WORK PROGRAMMES FOR EU REFERENCE LABORATORIES … · WORK PROGRAMMES FOR EU REFERENCE LABORATORIES ... MMP , formerly CRL ... determination of alkaline phosphatase activity in cheese

WORK PROGRAMMES FOR EU REFERENCE LABORATORIES

FOR 2011 **********

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

Milk and milk products

Salmonella Marine biotoxines

Bacteriological and viral contaminants of molluscs Transmissible Spongifiorm Encephalopathies

Listeria monocytogenes Coagulase positive Staphylococci

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

Parasites (in particular Trichinella, Echinococcus and Anisakis)

Antimicrobial resistance Animal proteins in feedingstuffs

EU REFERENCE LABORATORY FOR MILK AND MILK

PRODUCTS

WORK PROGRAMME 2011

French agency for food, environmental and occupational health safety – Maisons-Alfort laboratory for food safety 23, avenue du Général de Gaulle - F94706 Maisons-Alfort Cedex - Téléphone : + 33 (0)1 49 77 13 00 - Télécopie : + 33 (0)1 43 68 97 62 - www.anses.fr

Maisons‐Alfort laboratory for food safety

2011 Work Programme of the Reference Laboratory of the European Union

for Milk and Milk Products

Version 1 ‐ 11 August 2010

EU‐RL MMPEuropean Union Reference Laboratory for Milk and Milk Products

EU‐RL for Milk and Milk Products 2/9 11/08/2010 2011 Work Programme

The Maisons‐Alfort Laboratory for food safety of Anses (French agency for food, environmental and occupational health safety) –formerly Afssa‐Lerqap‐ foresees to undertake, as European Union Reference Laboratory (EU‐RL) for milk & milk products (EU‐RL MMP, formerly CRL MMP), the following works in 2011 according in particular to (a) the actions planned at the 12th Workshop of the National Reference Laboratories (NRLs) (28&29 May 2009), and (b) the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the EU‐RL for the period 2006‐2011.

These actions are part of the current mandate of the EU‐RL MMP, 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: o At the level of raw milk production & collection: for raw cow’s milk and raw

milk from other species milk (Chapter I, clauses I & III); o At the level of preparing dairy products (Chapter II, clause III‐criteria for the

use of raw cow’s milk for further processing). - Phosphatase activity:

o At the level of raw milk production (Chapter I, clause I.3): a reference is made to a negative phosphatase test to characterize the heat‐treatment to be applied to raw cow’s or buffalo’s milk coming from animals not meeting certain requirements on brucellosis or tuberculosis.

o At the level of heat treatment of raw milk or dairy products (Chapter II, clause II): the food business operators shall ensure that the heat‐treatment satisfies the requirements of Regulation 852/2004, Annex II, Chapter XI.

The EU‐RL 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 2011), either multi‐annual (on‐going programme on several years).


1. HYGIENE OF RAW MILK

Frame : The Regulation 1664/2006 prescribes the reference method for total flora enumeration at 30°C, Standard EN ISO 4833, and the reference method for somatic cells count, Standard EN ISO 13366‐1, as well as conditions for the use of alternative methods.

1.1. INTER‐LABORATORY PROFICIENCY TESTING FOR THE NRLS

The inter‐laboratory proficiency testing (PT) trials organised by the EU‐RL 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.

1.1.1. STUDY OF SAMPLE TYPES USED FOR INTER‐LABORATORY TRIALS ON TOTAL FLORA IN RAW GOAT’S MILK

(multi‐annual)

The EU‐RL (Unit HMPA) will complete in 2011 an investigation study (stability and homogeneity) to find a way, such as the addition of a chemical agent, to stabilize sufficiently the total flora (TF) contamination of raw goat’s milk, in order to organize PT trials on TF enumeration in raw goat’s milk samples. It is intended to select a formula adapted to TF; formula which would allow the bacteria to grow on plates after the dilution steps.

1.1.2. STUDY OF SAMPLE TYPES USED FOR INTER‐LABORATORY TRIALS ON SOMATIC CELLS IN RAW COW’S MILK

(multi‐annual)

The EU‐RL (Unit HMPA) will conduct in 2011‐2012 an investigation study (stability and homogeneity) to find a way, such as the addition of a chemical agent, to stabilize sufficiently the somatic cells contamination of raw cow’s milk, in order to organize PT trials on somatic cells count in raw cow’s milk samples.

1.1.3. ENUMERATION OF TOTAL FLORA AT 30°C IN RAW GOAT’S MILK

(annual)

The EU‐RL (Unit HMPA) will organize in 2011 an inter‐laboratory trial on the total flora enumeration at 30°C in raw goat’s milk by the reference method, the Standard EN ISO 4833 (total plate count method).


1.2. ANALYTICAL DEVELOPMENT

(multi‐annual)

1.2.1. DETERMINATION OF TOTAL FLORA AT 30°C AND SOMATIC CELLS IN RAW MILK BY AN INSTRUMENTAL METHOD

The EU‐RL (Unit HMPA) intends to complete in 2011 its experimental study for raw cow’s and goat’s milk, using a flow cytometer (Bactocount) purchased in 2007, as an alternative method to the bacterial total flora (TF) count and to the somatic cells count (SCC). This study aims at investigating the questions linked to the correlation of the Bactocount to the reference methods for TF and SCC, 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 cow’s and goat’s milk delivered at regular intervals of time will be analysed in parallel by the reference methods and by the Bactocount for TF and SCC.

Moreover, the EU‐RL (Unit HMPA) will launch in 2011 a study to characterize the microbial composition of raw milks by PCR‐DGGE (Denaturing Gradient Gel Electrophoresis).

1.2.2. DETERMINATION OF TOTAL FLORA AT 30°C IN COW’S COLOSTRUM

Frame: In the frame of Regulation 853/2004 modified, national hygienic requirements are referred to for colostrums.

In 2010, the EU‐RL will have conducted an enquiry to the NRLs to collect existing national hygienic requirements/microbiological criteria on cows’ colostrum. The synthesis of the enquiry will be prepared in 2011.

1.3. COORDINATION OF THE NRL ON DETERMINATION OF TOTAL FLORA

(multi‐annual)

Since the publication of the Standard EN ISO 21187 on the conversion factors between the routine method and the reference method for TF determination, the EU‐RL has been supervising 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.


This topic will be covered at the 2010 annual workshop of the NRLs which will be dedicated to TF in raw milk (30 September/1 October), and actions to be conducted in the future, in particular in 2011, will be defined at that workshop.

1.4. STANDARDIZATION ON VALIDATION OF ROUTINE METHODS FOR TOTAL FLORA IN RAW MILK

(multi‐annual)

IDF/ISO is conducting a revision of the Standard IDF 161 detailing the validation protocol of a routine method against a reference method for the TF determination in raw milk.

The EU‐RL will follow this standardization work and will ensure a liaison with the works undertaken as EU‐RL with the NRLs network.

1.5. DEVELOPMENT OF CERTIFIED REFERENCE MATERIALS FOR SOMATIC CELLS COUNT IN RAW MILK

(multi‐annual)

Given the deficiencies of the reference microscopic method for SCC in raw milk (lack of reproducibility) and the limited number of laboratories using it, it is of utmost importance to develop Certified Reference Materials, which are not currently available.

In 2011, the EU‐RL intends to bring its support to identify the possibilities to develop certified reference materials by the JRC/IRMM in Geel. For that purpose, DG SANCO is to contact JRC to organize a meeting.

1.6. DEVELOPMENT OF A REFERENCE SYSTEM FOR SOMATIC CELLS COUNT IN RAW MILK

(multi‐annual)

IDF/ISO has initiated the setting‐up of a reference system for SCC in raw milk, given the deficiencies of the microscopic reference method to provide reference values comparable between different laboratories. It is intended that this reference system, in addition to the reference method, would take account of reference materials and of instrumental methods used in routine. A network of expert laboratories is intended to be settled, to define assigned values associated to reference materials used for the calibration of instrumental methods.

The EU‐RL will go its participation to the IDF/ICAR working group in charge of developing this reference system as to envisage how it could be beneficial to the CRL/NRLs own works, to implement the requirements of Regulation 853/2004 concerning SCC.


2. DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY

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

2.1. INTERNATIONAL VALIDATION TRIAL AND, IN PARALLEL, INTER‐LABORATORY PROFICIENCY TESTING FOR THE NRL

(annual)

The EU‐RL (Team CAT‐AP) will organize in 2011 an international validation study on the determination of alkaline phosphatase activity in cheese by the fluorimetric method, in order to generate precision data according to ISO 5725‐2.

NRLS will be asked to participate to the study. The set of data submitted by NRLs will be extracted from the total data and submitted to a distinct statistical evaluation as an interlaboratory assessment trial.


(multi‐annual)

In 2011, the EU‐RL (Team CAT‐AP) intends to conduct the following activities.

2.2.1. STUDY OF THE IMPACT OF THE BETWEEN‐INSTRUMENTS VARIABILITY TO THE OVERALL VARIATION OF RESULTS OBTAINED BY DIFFERENT FLUOROPHOS UNITS

(multi‐annual)

The EU‐RL works with three different Fluorophos instruments. It is deemed important to evaluate the impact of the instruments themselves on the variation of the results obtained on the same test samples, when analysed using the same reagents and measured on the three instruments available.

In 2011, the EU‐RL will launch a study on different milk and cheese samples. The EU‐RL will share the conclusions of the study with NRLs and also with the instrument manufacturer.


2.2.2. DETERMINATION OF THE PHOSPHATASE ACTIVITY IN OTHER THAN COW’S MILK

The EU‐RL will continue the study of AP levels for species other than cow’s milk. The purpose of this work is to support DG SANCO in prescribing legal limits of AP activity in milk from different species.

EWE’S MILK

In 2011, the EU‐RL will work on the establishment of phosphatase inactivation curves in ewe’s milk and specifically on the time‐temperature conditions needed to inactivate AP in ewe’s milk. In fact, because of the high fat content of ewe’s milk, the heat load necessary to the pasteurisation of this type of milk is more important than for cow’s and goat’s milk.

Once the pasteurisation conditions for ewe’s milk will have been identified, the EU‐RL intends to start work on raw and laboratory‐pasteurised milk to initiate the project aiming to the setting up of AP limits in ewe’s pasteurised milk.

CAMEL’S MILK

A project to characterize the heat‐treatment of camel’s milk is developed within a technical collaboration between the Central Veterinary Research Laboratory (CVRL) of Dubai and the German NRL.

The EU‐RL is closely involved in this project at the request of DG SANCO: experimental work is to start only upon endorsement of the work program by EU‐RL, the EU‐RL is to be advised regularly on progress of the work and will scrutinize results obtained and conclusions drawn before their adoption.

2.2.3. DETERMINATION OF ALKALINE PHOSPHATASE IN CHEESES

REVISION OF THE INTERNATIONAL STANDARD ISO 11816‐2/IDF 155‐2

The EU‐RL will continue acting as Project Leader for this topic and will progress the item during all stages prescribed within the official procedures of standardisation. It is expected that in 2011 the text will get a positive vote to move to the Committee Draft stage.

Technical comments may still be forwarded at this stage of voting and this may result to some minor technical changes after the EU‐RL has studied the amendments proposed.


COORDINATION OF THE SURVEYS CONDUCTED BY NRL ON CHEESE MADE FROM

PASTEURISED COW’S MILK.

In 2011, the EU‐RL will continue the coordination of the experiments performed by NRLs on soft, hard and semi‐hard cheeses made from cow’s pasteurised milk.

This European study aims to collect information about the content of residual AP in pasteurised cow’s cheese so as to set up legal limits of AP allowing for the distinction between cheeses made from pasteurised cow milk and cheeses made from cow non‐pasteurised milk.

PARTICIPATION TO THE STUDY OF AP CONTENT IN PASTEURIZED COW’S CHEESE

The EU‐RL will participate to the above mentioned survey, particularly when difficult cases are raised by NRLs (for ex. AP content in French pasteurised Munster cheese).

2.2.4. COMPARISON OF THE CHEMILUMINESCENT/FLUORIMETRIC METHODS

In 2011, the EU‐RL intends to put into practice the approach of the accuracy profile (acceptability limits) to assess the equivalence of the chemiluminescent method versus the official fluorimetric method. Comparison will deal with cow’s skim and semi‐skim milk and, if possible, with goat’s whole milk.

2.2.5. HEAT TRACERS OTHER THAN AP, DEVELOPMENT OF ANALYTICAL PROTOCOLS

The EU‐RL will participate to an international project (Project leader: Dr George Ziobro, US/FDA) aiming to identify other pasteurisation tracers when AP is inappropriate, and to collect information on relevant analytical methods. Depending on the progress of the work item, preliminary assays on the identified protocols may be conducted in 2011.

2.2.6. REACTIVATED PHOSPHATASE

No experimental work is on the work programme but the EU‐RL will continue to keep an eye on the topic. In case FDA can accept the suggestion, an exchange of views with scientific experts of the US/FDA competent laboratory is envisaged.


3. ASSISTANCE TO THE NRL

Upon requests of NRLs, the EU‐RL may receive NRL staff for individual training on specific topics.

4. NRLS WORKSHOP

The EU‐RL will organise in 2011 the 14th NRLs Workshop of general scope. In particular, this workshop will enable:

1. to make a point of works undertaken by the EU‐RL since the last general workshop of 2009, in particular further to the 2010 Workshop dedicated to Total Flora;

2. to envisage the work programme for the following years.

5. TECHNICAL AND SCIENTIFIC ASSISTANCE TO THE EUROPEAN COMMISSION

(multi‐annual)

5.1. DG SANCO ACTIVITIES

Upon request of the services of DG SANCO in charge of food hygiene:

- Participation to the bilateral US/UE negociations on veterinary agreement (dairy hygiene, total flora, somatic cell count and phosphatase activity),

- And any other question which may arise during the year.

