of samples of compound feeds free of MBM

4.5.6 Test of the samples collected

4.5.7 Preparation of the samples for the storing

4.5.8 Storing of the samples

4.5.9 Maintenance of the samples bank

5 Workshops/trainings (2,5 p/m)

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

the 126/2003/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. (2 p/m)

5.1.1 Organisation of a workshop for the quantification of PAP using the

126/2003/EC method

5.1.2 Invitation of the attendees

5.1.3 Preparation and submission of the minutes of the workshop

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5.2 Provide specific workshop for the benefit of NRLs for the detection,

identification and/or quantification of PAPs in feed according new validated

method.

No activities forecasted in 2008

5.3 Provide specific workshop of experts from canditate member states for the

correct application of the 126/2003/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. (0.5 p/m)

5.3.1 Organisation of a workshop training for Croatia and Turkey for the

correct application of 126/2003/EC directive

5.4 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.

No activities forecasted in 2008.

work programmes for community reference laboratories for 2008 · frame: the regulation 1664/2006...

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