5.2. PARTICIPATION TO ISO/IDF STANDARDIZATION WORKS

On behalf of DG SANCO (and official nomination as EC representative to CEN/ISO meetings), participation to:

1. 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, and other pasteurisation

tracers. 2. The 2011 IDF/ISO Analytical Week (Lyon, France, May 2011) and the meetings of the

groups dealing with the topics mentioned above.

EU REFERENCE LABORATORY FOR SALMONELLA

WORK PROGRAMME 2011

Work-programme CRL-S 2011 27-08-2010 Page 1 of 6

CRL-Salmonella work-programme 2011 Introduction The working programme of CRL-Salmonella consists of the following activities (the duration of the activities are indicated between brackets): 1. Organisation of interlaboratory comparison studies (yearly); 2. Organisation of a workshop with the NRLs-Salmonella (yearly); 3. Performance of research (depending on the subject: yearly or for a limited period); 4. Giving assistance to the Commission and ad hoc activities (yearly); 5. Communication (every 3 months and yearly); 6. Training (duration dependent on the subject). 7. Missions (duration dependent on the subject); 1. Interlaboratory comparison studies For 2011 it is planned to organise 3 interlaboratory comparison studies; • One study on bacteriological detection of Salmonella in a veterinary matrix; • One study on bacteriological detection of Salmonella in a food or feed matrix; • One study on typing of Salmonella. The exact timing of the studies in 2011 will be planned with the NRLs-Salmonella, but an indication of the probable time period is given below. The general set-up of the CRL-Salmonella interlaboratory comparison studies on the bacteriological detection of Salmonella has remained the same for many years. In these studies a Salmonella negative matrix is artificially contaminated with Salmonella reference materials containing Salmonella Typhimurium or Salmonella Enteritidis at different contamination levels (low and high level). The differences over the years have been the choice of the matrix, the number of reference materials to be tested, the contamination level of these reference materials and the choice of the methods. Up to 2010 the reference materials consisted of capsules containing artificially contaminated milk powder. For the reconstitution of these capsules, a special procedure needs to be followed so that the treatment of the samples of the interlaboratory comparison studies is different from the treatment of routine samples. In 2010 several tests have been performed (or are still performed) for the use of lenticules as reference materials. The results of these tests are promising and may mimic better the treatment of routine samples. It is therefore planned to use lenticules instead of capsules in the interlaboratory comparison studies on detection of Salmonella in 2011. The choice of the serovars as well as the contamination levels will be decided per study. Interlaboratory comparison study on bacteriological detection of Salmonella in veterinary samples Like for former studies, a Salmonella-free matrix will be selected which will be artificially contaminated with Salmonella reference materials. The following is planned for the 2011 veterinary ring trial:


- Time period: February/March; - Matrix: animal faeces (most probably chicken faeces); - Number of samples and level of reference materials to be decided later; - Methods: Annex D of ISO 6579 (‘Detection of Salmonella in animal faeces and in

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

The results of each NRL will be evaluated and compared with the pre-set definition of ‘good performance’. In case of unexplainable ‘poor performance’, the follow-up will be discussed with the relevant NRL (e.g. sending extra samples which need to be tested according to a prescribed protocol, training at the CRL or visiting a NRL by members of the CRL-Salmonella staff). Since 2008, also reference laboratories of two third countries participated, being: Tunisia and Israel. These countries participated on request of DG-Sanco. It is foreseen that these (two) third countries will again participate in the study of 2011. If considered necessary by DG-Sanco, additional third countries may participate in the 2011 study. The results of the third countries will be analysed separately from the results of the NRLs of the European Member States. The justification for participation of these third countries was given in the work-programme of 2008 and is repeated below: Salmonella control programmes in live poultry are introduced in the European Member States by Regulation (EC) No 2160/2003. The control programmes in breeding hens include the monitoring of Salmonella by the testing of faecal materials in accordance with the provisions in Regulation (EC) No 1003/2005. Third countries, who want to remain or be added to the list of third countries from which Member States may import breeding hens or hatching eggs, should submit a control programme equivalent to the control programmes of the Member States. In order to evaluate the equivalence of testing in these third countries, they should participate in the ring trials organised by the CRL. Tunesia, Canada, Israel and the United States forwarded their control programme for breeding hens and should therefore be included in the ring trial. Interlaboratory comparison study on bacteriological detection of Salmonella in food or feed samples The choice of the matrix for the food/feed study in 2011 has not yet been made. However, if for this study indeed lenticule reference materials will be used for the first time, it may be good to repeat this study with a matrix which has earlier been used in a study with capsule reference materials (e.g. minced beef). The prescribed method will be ISO 6579 (2002: Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp.), with selective enrichment in RVS and MKTTn. The additionally requested method will be Annex D of ISO 6579, with selective enrichment on MSRV. The planning of the study is September/October 2011. The planning of the study is September/October 2011. Like for the veterinary study, the results of each NRL will be evaluated and compared with the pre-set definition of ‘good performance’. Actions will be taken in case of unexplainable ‘poor performance’ (see above).


Interlaboratory comparison study on typing of Salmonella Up to 2008, EnterNet laboratories (analysing human isolates) had the possibility to participate in the typing studies through a collaboration between the CRL-Salmonella and the Health Protection Agency (HPA), Colindale (UK). In fall 2008, this collaboration was formalised in a contract with the ECDC. This collaboration is also profitable for the NRLs, as it still gives them the opportunity to test their phage typing capacities additional to the serotyping of Salmonella in the interlaboratory comparison study on typing. Like in former studies the CRL-Salmonella will select twenty Salmonella strains for serotyping, including serovars with public health significance, serovars with antigens similar to those of public health significant strains and serovars that had caused typing problems in previous studies. The strains will be blindly coded and send to the NRLs for serotyping. The HPA will select twenty Salmonella strains for phagetyping (10 Salmonella Enteritidis strains and 10 Salmonella Typhimurium strains). These latter strains will only be sent to the NRLs who have indicated to perform phage typing as well. The planning of the study is November/December 2011. In 2007 a definition on the evaluation of ‘good performance’ of the serotyping was agreed upon with the NRLs and for the first time applied on the results of the study of 2007. The same criteria will be used for evaluating the results of the typing study of 2011, unless agreed otherwise with the NRLs and/or DG-Sanco. 2. Workshop The annual workshop is planned to be organised in Bilthoven in May/June 2011 and will last 1,5-2 days. The programme may contain the following items: - Introductory presentations (e.g., by EU representative and CRL-Salmonella); - Zoonoses in Europe (EFSA, DG-Sanco); - Results of research activities of CRL-Salmonella; - Results of interlaboratory comparison studies of 2010 and 2011; - Experiences, problems, results in relation to monitoring surveys for Salmonella; - Plans and results of research activities of the NRLs-Salmonella; - Discussion on methods (e.g. typing, molecular, serological); - Activities in ISO and CEN; - Future working plan of CRL-Salmonella. 3. Research Activities concerning ISO and CEN The CRL-Salmonella (Kirsten Mooijman) is involved (as project leader or as member of working groups) in several activities of ISO and CEN. More specific in: - ISO/TC34/SC9: International Standardisation Organisation, Technical Committee 34

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

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


Kirsten Mooijman of the CRL-Salmonella is convenor of three groups in ISO dealing with methods for Salmonella. The activities for these groups will be continued in 2011: Detection of Salmonella (ISO 6579-1): In fall 2010 the first draft of the amended

document will be discussed in the working group. In the mean time more research will be performed (literature research, as well as laboratory research). The results of the discussions as well as of the research will be used to further amend the document. This work will continue in 2011 and at least one meeting of the working group is foreseen in 2011 (to be organised by the convenor). Furthermore, the progress of the group needs to be presented at the plenary meeting of CEN and ISO by the convenor (Kirsten) of the working group.

Enumeration of Salmonella (ISO 6579-2): The finalisation of the document is foreseen in fall 2010. However, some unforeseen additional work may still be necessary in 2011.

Guide for serotyping Salmonella spp. (ISO 6579-3): It is expected that the New Work Item Proposal (NWIP) for this EN/ISO guidance document will be launched in fall 2010. After this voting, comments are expected after which the document needs to be amended. In 2011, Kirsten will organise one or two meetings to discuss the comments and the consequent amendments of the document in the ad hoc group. Furthermore, she will present the progress of the group at the plenary meeting of CEN and ISO in spring 2011.

Other activities related to CEN CEN mandate on validation of microbiological methods: In 2007 the CRL-

Salmonella was assigned as project leader for the validation of Annex D of ISO 6579. By then the CRL- Salmonella also submitted a work plan and budget forecast. In 2010 the consolidated work plan of 15 studies under the mandate will be submitted to the EC (by CEN). It is not clear when a contract for the performance of the study can be expected. As soon as this contract comes available, a further planning of the activities can be made, which will involve expertise of the CRL as well as of some NRLs for Salmonella.

Methods Literature researches and experiments are foreseen in relation to the ISO/CEN activities in the field of Salmonella (see above), especially for the revision of ISO 6579-1:

- Incubation time of the selective enrichment (24h or 48h); - Choice of the isolation medium; - Possibility of refrigerating BPW and/or selective enrichment media before

subculture; - Investigation of the usefulness of some biochemical tests; - Pooling of samples (dry-pooling as well as wet-pooling).

Furthermore, a report will be prepared in which all information and research results collected for revision of ISO 6579-1 will be summarised. In 2007 some artificially contaminated samples (chicken faeces, pig faeces, minced meat) were tested for Salmonella using a Real-Time Polymerase Chain Reaction technique. All


samples gave similar results with the RT-PCR and with the bacteriological detection method. In 2008 several naturally contaminated samples were obtained, with different contamination levels. The samples were frozen at -20 °C and it is planned to analyse these samples with the PCR technique in fall 2010. When necessary, additional analyses will be performed in 2011. Reference materials As indicated at 1. ‘Interlaboratory comparison studies’, it is planned to change from capsule reference materials to lenticule reference materials for the interlaboratory comparison studies on detection of Salmonella. If necessary, additional tests will be performed in 2011 to confirm the usefulness of the lenticules with the matrix chosen for a specific study. Matrices Research activities may be necessary for testing relevant matrices for the interlaboratory comparison study on the detection of Salmonella. The matrix will be tested with and without the addition of Salmonella reference materials. Also the amount of background flora will be analysed. 4. Giving assistance to the Commission and ad hoc activities Each year the CRL-Salmonella is contacted by various parties, i.e. institutes in Member States, Candidate Member States or third countries, with requests for information or for participation in activities being organised. Also, requests for support from the European Commission with respect to certain issues (e.g., methods, participation in working groups, advices) are raised. In these cases the CRL-Salmonella will in principle always react positively and will try to include the ad hoc work required in the working plan although it is difficult to plan the time needed to answer the different questions. Participation in Working Groups of DG-Sanco and EFSA Staff members of the CRL-Salmonella participate in the working groups of DG-Sanco and of EFSA for (among others) to give technical support in drafting EU legislation, for preparation of technical specifications of monitoring and control programmes, for drafting (EFSA) opinions for certain items. 5. Communication A staff member of the CRL-Salmonella has been trained to keep the CRL-Salmonella website up to date (www.rivm.nl/crlsalmonella). As it is foreseen that the RIVM website will be amended in 2011, it will be necessary to amend the CRL-Salmonella website as well. The newsletter of CRL-Salmonella is published every quarter with information from the CRL-Salmonella relevant for the NRLs-Salmonella or from NRLs-Salmonella relevant for the other NRLs-Salmonella. Also, a literature search is included in each newsletter covering the previous 3-month period.


Results on the interlaboratory comparison studies, the workshop and relevant research will be published in RIVM reports. The reports will be distributed to the EC and to the NRLs and other interested bodies. Furthermore they will also become available at the CRL-Salmonella website. Summaries of several intercomparison studies and related research will be published (if possible) in the scientific literature. 6. Training On request of an NRL, the CRL can give a training for a specific need of an NRL. It is also possible that the CRL will advice an NRL to follow a training at the CRL or that staff members of the CRL give a training at the laboratory of the NRL, especially in case of (repeated) poor performance of the NRL in interlaboratory comparison studies. 7. Missions Summarising the information as given in the chapters above, the following missions may be needed in 2011. Per mission one (occasionally two) staff member of the CRL-Salmonella will participate. - Visit to a poor performing NRL (if necessary, see information under chapter

‘Interlaboratory comparison studies’), approximately 2 days, country unknown. - Annual meetings of ISO/TC34/SC9 and CEN/TC275/WG6, planned in United

Kingdom, date not yet set (see information under chapter ‘Research’). Total duration of the meetings approximately 5 days.

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

Mrs. Drs. K.A. Mooijman Head CRL-Salmonella Bitlhoven, 27 August 2010

EU REFERENCE LABORATORY FOR MARINE BIOTOXINS

WORK PROGRAMME 2011

EU REFERENCE LABORATORY FOR BACTERIOLOGICAL AND VIRAL CONTAMINANTS OF MOLLUSCS

WORK PROGRAMME 2011

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]

1

WORK PROGRAMME FOR THE EURL FOR BACTERIOLOGICAL AND VIRAL CONTAMINATION OF BIVALVE MOLLUSCS, 2011 LEGAL FUNCTIONS AND DUTIES The functions and duties of the EURL 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 2011 work programme year 27 Member States and 3 candidate countries (Croatia, Turkey and Republic of Macedonia) are considered eligible for EURL assistance and invited to participate in EURL organised training programmes, comparative testing 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, 2011 Duration

1. Scientific advice and support

1.1. Assist DG Sanco in functioning and implementation of Community food hygiene legislation, in particular in 2011:

• Consideration of equivalence of US FDA and EU hygiene standards for live bivalve molluscs (LBM).

• Recommendations on harmonisation of standards for class A shellfish and end products in EU hygiene legislation with CODEX standards for LBM.

• Consideration of validation of alternative methods in legislation.

• Considerations of intensive purification for LBM harvested from class C areas.

And any other activities as required.

50 days

1.2. Participate in relevant EU and International scientific committees (ISO/CEN, WHO/FAO, ICMSS etc). In 2011 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.

40 days




2

• Lead the revision of the EU reference method for enumeration of E. coli in LBM for official control (ISO TS 16649-3) to establish the method as a full standard.

• Project leader for the revision of the ISO 6887 series part 3 initial preparation and dilutions for aspects of microbiology associated with LBM.

• Jointly lead the revision of ISO TS 21872-1 and 2 detection of Vibrio spp. in seafood.

• Participate in ISO/TC34/SC9/WG3 working group on validation of methods (revision of EN ISO 16140).

1.3. Contribute to the joint FAO/WHO expert group on risk assessment

tools for Vibrio parahaemolyticus and V. vulnificus associated with seafood.

1.4. Contribute to EFSA expert working group on foodborne viruses considering control options for viruses in LBM, to include risk based approached, virus standards, improvements in water quality, environmental legislation and improved depuration.

1.5. Assist DG Sanco with specialist assistance in relation to food and

veterinary inspections of Member States, Accession Countries and Third Countries as they arise.

10 days 15 days 10 days

1.6. Co-operate with, and assist DG TAIEX in the provision of training and advice to Accession Counties as required.

4 days

1.7. Undertake EURL missions in support of the above activities.

• During 2011 missions are foreseen in relation to the annual meetings of ISO and CEN (1 mission) the CEN/TAG4 working group on viruses in food (1 mission); CEN/TAG3 working group on vibrios (1 missions); ISO/WG3 working group on validation of methods (2 missions) and up to 6 missions in support of NRLs and DG Sanco activities (items 1.1 and 1.2).

• Mission associated with the joint EU/US FDA workshop on implementation and approaches to sanitary surveys (item 2.5).

• Mission to JRC/IRMM Geel, Belgium (item 3.4)

Up to a maximum of 30 days Included in above 3 days




3

1.8. Organise an annual review meeting between EURL representatives and designated DG SANCO representative in the area in Brussels or mutually convenient location.

1.9. Participation in relevant international scientific conferences. In 2011 it is foreseen that the EURL will present scientific research at the ICMSS conference in PEI, Canada, Health Related Water Microbiology, New Zealand.

5 days 10 days

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

technical assistance and training

2.1. Participate in annual EURL Director’s co-ordination meeting and other EURL co-ordination meetings/workshops as appropriate.

5 days

2.2 Organise, host, and participate in the tenth annual EURL workshop, produce resolutions and other workshop outputs (May 2011, EURL Weymouth). To include administrative assistance.

40 days

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

Up to 50 days

2.4 Produce web-based information and guidance on application of sanitary surveys - in accordance with the requirements of 854/2004 on official controls.

2.5 In collaboration with the US FDA organise and participate in the

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

4 days 15 days

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

Up to 6 days

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

5 days

2.8 Revise the EURL website (www.crlcefas.org) to ensure it is an effective means of dissemination of information to NRLs and other stakeholders.

10 days




4

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 EURL/HPA shellfish EQA scheme. Analyse results, produce report, advice and recommendations (by May 2011).

26 days

3.2 Organise norovirus and hepatitis A ring trials, in 2011 for both laboratory constructed and LBM matrix samples for quantitative and qualitative analyses. Analyse results, produce report and recommendations (by May 2011).

70 days

3.3 Undertake Vibrio spp. ring trials to assist in the elaboration of methods enabling detection/enumeration of human pathogenic Vibrio spp. associated with LBM. Analyse results, produce report and recommendations (by May 2011).

Included in item 5.2

3.4 In collaboration with JRC/IRMM Geel continue studies on the production of norovirus reference material using freeze-dried matrix samples.

30 days

4 Confirmatory testing

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

4.2 Re-accreditation to IEC ISO 17025 of the CEN method for detection of norovirus in bivalve shellfish.

30 days 30 days

4.3 Maintenance of EURL 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 the detection of Salmonella spp. and Vibrio parahaemolyticus.

4.4 Contribution to costs of the maintenance of EURL capability to perform analysis for human pathogenic strains of marine vibrios associated with LBM (e.g. serotyping V. parahaemolyticus, molecular characterisation of pathogenic strains of V. parahaemolyticus, V. vulnificus and) non01/0139V. cholerae).

50 days 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

5 Development of analytical methods (undertaken at EURL)

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. The deadline for a decision on funding of M/381 for full validation is September 30

th 2010.

10 days

5.2 Contribution as the project leader towards the elaboration and validation of the TAG3 molecular based standard for the detection of potentially pathogenic vibrios in foodstuff, including bivalve shellfish using molecular methods to include in 2011:

• Evaluation of a direct extraction and detection of V. parahaemolyticus and V. vulnificus in artificially and naturally contaminated samples.

• Performance characterisation of above method to evaluate linearity, limit of detection, limit of quantitation and repeatability.

• Limited comparative testing amongst NRLs with expertise in testing LBM for Vibrio spp. to examine method reproducibility and inform methodology development.

• Evaluation of PCR targets for inclusion in the revision of ISO TS 21872- parts 1 and 2 to enable the inclusion of a molecular confirmation step for vibrios of human pathogenic significance.

60 days

Rachel Rangdale EURL Co-ordinator August 2010




6

WORK PROGRAMME FOR THE EUROPEAN UNION REFERENCE LABORATORY FOR BACTERIOLOGICAL AND VIRAL CONTAMINANTS OF MOLLUSCS, 2011 Annex 1 Resources necessary to fulfil the listed activities I. LEGAL FUNCTIONS AND DUTIES The functions and duties are specified in Articles 3 and 4 of Council Decision 1999/313/EC (Official Journal of the European Communities No L 120 of 8.5.1999). II. PROGRAMME FOR THE PERIOD JANUARY – DECEMBER 2011 Item Baseline Activity Resources

required (£) 7% overheads

TOTAL budget requested

1 Scientific advice and support

22,510.70 1,575.75 24,086.45

2 Co-ordination of activities of NRL network and provision of technical assistance and training

33,766.05 2,363.62 36,129.67

3 Ring trials, comparative testing and quality assurance

112,553.50 7,878.75 120,432.25

4 Confirmatory testing

56,276.75 3,939.37 60,216.12

5 Development of analytical methods (undertaken at EURL)

Total Baseline Costs

225,107.00 15,757.49 240,864.49

Workshop

38,867.84 38,867.84

EU REFERENCE LABORATORY FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

WORK PROGRAMME 2011

EURL Work Plan 2011

2011 workplan.doc

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 EURL will organise proficiency tests for the interpretation of histopathology and immunohistochemistry (IHC) in 2011 (see section 2 for details).

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

1.3

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

1.4

Positive and negative material necessary for annual QA purposes (see section 2 below) will be requested ‘en bloc’ from the TSE Archive, and reserved for EURLpurposes. (This will require the continued maintenance of dedicated freezers. It was agreed in 2003 that the Commission would pay 1/5 depreciation costs each year.) We will request material in 2011, to prepare samples for EQA use in 2012.

1.5

As discriminatory tests become more widely used, it will be necessary for the EURL 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, 5 in 2006, 10 in 2007 and 5 in 2009. This provides material for test evaluation and sensitivity exercises, QA panels and species specific controls for STEG evaluations. However, some of these sheep did not succumb as expected, and were subsequently found to have the polymorphism T112, which has now been linked with resistance to BSE. Five of the sheep challenged in 2007 fell into this category and have been culled. Two sheep from 2005 and 2 from 2006 remain alive and carry this polymorphism. They offer an excellent opportunity to establish whether this polymorphism confers absolute resistance, or merely prolongs incubation period, and it is proposed that they are kept alive to address this question. These four sheep, plus the 5 surviving sheep from 2009 will incur maintenance costs in 2011 if they do not succumb to disease within the next 6 months. No challenges were undertaken in 2010. It is proposed to challenge a

EURL Work Plan 2011

2011 workplan.doc

2

further five sheep in 2011, with the recipients this time being of the AHQ/AHQ genotype, given that all existing supplies are of the single ARQ/ARQ genotype. We know that such sheep succumb to oral challenge with the same incubation period as ARQ/ARQ so we would predict a similar outcome, with clinical onset predicted in 2012.

1.6

Atypical scrapie, its definition and detection is of increasing importance across the Community. Ideally the EURL 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 five animals with ‘atypical’ scrapie was started in 2006, with a further five challenged annually, to provide a bank of such material exclusively for EU QA purposes. To date, seven animals have succumbed, but we have good evidence from parallel experiments now that the anticipated attack rate in this model will be 100%. The remaining 10 animals are currently healthy. Based on the incubation period of approximately 800 days observed in other research projects, it is predicted that up to five sheep may succumb within 2010, and that the remainder are predicted to incur maintenance costs into 2011. The amount of tissue required to include a sample in a QA round is approximately 65g (equivalent to two sheep). It is therefore not proposed to challenge further sheep in 2011, but tissue use will be monitored, and consideration will be given to the need for future challenges.

1.7

The identification and characterisation of H and L type BSE raises the need for reference material from such cases. Global supplies are currently very limited, and reserved for research. We have successfully challenged two cattle with H-type BSE and two with L-type BSE to generate a small bank of reference material for statutory testing purposes. If an initial analytical sensitivity study is performed successfully on approved rapid tests (see proposal in section 3.4) the EURL plansto include these variants in the 2011 discriminatory, rapid and confirmatory blot QAs, given sufficient availability of material . To ensure that sufficient material would be available for continuing with this type of activity in future years, it is proposed to use some of this experimentally generated material to challenge two further cattle with each variant – i.e. a further 4 cattle, to provide continuity of supply. Given that animals take between 550 and 650 days to develop disease (mean 579 for H-type and 614 for L-type) this would provide additional material towards the end of 2012.

2.0 Proficiency testing (see appended timetable)

2.1

The EURL 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 2011 and the second in October 2011. The format of these QA distributions will be based on a web-based QA system which will mean that we can use single existing slides to create electronic images, which can then be accessed by all readers at the same time. This will enable faster completion of distributions, and greater flexibility to include examples of unusual cases, challenging artefacts, different IHC protocols (drawn from the technical QA round – see below). This system will be administered through our QA Unit as

EURL Work Plan 2011

2011 workplan.doc

3

before, but the web images will be hosted by ‘Slidepath’, an external company which specialises in web-based imaging.

2.2

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

2.3

A proficiency test panel of ovine blood samples will be provided for the QA of laboratories undertaking genotyping for statutory purposes. Information will be requested about the methods used in each country. Reporting on 4 codons (136, 141, 154 and 171) of the ovine PrP gene will be required from all labs. Additionally, it is increasingly recognized that susceptibility to scrapie in goats is regulated very similarly to sheep through the PrP gene and its variations (polymorphisms). While, similar to the ovine gene, the caprine PrP gene is highly polymorphic, the polymorphisms are usually at different positions (codons) compared to sheep. Recently, several publications associated various polymorphisms in the goat PrP gene to susceptibility or resistance to scrapie (e.g. Goldmann et al., J Gen Virol (1996) 77:2885-2891; Vaccari et al, J Gen Virol (2006) 87:1395-1402; Papasavva-Stylianou et al., Vet Journal (2007) 173:459-462; Colussi et al., J Gen Virol (2008) 89:3173-3176; Barillet et al., J Gen Virol (2009) 90:769-776) and it is probably only a question of time until one of these findings is considered significant enough to find its way into regulations. In the light of these developments, we propose a QA pilot scheme for the caprine PrP gene. This would include sourcing of blood with different polymorphisms from other countries in the EU, validation of the test for caprine polymorphisms at the EURL, and the distribution of the blood to 5 laboratories (including the EURL), who would voluntarily take part in this QA pilot scheme.

2.4

The EURL 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. Following successful transmission of H & L type BSE to cattle (see 1.7), and dependent on the demonstration of appropriate test sensitivity levels (see 3.4) it is proposed that this round in 2011 will include an H and/or an L type BSE sample in addition to the bovine classical BSE

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

in ovine brainstem tissue. In previous years this covered classical scrapie only. [Please note that it is not possible to use samples from atypical scrapie animals at present because of signal degradation in homogenates. However this situation will be kept under investigation with a view to obtaining a suitably stable homogenate. At the same time we will also issue a proficiency test panel for confirmatory blotting methods to assess PrP

EURL Work Plan 2011

2011 workplan.doc

4

detection in ovine brain tissue. This will include an atypical sample as well as classical scrapie. We further intend to provide scrapie positive and negative goat samples in the rapid test round for 2011, which will have been trialled already in the 2010 round.

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

those NRLs which are operating such methods. Please note that atypical scapie, ovine BSE and bovine H & L type BSE samples and goat tissues are of limited availability and will not necessarily be included in subsequent years, unless challenged animals continue to succumb, and goat scrapie material can be sourced from outside the UK. The decision on whether to include this type of sample each year will depend partly on the performance of laboratories in receipt of the samples in the 2010 QA exercises, the outcome of which will not be known until near the end of 2010.

2.5

The EURL will monitor proficiency testing practices to ensure that they remain relevant, through discussion at the EURL 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 EURL, 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 EURL 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 EURL 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 EURL will maintain an up-to-date database of all relevant NRL contacts and contact details.

2.6

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

3.0 Provision of diagnostic and confirmatory testing and advice

3.1

The demand for diagnostic testing will depend on individual countries. Most Member States have adequate arrangements and do not require significant help with routine diagnostic testing. However, confirmation of results may be an important task for EURL, 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 EURL will continue to attempt to collect data on cases which are in some way ‘unusual’, to enable comprehensive cross-referencing and collation of information

EURL Work Plan 2011

2011 workplan.doc

5

on such cases for the Commission. Full instructions for sheep have been issued as part of the manual on Discriminatory Testing. The success of any parallel system for cattle data will be dependent on the willingness of MS to comply with such a request if our diagnostic opinion is not sought initially. Additionally, as part of the approval for the heat treatment protocol for the IDEXX test, NRLs must send any samples which are initially reactive on the IDEXX tests, but where the signal is ablated by the heat treatment protocol, to the EURL.Depending upon the amount of such samples which arrive, investigations into the nature of these samples will be conducted by the EURL. No costs have been explicitly planned for this purpose on the assumption that the submission rate will be very low. However, if significant numbers of referrals are made, a separate plan of work will be proposed for this group of samples.

3.2

The EURL will provide expert advice on the epidemiology and clinical manifestations of BSE and scrapie. In 2011 this will include clinical data generated from the experimental challenges with H- and L-type. The EURL will also provide scientific supervision of certain studies funded by the European Commission on request (see section 3.5)

3.3

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

3.4

Following production of H- and L-type BSE materials, the EURL proposes to create homogenates (as produced for existing proficiency tests) and run a limited analytical sensitivity test on all manufacturers’ approved BSE rapid test kits, to determine ability to detect these BSE variants. Once this has been completed, it is proposed to use the same materials (subject to availability – see 1.7) in proficiency tests as stated in section 2.4.

3.5 In continuation of the collaboration between the Cypriot veterinary services and the RL, we will provide advice and support to the ongoing work on the ‘TSE resistance in goats in Cyprus’ protocol. Bioassay components of the protocol will continue into 2011 at the EURL, with the remaining practical work undertaken by the Cypriot veterinary services; the costs for this have been submitted separately. However, the collation and interpretation of the field case data already generated, the epidemiological modelling using this data, and the integration and interpretation of this and the experimental data as it is gathered, will still require significant input and support from a range of disciplines within the RL.

4.0 Provision of training

4.1 A workshop for National experts will be arranged in the first half of 2011. This workshop will cover all aspects of NRL functions, with the exception of 4.3 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.

EURL Work Plan 2011

2011 workplan.doc

6

Provision has been made in 2011 for three laboratory visits to assist with technical issues should the need arise.

4.3

Training in rapid diagnostic techniques will not be provided. All the evaluated tests are commercially available and it is assumed that the manufacturers will provide training/guidance on the use of the tests. Similarly, should problems be encountered then it is appropriate that the manufacturers address these directly with the test users. Feedback from the national laboratories will alert EURL to any problems and the EURL will liaise closely with the national laboratory and the test manufacturer. General advice and information will be posted (where relevant) on the website. Rapid test manufacturers are now invited to participate in a specific session at the EURL workshop (see 4.1) each year where issues can be discussed directly with NRL representatives.

5.0 Strain typing

5.1

The EURL 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 EURL and NRLs, and partly from other sources. This group plans to meet twice in 2011. Discussion will continue to focus on the validation/interpretation of the increasing range of Tg bioassay methodologies. Data emerging from the spiked pool study (5.4) will also be considered by the group. 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. None are anticipated at present.

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 EURL strain typing group for subsequent investigation by ring-trial, the EURL will be liable for any laboratory costs incurred (see section 7.4).

5.3 Any isolate still considered BSE-like following ring trial (see section 7.4) will be forwarded for bioassay in mice. Historically, only conventional mice were sufficiently evaluated and defined for this purpose, and interpretation was based on a full panel (i.e. RIII, VM and C57Bl6). However, the choice of mouse strains has been under active discussion (see section 5.1) including the use of Tg strains. Although TSE strains have traditionally been characterised using wild-type mice, recently a number of transgenic lines have been used extensively in several European laboratories, and a large amount of data regarding their usefulness in identifying different TSE strains is now available, including unpublished data which is available to STEG. Indeed, some have already been adopted by STEG for the bioassay of demanding referred samples. A major advantage of these Tg lines

EURL Work Plan 2011

2011 workplan.doc

7

over the wild-type lines is their enhanced susceptibility to certain TSE isolates e.g. transgenic mouse lines are susceptible to atypical scrapie when conventional lines are not. Consequently we propose to change the STEG referral bioassay panel from 3x20 mice of three conventional lines to 10x Tg338 (VRQ ovine), Tg110 (bovine) and TgShpXI (ARQ ovine). The use of TgShpXI line is advisable because it is susceptible to certain classical scrapie isolates where Tg338 mice show prolonged incubation periods (e.g. Suffolk and Italian scrapie isolates). The cost estimates are based on the assumption that a maximum of 2 isolates will require further characterisation by bioassay.

5.4 A panel of samples representing mixtures of ovine BSE with various distinct scrapie sources (previously referred to as ‘spiked pools’) were subject to currentdiscriminatory testing methods in 2010. These were used as a crude proxy for co-infected animals. Preliminary results show that while the discriminatory WB performs reasonably well in most mixtures for the detection of BSE, the ELISA is less successful. None of the tests identified all the samples containing BSE. Phase 2 of this study proposes the use of Tg338 and Tg110 mouse panels to determine whether increased discriminatory sensitivity can be achieved using this in vivo model, by examining 2 mixed samples from each combination, at the point at which the biochemical tests lost discriminatory potential.

6.0 Rapid diagnostic methods

6.1 The EURL 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 EURL work cannot be readily predicted - if for example we have to undertake laboratory work to investigate a problem that arises in year. Additional costs may therefore arise in-year that would require additional funding, or a reassessment of EURLcommitments to enable delivery within the agreed annual budget. The EURL 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 EURL to confirm adherence to the manufacturer’s Quality System. An annual statement will be sent to the COM confirming which manufacturers continue to comply with these requirements, so that the listing in the regulation is kept up to date.

6.2

The EURL will continue investigations into a sustainable standardised source of positive control material. Currently, this is based upon a macerate or homogenate

EURL Work Plan 2011

2011 workplan.doc

8

of bovine brain material to provide all NRLs with the same material, which could be used to compare test performance. However, attempts will also be made to assess more sustainable source materials for the long term. (This issue is partially addressed under sections 1.5 –1.7). Homogenates will have been prepared in 2010 for QA testing in 2011. There will be requirement to prepare further supplies of homogenate in 2011 for use in 2012.

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 EURL website. The document will continue to be subject to annual review.

6.4

Batch testing of approved rapid tests for the detection of BSE in bovine samples was introduced in 2008. Nominated NRLs are responsible for testing to an agreed protocol and the EURL approves batches for release and communicates this information to NRLs for cascade to testing labs throughout the EU. Costs are based on current test usage, and an estimate of 40 batch releases per year. All documents are placed in the secure web areas within TSE-LAB-NET. Maintenance of this web area will incur external contractor charges from October 2011.

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

7.0 Discriminatory testing

7.1 The Discriminatory Testing Handbook detailing precise methods for discriminatory blotting was produced in 2005, updated in 2007 and again in 2009. This Handbook will be reviewed annually, revised and updated as necessary to include additional information of relevance to the surveillance of TSE, and re-issued online (.http://www.defra.gov.uk/vla/science/docs/sci_tse_rl_handbookv2mar07.pdf) A protocol, as presented at the STEG meeting in April 2008, for the discrimination of H and L type BSE from C type has been made available on line. It should be noted that the material to perform initial QA or QC (see section 1.7) is now available and will be used for basic test assessment (see 3.4). Until this has been done, any testing performed in NRLs using this protocol will, by definition, remain‘out of control’. (A referral system will be recommended, but cannot be enforced).

EURL Work Plan 2011

2011 workplan.doc

9

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

7.3 The EURL will continue the evaluation of cervinised mice (developed by Glenn Telling, University of Kentucky) for susceptibility to BSE relative to experimental cervid passaged BSE, to establish the suitability of this model for investigation of strain should any European cervid be identified through the surveillance programme. The experimental challenge of these mice was started mid –2008. To date (June 2010) the cervid-passaged BSE has resulted in disease, but the bovine BSE source has yet to result in disease in these mice. This would be anticipated from first principles given the presence of a species barrier). Maintenance of the Tg line at the EURL by minimal breeding, if this is considered necessary, will incur some cost. Once the evaluation of the cervinised mice for susceptibility to BSE is carried out, the continued maintenance of the cervinised mice at the EURL will depend on the eventual opinion of the European Food Safety Authority (EFSA) on the results of the EU cervid survey which has now been completed. The survey lasted three hunting seasons and some 12,000 analyses were made and all were negative. The results have been sent to EFSA by DG SANCO and additional information was requested by EFSA in order to provide a more complete answer to the terms of reference of the mandate. The deadline for the EFSA opinion is now 30th September 2010. Costs have been included in this workplan for a nucleus colony to be maintained, but this can be reviewed on the basis of the EFSA opinion.

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

7.5 One of the evaluated discriminatory tests (CEA discriminatory ELISA) has been carried out, to date, by Dr Jacques Grassi’s group in Gif sur Yvette, Saclay, France. This group are no longer active in TSE work and will be stopping provision of this assay in 2010. It is proposed that the EURL imports this test for continued use within any discriminatory ring trials in 2011. Whereas there are several validated discriminatory Western blot tests and two discriminatory IHC tests available this is the only validated ELISA based discriminatory test. It has been instrumental in ruling out BSE in sheep from the unusual cases (detected by Western blot screening methods) that have been referred to STEG in the past.

EURL Work Plan 2011

2011 workplan.doc

10

Appendix 1

PROVISIONAL TIMETABLE FOR TSE EURL QA EXERCISES IN 2011

Intended Start

Date i QA activity

February 2011

Ovine genotyping

March 2011 Immuno-histochemical technique

April 2011 Histopathology and immunohistochemistry interpretation (round 1)

October 2011 Histopathology and immunohistochemistry interpretation (round 2)

July 2011 Bovine rapid testing incorporating confirmatory blotting if appropriate

November 2011 Ovine rapid testing, incorporating confirmatory blotting if appropriate

September 2011 Ovine discriminatory Western blotting i Some QA exercises (such as the technical and slide interpretation) take several weeks or months to complete. Any follow-up activities will also lengthen the duration. It is not therefore possible to accurately predict completion dates for these activities.

EU REFERENCE LABORATORY FOR LISTERIA

MONOCYTOGENES

WORK PROGRAMME 2011

French agency for food, environmental and occupational health safety – Maisons-Alfort laboratory on food safety 23, avenue du Général de Gaulle - F94706 Maisons-Alfort Cedex - Téléphone : + 33 (0)1 49 77 13 00 - Télécopie : + 33 (0)1 43 68 97 62 - www.anses.fr

Maisons‐Alfort laboratory on food safety

2011 Work Programme of the Reference Laboratory of the European Union

for Listeria monocytogenes

Version 1 ‐ 10 August 2010

EU‐RL LmEuropean Union Reference Laboratory for Listeria monocytogenes

EU‐RL for Listeria monocytogenes 2/10 10/08/2010 2011 Work Programme

In May 2006, the Maisons‐Alfort laboratory for food safety of Anses (French agency for food, environmental and occupational health safety) ‐formerly AFSSA‐LERQAP‐ has been nominated European Union ‐ Reference Laboratory for Listeria monocytogenes (EU‐RL Lm, formerly CRL Lm) (see Regulation 776/2006).

The EU‐RL Lm foresees to undertake the following actions in 2011, according to the actions planned at the 4th Workshop of the National Reference Laboratories (NRLs) (15&16 May 2010), as well as to the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the EU‐RL for the period 2006‐2011.

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.

NB: in brackets under each item, the scheduled duration of the action is indicated: either annual (limited to 2011), either multi‐annual (on‐going programme on several years).


1. DETECTION AND ENUMERATION OF L. MONOCYTOGENES IN FOOD

1.1. INTER‐LABORATORY PROFICIENCY TESTING

(annual)

The inter‐laboratory proficiency testing (PT) trials organised by the EU‐RL 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.

1.1.1. STUDY OF SAMPLE TYPES FOR INTER‐LABORATORY TRIALS

The EU‐RL (Unit HMPA) will conduct a study to develop the sample types to be used for the PT trial to be organized in 2011, using as matrix powdered infant food formula (PIF). In particular, the homogeneity and stability of this type of samples will be studied, by mainly taking into account studies performed in 2010.

1.1.2. ENUMERATION OF L. MONOCYTOGENES

The EU‐RL (Unit HMPA) will organize in 2011 a PT trial for the NRLs on the enumeration of L. monocytogenes by the reference method EN ISO 11290‐2, using PIF as matrix.

From a regulatory point of view, Regulation 2073/2005 defines a quantitative food safety criteria (limit at 100 cfu/g) on this type of product (criteria 1.2&1.3 in Annex I). Moreover, PIF is not a sterile product, its microflora belongs mainly to Gram‐positive cocci and Bacilli, which can cause false‐positive results on ALOA agar.

1.1.3. COMPARISON OF INOCULATION TECHNIQUES OF SOLID FOOD MATRICES

Solid food matrices can be contaminated in depth or on surface, with various techniques, according to packaging in particular.

Different inoculation techniques of solid food matrices will be tested and compared so as to optimise the combination solid food matrix/inoculation technique.

This study would be used by the EU‐RL for its future PT trials and could help NRLs for the organization of their interlaboratory PT trials.



(multi‐annual)

Frame: The Standard methods 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.

1.2.1. 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 EU‐RL (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 at low levels.

Since 2008, the EU‐RL has tested the applicability of this membrane filtration method to various foods.As agreed at the 2009 workshop, in a second step, from 2011, dairy products and vegetable products will be studied.

1.2.2. REDUCTION OF THE SECOND ENRICHMENT STEP IN FRASER BROTH FROM 48 TO 24H

The Standard method for the detection of L. monocytogenes in foods (EN ISO 11290‐1) consists in a double enrichment in Half Fraser broth and Fraser selective broth. The initial incubation in Half Fraser is carried out for 24 hours at 30oC, followed by a 48h enrichment in Fraser broth at 37oC. The EU‐RL study, conducted in 2007‐2008 on the enrichment phases of the detection method, suggested that a 24h‐incubation in Fraser broth could be sufficient to reach the maximum population, instead of the current practice of 48h. This would enable to shorten the total duration of the Standard detection method by 24h, which would represent a significant improvement in its practicability. However, the reduction of the 2nd enrichment step requires the analysis of additional naturally contaminated samples from various origins, comparing the modified method with the current Standard method. In case of positive outcome of the study, the EU‐RL would transmit its outcome to the CEN and ISO structures in charge of EN ISO 11290‐1, with a recommendation to reduce the 2nd enrichment phase of this standard detection method.


In 2011, the EU‐RL (Unit HMPA) will go on this study on naturally contaminated samples, in collaboration with some NRLs and Canadian regulatory laboratories. To conduct this stage of the study, there is a need to receive from NRLs naturally contaminated samples from various origins. A call for samples will be launched.

1.2.3. ENVIRONMENTAL SAMPLING TECHNIQUES

After having reviewed the ISO 18593 Standard, the EU‐RL (Unit HMPA in collaboration with Unit SRPI) conducted in 2009 a bibliographic study in order to check whether the ISO Standard fully covers 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.

Given the outcome of this bibliographic review, it was agreed at the 2010 workshop that the EU‐RL, in association with a working group of volunteering NRLs and other FBOs, would draft a technical guidance document (TGD) on environmental sampling techniques specific to Listeria monocytogenes. The EU‐RL intends to complete this study in 2011.

1.2.4. MEASUREMENT UNCERTAINTY

EU‐RL GUIDE ON MEASUREMENT UNCERTAINTY

The EU‐RL Lm will go on the preparation of Version 2 of the EU‐RL Guide on measurement uncertainty (MU) in the case of Lm enumeration, with the NRLs working group. The EU‐RL will also participate to the revision of ISO/TS 19036 launched by ISO/TC 34/SC 9.

IMPACT OF TEST PORTION SUB‐SAMPLING

In the series of Standards EN ISO 6887‐2 to 5 on the preparation of test samples for microbiological analyses, it is not specified how to sub‐sample the test portion in the laboratory sample (sample that is sent to the laboratory), depending on the different types of food matrices to be submitted to microbiological analyses. This stage is however recognized as a major source of measurement uncertainty, in particular for solid matrices characterized by heterogeneous bacterial contaminations, such as matured cheeses.

The EU‐RL has launched a study to assess the impact of test portion sub‐sampling on measurement uncertainty, which will be continued in 2011. This requires the analysis of naturally contaminated samples from various origins. This study could be conducted in collaboration with some NRLs.

1.3. TRAINING OF THE NRLS

Upon request, the EU‐RL could receive NRLs for individual training.


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 its 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 EU‐RL Lm envisages conducting the following works.

2.1. TECHNICAL GUIDANCE DOCUMENT ON SHELF‐LIFE STUDIES

A Technical Guidance Document (TGD) on shelf‐life studies for L. monocytogenes in ready‐to‐eat foods prepared by the EU‐RL (Unit MQER) in 2008 describes the microbiological procedures for determining growth of L. monocytogenes using challenge tests and durability studies.

In 2010 a questionnaire was sent to the NRLs to assess the opportunity to prepare in 2011 a new version of the EU‐RL TGD. Few NRLs have performed challenge tests or durability studies since the publication of the TGD:

• 6 NRLs: challenge tests assessing the parameter « δ »

• 3 NRLs: challenge‐tests assessing the parameter « µmax »

• 4 NRLs: durability studies.

Considering the low number of NRLs having implemented these tests, it was agreed at the 2010 workshop to postpone the preparation of a new version of the TGD. Nevertheless, it was planned to deal with a specific point of the TGD: to harmonize the preparation of the inoculum. For that purpose, in 2011, a working group including scientists of the EU‐RL Lm and 3 NRLs will select a set of well documented strains, set which could be used to perform challenge tests. This WG will meet once in 2011.

2.2. EXPERTISE OF CHALLENGE TESTS/DURABILITY STUDIES

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


2.3. NRL TRAINING

Further to the sessions organized in 2008, 2009 and 2010, the EU‐RL (Unit MQER) will organize in 2011 a 4th session of the 4‐day theoretical and practical training for the NRLs on:

• durability studies;

• challenge tests;

• predictive microbiology softwares;

• shelf‐life of foods related to L. monocytogenes.

Two external experts will have an input in this training to present the 2 predictive microbiology softwares: Combase and Symprevius.

2.4. PROFICIENCY OF LABORATORIES IMPLEMENTING CHALLENGE TESTS AND DURABILITY STUDIES

The need has arisen from DG SANCO WG on microbiological criteria and from NRLs to give guidance on how to define the proficiency of laboratories conducting challenge tests and durability studies.

In 2010, a WG gathering 11 members from EU‐RL and NRLs met on 4 and 5 May in Porto (Portugal), in conjunction with the ISOPOL conference. The preparation of a document on the proficiency of laboratories conducting challenge‐tests assessing growth potential was launched.

In 2011, 3 sub‐WGs, working by electronic way, will draft the 3 parts of the document: requirements on the general organization of the laboratory, data needed in the application file, and evaluation criteria. The WG will meet once in 2011.

It is scheduled that this document could be finalized in 2012.

2.5. BEHAVIOUR OF LISTERIA IN BUTTERS AND MARGARINES

L. monocytogenes in butter recently created public health concern and raised questions about the development of L. monocytogenes in butters and margarines. Different factors may be taken into consideration as fat content, salt content, preservatives, pH, size of water droplets, storage temperature.

In 2011, the EU‐RL Lm (Unit MQER) will launch a study to assess the evolution of Listeria spp. in butter and margarines. The objectives are:

i. To acquire data related to the evolution of strains of Listeria innocua (as of surrogate of L. monocytogenes) in butters and margarines;

ii. To classify butters and margarines according to EC Regulation 2073/2005;


iii. To investigate interactions between the factors impacting the growth of the bacteria iv. To validate growth models.

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 EU‐RL 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. SETTING UP A CENTRAL EUROPEAN MOLECULAR DATABASE

(multi‐annual)

This database will aim to gather Lm molecular typing profiles from the MSs and from different non‐human sources. For submitting profiles to the central database, NRLs will have to satisfactorily participate to the PFGE and molecular serotyping PT trials organized by the EU‐RL.

The EU‐RL (Unit CEB) will establish this central database, to be implemented with the BioNumerics software and requiring a special server and assistance from Applied Maths for its functioning. After submission by the NRLs of molecular PFGE profiles to this database, the EU‐RL will check and register these profiles, then compare the profiles from the different participating NRLs.

In 2011, a working group of interested NRLs will be settled and will specify the details of profile submission.

3.2. TECHNICAL & SCIENTIFIC ASSISTANCE TO NRLS, DISPATCH OF STRAINS

(annual)

Upon request of the NRLs, the EU‐RL (Unit CEB) would provide technical and scientific assistance (in particular to perform PFGE and PCR), and would send them Lm field strains from its collection, as well as the control strain Salmonella Branderup H9812.

The EU‐RL will visit one NRL in 2011, depending on the needs.

3.3. INVESTIGATION OF RECENT MOLECULAR TYPING TECHNIQUES

(multi‐annual)

The EU‐RL (Unit CEB) will go on the study for the evaluation of recent molecular typing techniques, mainly Multi Locus Variable number tandem repeat Analysis (MLVA), in comparison with PFGE as reference method (collaboration with SSI, Denmark). The EU‐RL


will use MLVA sub‐typing methods in addition to PFGE for some investigations of listeriosis cases.

Molecular serotyping using a real‐time PCR method will be explored and compared to the conventional PCR one.

In addition, the EU‐RL will go on the study for the comparison of a new Fluorescent AFLP (Amplified Fragment Length Polymorphism) protocol with PFGE for the typing of L. monocytogenes (collaboration with the UK‐NRL, HPA, London).

A progress report on these studies will be given at the 2011 annual workshop.

3.4. PARTICIPATION TO THE EUROPEAN LM BASELINE STUDY IN RTE FOOD PRODUCTS ‐ SUB‐TYPING STEP OF FOOD ISOLATES

Once the coordinated monitoring programme on the prevalence of Listeria monocytogenes in certain ready‐to‐eat foods will have been carried out by MSs, it is envisaged that the EU‐RL (Unit CEB) will coordinate, in liaison ECDC, the molecular sub‐typing of the Lm isolates.

The EU‐RL would provide a technical and scientific assistance to the NRLs willing to sub‐type the food isolates from their respective countries. PFGE would be performed by NRLs that have demonstrated their competence according to their participation to the EU‐RL PFGE PT trials. NRLs should submit PFGE profiles to EU‐RL for comparison and further interpretation. PFGE profiles should be transmitted to EU‐RL through a defined protocol. Molecular profiles would be entered and saved into the European central database (see 3.1).

4. WORKSHOP OF THE NRLS

(annual)

The EU‐RL will organise the 5th Workshop of the NRLs in 2011, of general scope:

‐ to make a progress report on works undertaken by the EU‐RL since the 2010 Workshop; ‐ to envisage the work programme for 2012 and further.



(multi‐annual)


‐ participation of the EU‐RL coordinator, for the analytical aspects, to the update of Regulation 2073/2005 on microbiological criteria related to Lm (in particular to the on‐going addition to the existing criteria on Lm);


‐ technical and scientific assistance of the Unit MQER for the implementation of the Annex II on studies to verify compliance with the 100 cfu/g‐ml limit at the end of shelf‐life;

‐ and any new question which may arise during the year.

5.2. PARTICIPATION TO CEN/ISO STANDARDIZATION ACTIVITIES

(multi‐annual)

On behalf of the EU‐RL 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 (scheduled in Bournemouth, UK, June 2010), in particular 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).

Meeting with USDA/FSIS to investigate possibilities of harmonization of reference methods (FSIS official method and Standard methods EN ISO 11290‐1&2).

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 »

EU REFERENCE LABORATORY FOR COAGULASE POSITIVE STAPHYLOCOCCI

WORK PROGRAMME 2011

French agency for food, environmental and occupational health safety – Maisons-Alfort laboratory f or food safety 23, avenue du Général de Gaulle - F94706 Maisons-Alfort Cedex - Téléphone : + 33 (0)1 49 77 13 00 - Télécopie : + 33 (0)1 43 68 97 62 - www.anses.fr

Maisons‐Alfort laboratory for food safety

2011 Work Programme of the European Union Reference Laboratory for Coagulase Positive Staphylococci

Version 1 – 10 August 2010

EU‐RL CPSEuropean Union Reference Laboratory for Coagulase Positive Staphylococci

EU‐RL for Coagulase Positive Staphylococci 2/10 10/08/2010 2011 Work Programme

In May 2006, the Maisons‐Alfort laboratory for food safety of Anses (French agency for food, environmental and occupational health safety) –formerly AFSSA‐LERQAP‐ has been nominated European Union Reference Laboratory (ex‐CRL) for Coagulase Positive Staphylococci (EU‐RL CPS, formerly CRL CPS), including Staphylococcus aureus and their toxins (see Regulation 776/2006).

The EU‐RL CPS foresees to undertake the following actions in 2011, according to the actions planned at the 4th Workshop of the National Reference Laboratories (NRLs) (17&18 June 2010), as well as to the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the EU‐RL for the period 2006‐2011.

Most of these activities aim at implementing, from an analytical point of view, the EC Regulation 2073/2005 on microbiological criteria for foodstuffs, modified by the Regulation 1441/2007, 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.

NB: In brackets under each item, the scheduled duration of the action is indicated: either annual (limited to 2011) , either multi‐annual (on‐going programme on several years).


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

(multi‐annual)

As to be able to organize an inter‐laboratory proficiency testing (PT) trial on a new matrix compared to the former PT trials, the EU‐RL (Unit HMPA) will complete and finish an investigation study (stability and homogeneity) launched in 2009 to prepare an artificially contaminated freeze‐dried milk powder which could be used to prepare and dispatch samples for quantitative CPS determinations.

1.2. COMPARISON OF INOCULATION TECHNIQUES OF SOLID FOOD MATRICES

(multi‐annual)

Solid food matrices can be contaminated in depth or on surface, with various techniques, according to packaging in particular.

Different inoculation techniques of solid food matrices will be tested and compared so as to optimise the combination solid food matrix/inoculation technique. A laboratory, AINIA (ES), having developed a spray‐drying technique, will be visited.

This study could in particular help NRLs to organize their interlaboratory PT trials at national level.

1.3. MEASUREMENT UNCERTAINTY: IMPACT OF SUB‐SAMPLING OF THE TEST PORTIONS

(multi‐annual)

In the series of Standards EN ISO 6887‐2 to 5 on the preparation of test samples for microbiological analyses, it is not specified how to sub‐sample the test portion in the laboratory sample (sample that is sent to the laboratory), depending on the different types of food matrices to be submitted to microbiological analyses. This stage is however recognized as a major source of measurement uncertainty, in particular for solid matrices characterized by heterogeneous bacterial contaminations, such as matured cheeses.

In July 2010, an enquiry was sent to the NRLs on sub‐sampling of the test portions in a first step for cheeses, in order to achieve an inventory of practices in the NRLs/Member States.


The Standard applicable for the preparation of test sample for milk and dairy products is EN ISO 8261, to be soon replaced by EN ISO 6887‐5.

In 2011, the EU‐RL (Unit HMPA) will launch an experimental study on sub‐sampling of the test portions in cheeses. This will require the analysis of naturally contaminated samples from various origins. This study could be conducted in collaboration with some NRLs.

Moreover, the EU‐RL (Unit HMPA) will send an enquiry to the NRLs on the sub‐sampling of the test portions for other solid matrices than cheeses.

1.4. ALTERNATIVE ENUMERATION METHODS OF COAGULASE POSITIVE STAPHYLOCOCCI IN FOOD

(multi‐annual)

At the 2009 workshop, some NRLs asked whether it was possible to have guidance on other more recent methods than a colony‐count technique to enumerate CPS, including Real Time PCR.

In 2011, the EU‐RL (Unit HMPA) will complete a bibliographical study on these alternative methods, including their degree of validation.


2. DETECTION OF STAPHYLOCOCCAL ENTEROTOXINS IN FOOD

Frame: The European screening method of the EU‐RL CPS is cited as reference method in the criterion for staphylococcal enterotoxins (SETs) by the EC Regulation 2073/2005 modified.

2.1. OPTIMIZATION OF THE EUROPEAN SCREENING METHOD

Given the need to suspend the use of the Transia kit in the European Screening Method (ESM) of the EU‐RL CPS, the EU‐RL (Team CAT‐BAC) has conducted in 2010 an intra‐laboratory validation study of the method using 2 other kits, the Ridascreen SET Total kit and the Vidas SET2 kit.

It will organize in 2011 an inter‐laboratory validation trial to fully validate the modified method.

2.2. INTER‐LABORATORY PROFICIENCY TESTING TRIAL

(annual)

The inter‐laboratory proficiency testing (PT) trials organised by the EU‐RL 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 modified.

The EU‐RL (Team CAT‐BAC) will organize in 2011 an inter‐laboratory PT trial on the SE detection in cheese matrices by the updated version of the ESM.

Other food type matrices could be also tested if the studies performed on other matrices than milk products performed in 2010 would give satisfactory results.

2.3. EVALUATION OF COMMERCIALLY AVAILABLE ANTIBODIES AGAINST SE

(multi‐annual)

In order to transfer to the NRLs a quantitative method, the EU‐RL (Team CAT‐BAC) evaluated commercially available antibodies to perform a specific ELISA test to quantify SE types A to D (and/or E) in milk‐based products.

In 2011, the EU‐RL will complete the evaluation of commercial available antibodies against SEs and will propose to the NRLs training sessions on the detection of SEA to SED (and/or SEE) in milk and milk products using the quantitative ELISA developed with commercial available antibodies (see 2.6).


2.4. USE OF MASS SPECTROMETRY FOR SE CHARACTERISATION AND QUANTIFICATION IN FOOD

(multi‐annual)

The EU‐RL (Tem CAT‐BAC) has launched in collaboration a project to investigate alternative tools to immunology, such as the use of quantitative mass spectrometry (MS), to confirm and quantify SEs presence in food matrices.

In 2011, this methodology should be implemented in the laboratory. The team CAT BAC will optimise and evaluate a multiplex quantitative MS methodology using protein standard absolute quantification (PSAQ) for its application in food matrices, at the condition that this PSAQ is made available at the laboratory.

2.5. DEVELOPMENT OF CERTIFIED REFERENCE MATERIALS

(multi‐annual)

The need of certified reference materials (CRMs) for SEs in food is one of the priorities of the EU‐RL for CPS and a major need for the NRLs.

Further to an official request sent in 2010 by DG SANCO to JRC/IRMM (Geel, Belgium) to develop such CRMs, JRC promised to organise a meeting with DG SANCO and the EU‐RL to envisage a collaboration in this field.

2.6. DEVELOPMENT AND OPTIMIZATION OF IMMUNO‐QUANTITATIVE PCR FOR SE DETECTION IN FOOD

(multi‐annual)

The immuno‐quantitative PCR (iqPCR) is a procedure similar to conventional ELISA (used in the ESM) but allows for a more sensitive SE detection in food, as well as for SE quantification. Compared to the current ESM, the purpose is to improve the sensitivity of SE detection (10 to 1000 fold), to reduce the duration of the test, and in addition to allow for SE quantification.

The EU‐RL (Unit HMPA and Team CAT‐BAC in collaboration) has started in 2009 to develop and to apply this technique to detect and quantify SEA and SED in food. This development and evaluation will be continued in 2011:

• To test the reproducibility of the method (using optimized conditions);

• To test the specificity of the method against other major enterotoxins (SEB, SEC, SED, SEE) in order to assess possible cross‐reactions;

• To determine the limit of detection (LOD) and of quantification (LOQ) on standard curves, culture supernatants and on food matrices.


2.7. NRL TRAINING

(multi‐annual)

The EU‐RL (Team CAT‐BAC) will organize in 2011 one (or two) training session(s) on the detection of SEs for the NRLs, using dialysis‐extraction and a detection by both Vidas SET2 and Ridascreen SET Total detection kits.

Moreover and depending on the needs, complementary training sessions could be organized on a quantitative SEA to SEE ELISA detection method recently developed in the Team CAT‐BAC using commercial available antibodies (see 2.2).

2.8. TECHNICAL AND SCIENTIFIC ASSISTANCE

(multi‐annual)

Depending on the needs, the EU‐RL for CPS (Team CAT‐BAC) will collaborate and provide scientific and technical assistance to DG SANCO and to the NRLs, especially to perform confirmation analysis of positive results obtained by the NRLs with the ESM, in the frame of official controls performed according to the Regulation 2073/2005 modified.


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 EU‐RLs (SANCO/2214/2005), the Annex 1 describes the specific functions of the EU‐RL 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.

3.1. DISPATCH OF STRAINS

(multi‐annual)

Upon request of the NRLs, the EU‐RL (Unit CEB) would send them CPS field strains from its collection.

3.2. STRENGTHENING OF MOLECULAR PFGE DATABASE

(multi‐annual)

The EU‐RL (Unit CEB) will go on the PFGE typing of ST398 “not typable” strains collected in the frame the EU 2008 baseline survey on S. aureus ST398 prevalence in breeding pig farms and other ST 398 strains (n=200).

The EU‐RL (Unit CEB) will go on the spa typing of all PFGE database strains (n=350).

3.3. DEVELOPMENT OF SE GENES DETECTION BY MULTIPLEX PCR AND OTHER MOLECULAR TECHNIQUES

(multi‐annual)

3.3.1. MULTIPLEX PCR

Upon request of the NRLs, the EU‐RL (Unit CEB) will perform the detection of se genes on CPS strains sent by the NRLs.

3.3.2. OTHER MOLECULAR TECHNIQUES

At the 2010 annual workshop, it was agreed to settle in 2011 a working group on the harmonization of an RT‐PCR scheme for se genes detection, based on a revue of the different schemes available to be conducted by the EU‐RL. An harmonized protocol scheme will be submitted to the NRLs at the 2012 annual workshop. Working group’s activities will be supported by preliminary analysis performed at the EU‐RL on its RT‐PCR equipment.


3.4. INVESTIGATION OF RECENT MOLECULAR SUB‐TYPING TECHNIQUES

(multi‐annual)

Further to the 2010 annual workshop, the EU‐RL will launch in 2011 a collaboration with interested NRLs to develop new molecular sub‐typing methods.

New subtyping techniques, developed by the NRLs, would be compared with the PFGE sub‐typing method performed at the EU‐RL. In the frame of this collaboration, a common strain panel, mixing local strains from the NRLs and EU‐RL strain collection, will be created. Comparison of typing data will allow the assessment of different methods on a common strain panel.

3.5. PFGE TRAINING SESSION

(Annual)

Upon NRL request, the EU‐RL may organize a training session on PFGE sub‐typing of CPS.


4. WORKSHOP OF THE NRLS

(annual)

The EU‐RL will organise the 5th Workshop of the NRLs in 2011, of general scope :

‐ to make a progress report on works undertaken by the EU‐RL since the 2010 Workshop; ‐ to envisage the work programme for 2012 and further.



(multi‐annual)


‐ If needed, participation of the EU‐RL 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 EU‐RL and as EC representative, follow‐up by the EU‐RL 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 EU‐RL L. monocytogenes).

In particular, participation to the works of one working group of ISO/TC 34/SC 9 of specific interest for the EU‐RL activities and for DG SANCO: WG 3 “Method Validation” (2 meetings).

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 »

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

(VTEC)

WORK PROGRAMME 2011

Page 1 of 11

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

Unit of Foodborne Zoonoses and Veterinary Epidemiology Istituto Superiore di Sanità

EU Reference Laboratory (EU-RL)

for Escherichia coli,

including Verotoxigenic E. coli (VTEC)

Work Programme

1st January - 31st December, 2011

Page 2 of 11

Introduction

The work programme of EU-RL for VTEC (EU-RL VTEC) for the year 2011 will

consist of the following activities:

1. Consolidating the EU-RL structures

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

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

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

2.4. Characterization of VTEC strains isolated by the NRLs 3. Implementation of the EU-RL-VTEC web site 4. Co-operation with other EC or non-EU structures and projects

4.1. The European Food Safety Authority (EFSA)

4.2. The European Committee for Standardization (CEN) 4.3. The European Centre for Disease Control (ECDC) 4.4. The FP7 Coordination Support Action “The Disease Control Tools” (DISCONTOOLS) 4.5. U.S. structures and initiatives active in the field of VTEC infections

prevention and control 5. Inter-laboratory comparison studies

5.1. PCR typing of vtx genes variants 5.2. Detection of VTEC non-O157 in food

6. Research activities on VTEC 7. Missions

The duration of each action is indicated, and will be either limited to 2011 or multi-

annual (ongoing program).

Page 3 of 11

1. Consolidating the EU-RL structures

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

RL’s activities. Four persons, hired with EU-RL funds, will continue to work full time

at the EU-RL-related activities with the status of “temporary staff employees”.

(Duration: ongoing)

1.2. Accreditation of the laboratory The EU-RL VTEC is constantly improving the effectiveness of its management

system through the use of the quality policy, according to its accreditation UNI

EN/ISO 17025:2005 (N. 0779) obtained in 2007 from SINAL, the Italian accreditation

body. Beside the management of the laboratory, the accreditation covers the

methods for detection and typing of VTEC related with EU-RL’s tasks and activities.

The possibility to submit additional methods for accreditation will be evaluated.

The EU-RL has decided to organize its inter-laboratory comparison and proficiency

studies according to the International Standard ISO/IEC 17043:2010 “Conformity

assessment – General requirements for proficiency testing”, which has recently been

issued (First edition 01/02/2010). The studies carried out in 2010 were organized

according to the requirement defined in the above mentioned standard, and the

same will apply to the studies for 2011 (see point 5). The Laboratory has initiated the

process to get a formal accreditation ISO/IEC 17043:2010 as proficiency tests

provider, which should be completed within the year 2011.

(Duration: ongoing)

1.3. MLVA-typing of VTEC Most bacterial genomes contain tandem duplications of short DNA sequences,

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

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

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

to pulsed-field gel electrophoresis (PFGE). MLVA for VTEC O157 was implemented

and is available at the EU-RL VTEC, and a Standard Operating Procedure will be

published in the “methods” section on the EU-RL website for the benefit of the NRLs

institutions (see also point 2.4).

Page 4 of 11

Although first developed for VTEC O157 typing only, the possibility to generate

MLVA profiles useful for strain typing from VTEC non-O157 is under investigation at

EU-RL VTEC. Two different strategies will be attempted, involving either the

possibility to use the primers for VTEC O157 loci for non-O157 VTEC typing or the

study of new loci specific for top five VTEC serogroups. The latter strategy will

involve research studies in collaboration with European and non-EU institutions (see

points 4 and 6).

Duration: ongoing

2. Coordination of the NRLs network and provision of technical assistance and training 2.1. Annual workshop with NRLs The 6th annual workshop will be held in the second half of 2011 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 2011 inter-laboratory studies will be

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

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

the NRLs needs. The program will also include updates on the surveillance and

monitoring activities of VTEC infections carried out in the EU, information on new

diagnostic tools, research results, recommendations, and exchange of experiences

with presentations made from the NRL representatives. Representatives from EFSA

and from the European Centre for Disease Control (ECDC) will be invited.

2.2. Assistance to NRLs The EU-RL-VTEC will continue to assist the NRLs in the field of VTEC detection and

typing, providing methods and standard operating procedures via the web site, and

reference materials, in particular VTEC strains. Drafts of other standard operating

procedures for detection of other pathogenic Escherichia coli in animals, food, and in

other relevant matrices and for typing of the isolated strains will be developed and

discussed with the NRLs. If needed, the EU-RL-VTEC will visit NRLs to help in

solving problems.

Duration: ongoing

Page 5 of 11

2.3. Training

Upon request from NRLs within EU or from governmental institutions of third

countries, the EU-RL will be available to receive visits of staff for individual training

on specific topics related with detection and typing methods.

Duration: ongoing

2.4. Characterization of VTEC strains isolated by the NRLs The phage-typing and MLVA-typing techniques for subtyping VTEC O157 strains

have been implemented and are available at the EU-RL VTEC. The original project

was to applying these techniques to representative subsets of VTEC O157 strains

isolated in different countries in order to draw a picture of the VTEC O157 clones

circulating in the animal reservoirs and the food vehicles in the EU. However, this

project has been hampered by the difficulties and the high costs of the shipment of

VTEC O157 strains from the NRLs to the EU-RL. Therefore, we will evaluate the

possibility to perform the MLVA characterizations at the EU-RL, using DNA samples

extracted from representative strains and sent by the NRLs. The MLVA protocol

could also be transferred to the NRLs interested in performing this typing method in

their own laboratories. For this purpose, the EU-RL will be available to provide

specific training and external quality assurance of the results. The MLVA profiles

obtained will be collected in a database for VTEC isolated from food and from

animals held at the EU-RL VTEC, that will be available for comparison with the

profiles of the strains causing human infections throughout Europe. Phage-typing

could be applied to a limited number of strains, representative of clusters of strains

identified by MLVA.

Duration: ongoing

3. Maintaining and implementing the EU-RL VTEC web site

The web site of the EU-RL VTEC (http://www.iss.it/vtec) will be maintained, and

updated on a regular basis with documents, methods, workshops reports,

information on the NRLs, and links. The “Restricted Area” will be used for the on-line

submission of the results of the inter-laboratory studies.

Duration: ongoing

Page 6 of 11

4. Co-operation with other EC or non-EU structures and projects

The EU-RL will continue the cooperation with EC structures or EC-supported

projects active in the field of human and animal health and food safety. Cooperation

will also involve non-EU initiatives on the prevention and control of VTEC infections.

The following liaisons will be maintained and implemented:

4.1. The European Food Safety Authority (EFSA) The EU-RL will contribute as reference laboratory (scientific and technical advice,

elaboration of methods, sub-typing of VTEC O157, etc.) to any program on VTEC

monitoring that will be implemented by the EFSA Task Force on Zoonoses Data

Collection, according to the “Technical specifications for the monitoring and reporting

of VTEC on animals and food on request of EFSA” (EFSA Journal; 7(11): 1366).

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

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

The EU-RL VTEC presented in 2007 a validation project for the EN/ISO 16654:2001

in response to the Mandate for standardization M/381 addressed to CEN. The

project is based on a collaborative study involving 13 NRLs for E. coli and has been

approved by CEN for the final submission. Although the project had positive

feedback, the process of enrolling the laboratories is still in stand-by, waiting for the

EC decision on the release of funds.

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

The project for the development of an international standard on the detection of

VTEC in food and animal feed, chaired by the EU-RL VTEC, has been successfully

addressed. The draft method has been formally endorsed by the CEN TC 275 WG6

members during the last plenary meeting held in Buenos Aires on June 2010. The

document has been sent for the final vote before publication as an ISO Technical

Specification.

Meanwhile, a joint study between the EU-RL VTEC and the Institute for Health and

Consumers Protection of the Joint Research Centre (JRC) in Ispra (VA, Italy) for the

validation of the method is ongoing. The preliminary phase, which addressed the

determination of the performance characteristics of the different steps of the method,

was concluded. The following steps will include the drafting of a technical report on

Page 7 of 11

the first part of the study, as well as the designing of the collaborative study needed

for the validation. The experimental design of this collaborative study should be

drafted within the first half of 2011. The study could be carried out in 2012 and will

involve the NRLs network.

4.3. The European Centre for Disease Control (ECDC) Food- and Waterborne

Diseases (FWD) Network (formerly Enter-net) The EU-RL will continue to take part into the Coordination Group of the ECDC FWD

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

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

safety fields (Regulation (EC) No. 882/2004). The EU-RL-VTEC will also continue

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

International Escherichia and Klebsiella Centre of the Statens Serum Institut,

Copenhagen), which is in charge of the external quality assurance activities for the

network. This will allow the harmonization of the identification and typing schemes,

making the respective monitoring programs and databases compatible for

comparison of human and non-human data. In the framework of this collaboration,

the refinement of a PCR-based method for the characterization of the different vtx

genes subtypes, as defined by the nomenclature scheme proposed in 2009, is

ongoing. In 2010 the Statens Serum Institut provided a group of laboratories

specialized in VTEC diagnostics with a shared reference collection, constituted by

48 VTEC strains, with the aim of applying the draft protocol for vtx genes subtyping.

The analysis of the results is currently ongoing and the data obtained will be useful

for the evaluation of the method. Typing of vtx gene variants will also constitute the

subject of an inter-laboratory study with the NRLs in 2011 (see point 5.1).

4.4. The FP7 Coordination Support Action “The Disease Control Tools” (DISCONTOOLS)

The DISCONTOOLS project, funded by DG Research and led by IFAH-Europe, is a

joint initiative of industry and a wide range of stakeholders, including the research

community, to provide a mechanism for focusing and prioritising research that

ultimately delivers new and improved vaccines, pharmaceuticals and diagnostic

tests for animal diseases and zoonoses (http://www.discontools.eu).

DISCONTOOLS operates in the framework of the European Technology Platform for

Page 8 of 11

Global Animal Health (ETPGAH) and deals with 47 animal diseases and/or

zoonoses. The EU-RL VTEC coordinates the Expert Group on VTEC infections,

which has already drafted documents on the current knowledge on this zoonosis and

on the gaps in terms of research and product availability. As group coordinator, the

EU-RL will continue to cooperate with the initiatives of the project.

4.5. U.S. structures and initiatives active in the field of VTEC infections prevention and control The EU-RL has established collaborations with laboratories and initiatives in the

U.S. that are dealing with VTEC in a food safety perspective. The following liaisons

will be maintained and implemented:

4.5.1. The U.S. Department of Agriculture (USDA)

The collaboration with the Eastern Regional Research Center of USDA (Wyndmoor,

PA, USA) will continue. The collaboration aims at identifying genetic markers that

could represent candidate targets for the detection of VTEC in food. A joint research

program is being developed aiming at studying VTEC strains belonging to the main

sero-pathotypes causing severe disease in humans by means of high throughput

technologies such as microarray and whole genome automated sequencing (see

also point 6).

4.5.2.The CAP STEC Consortium (Coordinated Approaches to Prevent Shiga Toxin-

producing E. coli in Beef)

The CAP STEC Consortium consists of a multi-institutional and multi-disciplinary

team of investigators, from leading institutions within the U.S. and abroad, who will

work together to integrate cutting-edge, basic and translational research on STEC in

beef. The CAP STEC consortium was formed in April of 2010 to find solutions to

better diagnose, treat, prevent and control STEC in beef in response to a request for

proposal from USDA National Institute of Food and Agriculture Competitive

Programs. Dr. Stefano Morabito has been invited to take part in the External

Advisory Board and will participate in the activities of this body, which are: review of

the program activities, advice to the Scientific Advisory Board, participation in the

annual CAP STEC meeting.

4.5.3.The US STEC Research

The US STEC Research is a team of US researchers created in response to the

Page 9 of 11

request for proposals from USDA National Institute of Food and Agriculture

Competitive Programs. The research team is focused on proposing research

activities on STEC non-O157 in the framework of the granting program. Following a

request to the EU-RL VTEC to join the Scientific Advisory Panel of this research

group, Dr. Morabito has been designated as contact person. The first meeting of the

advisory panel is scheduled for November 2010, and Dr. Morabito will attend that

meeting. During the event, the work-plan of the panel activities and the 2011 annual

meeting will be scheduled. In particular, the tasks of the panel, to be carried out

during 2011 will be:

• Critical review of the proposal.

• Participation in the annual meeting.

• Provide update on research on STEC.

Duration: ongoing

5. Inter-laboratory comparison studies Two studies are planned for 2011. A third for the validation of the method EN/ISO

16654:2001 for the detection of E. coli O157 in food could be carried out upon

availability of CEN funding (see points 4.2.1).

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

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

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

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

same panel of VTEC strains harboring different vtx gene subtypes will be sent to the

NRLs of both the medical and veterinary/food networks. The NRLs will be provided

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

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

and typing schemes and the compatibility of the respective monitoring programs and

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

5.2. Detection of VTEC non-O157 in food samples

According to the EFSA “Technical specifications for the monitoring and reporting of

VTEC on animals and food on request of EFSA” (EFSA Journal; 7(11): 1366), the

Page 10 of 11

study will consist on the detection of VTEC non-O157 strains belonging to

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

food matrices considered as likely sources of infection. According to the above

mentioned EFSA guidance, the detection method will be the draft ISO-CEN

Technical Specification “Horizontal method for the detection of Shiga toxin-producing

Escherichia coli (STEC) belonging to O157, O111, O26, O103 and O145 serogroups

- Qualitative Method” (see point 4.2.2). Duration: 2011

6. Research activities on VTEC Research studies on the different issues concerning VTEC, including pathogenicity,

phylogenesis and molecular epidemiology, represent the rationale for developing

tools for the detection of these pathogens in the reservoirs and vehicles as well as

for the study of the epidemiology of the infections. Such studies are therefore carried

out at the EU-RL VTEC, which is involved in different research programs also in

collaboration with European and non-EU scientific partners (see points 4.3 and 4.5).

The topics currently under investigation include the characterization of the mobile

genetic elements conferring virulence to the most pathogenic VTEC strains, the

genetic determinants shared between atypical EPEC and VTEC, as well as the

phylogenetic relationships between these two E. coli pathogroups. Other research

areas will include the study of the whole genome of VTEC strains belonging to the

main sero-pathotypes causing severe disease in humans by means of high

throughput technologies such as microarray and whole genome automated

sequencing. Such studies will allow unraveling the molecular bases of the virulence

of these pathogens and will help in filling the gap in the knowledge on the biology of

VTEC non-O157.

Duration: ongoing

7. Missions The following missions may be needed in 2011:

• Participation of a EU-RL representative in 2 meetings of the Coordination Group

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

Page 11 of 11

Network, presumably in Stockholm.

• Participation of a scientist at the 18th CEN/TC275 WG6 annual plenary meeting,

to be held in the UK. • Participation of a scientist at a EU-RL-VTEC/JRC meetings for drafting the

preliminary validation report and defining and managing the collaborative study

for the ISO/TS validation study. • A visit to one NRL can be planned for 2009, upon agreement with the EC and the

interested country.

August 25th, 2010

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

EU REFERENCE LABORATORY FOR CAMPYLOBACTER

WORK PROGRAMME 2011

EU-RL- CAMPYLOBACTER WORK PROGRAMME FOR 1st OF JANUARY 2011 TO 31st OF DECEMBER 2011 Introduction The activities in the work programme for 2011 for the EU Reference Laboratory (EU-RL) - Campylobacter will follow EU legislation on CRLs (now EU- RLs) functions, duties and designation (Regulation (EC) No 882/2004 and Commission Regulation (EC) 776/2006). The work programme for 2011 will consist of the following key activities, repeated annually:

1. Proficiency test(s) 2. Workshop 3. Research and development 4. Scientific and technical assistance to the European Commission and to the NRLs-

Campylobacter, including ad hoc activities 5. Missions 6. Communication 1. Organisation of proficiency test(s) in 2011 Short background The EU-RL has so far organised 7 proficiency tests (“ring tests”) for the NRLs. Three tests have included both detection and enumeration of Campylobacter in broiler skin or meat. Basically, the protocol for analysis (the SOP) has followed the standardised protocols of ISO 10272 Part 1 and Part 2: 2006 “Microbiology of food and animal feeding stuffs – Horizontal method for detection and enumeration of Campylobacter spp”. The majority of NRLs have performed very well with the analyses. The tests have been developed to correspond to the type of analyses that are common in official control of Campylobacter in the food chain in the EU MSs. 1.1. Detection and enumeration of Campylobacter in meat matrices The planned proficiency test in 2011 will consist of detection and enumeration of Campylobacter in meat matrices, basically using the abovementioned ISO standards. The ISO 10272 standards are under revision and therefore other enrichment broths and incubation times and temperatures may be tested in the proficiency test. Suitable modifications to be tested will be discussed at the EU-RL-Campylobacter workshop in October 2010. Freeze dried bacterial cultures will be used as reference material. Meat matrices will be purchased and thoroughly tested to ensure that they are free from Campylobacter and suitable to be used for the test. The EU-RL will prepare a test protocol (SOP) and test report. The ring test is planned to be distributed by courier in the spring 2011.

1.2 Identification of Campylobacter in swab samples. Ad hoc proficiency test An additional proficiency test may be organised with identification of Campylobacter in swab samples. The need and extent of an additional proficiency test will be discussed at the workshop in October 2010. 2. Organisation of a workshop A workshop will be organised in the fall (probably in October) in 2011. Representatives from the Member States´ NRLs for Campylobacter will be invited. The EU Candidate Countries and Iceland, Norway and Switzerland will also be invited to participate at own costs. Candidate Countries are advised to apply for financial assistance by TAIEX. As in previous years, experts from DG- SANCO, the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) will be invited. The agenda will include presentations and discussions on:

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

- Results of proficiency test(s) - Updates on analytical methods, including validation/assessment of methods for

detection and enumeration of Campylobacter and molecular methods for identification and characterization of Campylobacter strains

- Campylobacter activities in MSs, including national monitoring and research studies

- Information about proficiency tests to come - Information from meetings and activities within working groups of ISO/CEN,

ISO/TC34/SC9 and CEN/TC 275/WG6 - Information about revision of ISO standards - Future EU-RL-Campylobacter- NRL collaboration and activities, depending

on recent and urgent matters of common interest

3. Research and development 3.1. Studies on bacterial and matrix reference materials for the proficiency test(s). 3.1.1. Detection and enumeration of Campylobacter in meat matrices As for the proficiency test in 2010, the EU-RL plans to purchase vials with freeze dried reference material consisting of Campylobacter and other bacterial species of different and specified concentrations. The EU-RL will test batches of the freeze dried reference material with selected matrices and different conditions (enrichment broths, incubation times and temperatures) to ascertain the stability of the test procedure. The test will be delivered by courier. Although the test should arrive at the NRL within 48 hours, it could take longer time to some destinations and therefore the EU-RL prepares samples that are stable for at least 4 days. 3.1.2. Identification of Campylobacter in swab samples. Ad hoc proficiency test For an ad hoc proficiency test, selected isolates will be thoroughly tested with phenotypic tests and PCR-based assays. The isolates will be tested for stability and sent as swab

samples in Amies transport medium containing charcoal. They will be transported by courier. 3.2. Validation study. ISO 10272 Part 1 and Part 2:2006 have not been validated in collaborative tests. The proficiency test 2010 served as a pre-study for validation. The results will be discussed at the workshop in October 2010 and the need for further validation studies will be investigated. 3.3. DNA-based (molecular) methods for detection, species identification and strain characterization of Campylobacter. Short background The traditional methods for detection and identification of Campylobacter include culture in selective media, followed by phenotypic methods for genus and species identification. Although molecular or DNA based methods are considered more reliable, there are no ‘golden standard’ methods for detection, species identification or strain characterization (subtyping/fingerprinting). PCR-based tests for detection of Campylobacter in food items have recently become commercially available. An increasing number of NRLs are now using PCR-based assays for identification of Campylobacter. The EU-RL is often asked by the NRLs for guidance which test they should choose and there are requests from the manufacturers to evaluate their tests too. 3.3.1. PCRs for detection and identification of Campylobacter. The EU-RL will perform comparative studies and assess PCR-based methods in order to assist the NRLs. Both traditional PCR and realtime PCR assays will be reviewed and selected for further testing, preferably in collaboration with some NRLs on a voluntary basis.

3.3.2. Subtyping/fingerprinting For subtyping by Pulsed-field gel-electrophoresis (PFGE), the EU-RL routinely uses the standardised Campynet protocol (http://campynet.vetinst.dk/PFGE.html). In 2010, the protocol developed by PulseNet (USA- PulseNet) (http://www.cdc.gov/PULSENET/protocols.htm) was compared with the Campynet protocol. The PFGE typing method according to the PulseNet protocol has now been set up at the EU-RL- Campylobacter. In 2010, a 3-year research project “The distribution of putative virulence factors among Campylobacter isolates from poultry and humans” was initiated in collaboration with Uppsala University, Sweden. One of the project aims is to study occurrence of putative virulence factors in chicken and human Campylobacter jejuni strains of certain PGFE types. For this purpose, PFGE-typing will be performed on selected human and broiler strains collected during the same time period in 2010-2011. The MLST (Multilocus sequence type) method according to the protocol of Dingle et al. (J. Clin. Microbiology, 2001, 39: 14-23) was set up at the laboratory in 2010 and is used for studying populations of Campylobacter jejuni. Subtyping of the the EU baseline

survey (2008) isolates by PFGE and MLST will continue in 2011. The typing will provide information about prevalent strain types and degree of strain diversity among C. jejuni in broilers in Europe. 3.4. Participation in international networks and scientific meetings In 2011, EU-RL staff members will participate in: MedVetNet Association. The European Network of Excellence MedVetNet ended in 2009 and an Association with the same name was formed. The EU-RL will participate in relevant activities of the new Association. ISO/CEN standardization activities:

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

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

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

continue in 2011

- Other relevant national and international seminars and research meetings in order to assure competence and knowledge on recent advancement within the Campylobacter area, for example The 16th International Workshop on Campylobacter, Helicobacter and Related Organisms (CHRO) in Vancouver, British Columbia, August 28 - September 1, 2011

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

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

- As leader for an Expert Group on Campylobacteriosis for the DISCONTOOLS

project. The project is funded by the European Commission services and was started in 2008 (http://www.discontools.eu/home/index)

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

The EU-RL will provide scientific and technical assistance to the Commission and the NRLs on issues regarding Campylobacter. The EU-RL has collected information about the NRLs´ needs for laboratory training of molecular methods for identification and characterization of Campylobacter. The EU-RL plans to organise a training course in PFGE with up to 6 participants/occasion. The course, which may be organised twice, will last one week and include theoretical parts and laboratory exercises. Practical training in PCR-techniques and study visits to the EU-RL will be organised on an ad hoc basis for 1-2 participants at a time.

EU- RL staff will lecture on the topic of Campylobacter at a training course in microbiology for third countries. The course is part of the EU program Better Training for Safer Food (DG Health and Consumers) and is organised by National Food Institute in Denmark. Request from the NRLs and the Commission for support and advice will be handled by the EU-RL scientific staff as soon as possible. Assistance to the Commission and EFSA services will have priority. 5. Missions

The following missions are planned to be made in 2011 to:

- The 30th meeting of ISO/TC34/SC9 and the 18th meeting of

CEN/TC275/WG6, which will be held in UK in 2011. Total duration of the two meetings will be 5 days.

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

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

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

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

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

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

- Participation in The 16th International Workshop on Campylobacter,

Helicobacter and Related Organisms (CHRO) in Vancouver, British Columbia, August 28 - September 1, 2011

6. Communication

The EU-RL- Campylobacter webpage will be improved in order to increase the utility for the NRLs. Information about analytical methods, proficiency tests, validation studies, and other relevant information will be made available in a user friendly style. The content will be adjusted to the needs of the NRLs in their daily activities. The EU-RL will to co-operate with EU Commission Services and other organisations and authorities working in the field of human and animal health.

EU REFERENCE LABORATORY FOR PARASITES (IN PARTICULAR TRICHINELLA,

ECHINOCOCCUS AND ANISAKIS)

WORK PROGRAMME 2011

European Union Reference Laboratory for Parasites

Istituto Superiore di Sanità

page 1 of 12

EUROPEAN UNION REFERENCE LABORATORY

FOR PARASITES

WORK PROGRAMME

2011



page 2 of 12

The 2011 working programme of EURL for Parasites (EURLP) consists of the following activities: 1. Ad hoc activities 1.1 Trichinella

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

o To establish a Trichinella-positive standard pig serum (multi-years)

o To increase and maintain the serum bank of Trichinella-infected 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 Screening of commercial kits to detect anti-Trichinella IgG in pig sera (multi-years)

o Diagnostic activity with accredited methods (multi-years)

1.2 Anisakidae worms

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

o Development and validation of a multiplex PCR assay to identify Anisakidae worms at the species level (one year)

o Diagnostic activity with the accredited method (multi-years)

1.3 Echinococcus

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

o To establish a serum bank of Echinococcus-infected humans (multi-years)

o Diagnostic activity with the accredited method (multi-years)

1.4 Other Cestodes

o To establish a genetic bank of zoonotic cestodes such as those of the genus Taenia and Diphyllobotrium (multi-years)

1.5 Opisthorchidae trematodes

o To establish a genetic bank of zoonotic Opisthorchidae trematodes (multi-years)

o To establish a serum bank of Opisthorchis-infected humans (multi-years)

1.6 Cryptosporidium

o To establish a genetic bank of protozoa of the genus Cryptosporidium (multi-years)



page 3 of 12

1.7 Giardia

o To establish a genetic bank of protozoa of the genus Giardia (multi-years)

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

domestic animals and contaminating foodstuffs (multi-years)

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

2.3 Identification of polymorphic microsatellites in Trichinella spiralis and Trichinella britovi (multi-years)

2.4 Identification of Trichinella-specific antigens most frequently recognised by human sera by Western blot (multi-years)

2.5 Development of a molecular test to identify Alaria spp. mesocercariae (one-year)

2.6 Study of the genetic polymorphisms of Echinococcus granulosus sensu stricto (multi-years)

2.7 Development of analytical methods for the species identification of parasites of the genus Cryptosporidium (one-year)

3 Interlaboratory comparison study 3.1 Trichinella

o PT on Trichinella larva detection in meat samples (multi-years)

o PT on Trichinella larva identification

3.2 Echinococcus

o PT on Echinococcus adult worms detection

4 Workshop

5 Visit to NRLs

6 Training for Personnel of NRLs and of developing countries

7 Further development and update of the web site of the EURL for parasites

8 Production of videos on diagnostic methods (multi-years)

9 Standardization of methods for the detection of parasites in food

10 Development of reference materials for diagnostic methods to detect Trichinella infections

11 Validation of apparatuses for the detection of Trichinella larvae in meat samples

12 Quality Assurance System



page 4 of 12

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

Serum and/or meat juice samples will be collected from Trichinella-infested pigs, from 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 updated. Pig serum samples from different world regions and pig races (infested and not infested with Trichinella) will be collected, in order to obtain control sera and to refine the most appropriate cut-off value which will be useful for serological studies on different swine races. If the EURLP will receive a high request of reference serum samples, SPF pigs will be experimentally infested with Trichinella spp. larvae. Before the infestation, sera will be collected from each pig. After the infestation, the kinetics of anti-Trichinella antibodies will be followed 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

The EURLP aims at establishing a collaboration with the Institute for Reference Material and Measurements (IRMM), Joint Research Centre of the European Commission, to develop an international standard Trichinella-positive pig serum. The potential reference material, already available at the EURLP, needs to be further characterized with respect to several parameters of relevance. Indeed, if the reference material is to be used as an arbitrary standard, it must be ensured that its production is reproducible, i.e. the parameters to be reproducible and characterized, must be known. Thus, the ELISA target compounds would have to be quantified in the candidate reference material through collaborative studies.

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

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

1.1.4 To produce reference Trichinella antigens for serology

Excretory/secretory (E/S) antigens will be produced from Trichinella spp. larvae in order to supply NRLs with the reference antigens 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 infected with Trichinella species/genotypes will be provided to laboratories for training and for typing of wild isolates. Trichinella spp. larvae from reference strains will be stored in ethyl alcohol and forwarded to laboratories as reference material. To increase the quality and the reliability of the maintenance of these reference strains in vivo,



page 5 of 12

the identity of each infected mouse will be monitored by a microchip inserted under the mouse skin.

1.1.6 Screening of commercial kits to detect anti-Trichinella IgG in pig sera

According to the Commission Regulation (EC) 2075/2005, a serological test may be used once a suitable test is validated by the Community reference laboratory to monitor the circulation of Trichinella infections in pig herds. The ELISA test has been validated by the EURLP and can be used to monitor the circulation of Trichinella parasites in pig herds. A plethora of commercial kits to detect anti-Trichinella IgG in swine are now commercially available, but none of them has been validated by the EURLP. Since one of the core duties of the EURLP is to give critical advices, we plan to invite the companies producing ELISA kits for the detection of anti-Trichinella IgG in swine sera to provide us with their kits in order to determine their performance and, in particular, their sensitivity, specificity, inter- and intra-assay variation, reproducibility and robustness, using a panel of known pig sera with different levels of IgG. The results will be spread by publication on a peer-reviewed international journal.

1.1.7 Diagnostic activity with accredited methods

Diagnostic samples provided by NRLs will be tested with the following accredited tests:

i. Identification of anti-Trichinella IgG antibodies in swine sera ii. Identification of anti-Trichinella IgG antibodies in human sera iii. Detection of Trichinella larvae in meat samples iv. Identification of parasites of the genus Trichinella by a multiplex-PCR

analysis. 1.2 Anisakidae worms 1.2.1 To increase and maintain the collection of Anisakidae worms and/or their

genomic DNAs Reference larvae and/or DNA will be directly collected from naturally infected fish or will be requested to European and extra-European laboratories experienced on this subject.

1.2.2 Development and validation of a multiplex PCR assay to identify Anisakidae worms at the species level In order to improve the test method for the species identification of Anisakidae larvae by PCR and to easily analyze a large number of sample in a short time, a multiplex PCR method will be developed. Multiplex-PCR is a modification of the “standard PCR” where two or more oligonucleotide pairs are used. In this case, it is possible to amplify with a single PCR analysis more than one sequence at the same time. The method will allow to distinguish between Anisakis pegreffi, A. simplex s.s., A. physeteris, A. typica, Pseudoterranova spp, Hysterotilacium spp. and Contarcoecum spp. Multiple sequence alignment of the nucleotide portion encompassing the 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA, deposited in GeneBank, have shown that high sequence differences occurs in the ITS region between different anisakidae species, except for the sibling species A. pegreffi and A. simplex, that differs only by two base pairs. A combination of six forward



page 6 of 12

primer, specific for A. pegreffi, A. physeteris, A. typica, Pseudoterranova spp, Hysterotilacium spp. and Contarcoecum spp, and one primer recognizing both A. pegreffi and A. simplex, together with a ribosomal universal reverse primer will be used. The differences in the molecular size of the amplification fragments will allow, with a single amplification assay, the unambiguous identification of single larvae at the species level. The collected reference material will be used to test the multiplex PCR.

1.2.3 Diagnostic activity with the accredited method Anisakidae worms isolated from fish products by NRLs will be identified using the accredited PCR-RFLP test.

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 host specificities and transmission patterns which play an important role in their epidemiology. Furthermore, not all species/genotypes belonging to the Echinococcus granulosus complex play a zoonotic role. The main zoonotic species circulating in Europe are E. multilocularis, E. ortleppi and the E. granulosus complex with several related genotypes/species. DNA from adult, larval and egg stages will be collected to establish and increase a genetic bank of cestodes of the genus Echinococcus.

1.3.2 To establish a serum bank of Echinococcus-infected humans Serum samples from E. granulosus and E. multilocularis infected humans with a confirmed diagnosis will be collected, aliquoted and stored at -80°C.

1.3.3 Diagnostic activity with the accredited method Echinococcus granulosus larvae, adult worms or eggs detected in intermediate and final hosts by NRLs, will be analysed using the accredited PCR method for the identification at the species/genotype level.

1.4 Other Cestodes 1.4.1 To establish a genetic bank of zoonotic cestodes such as those of the genus

Taenia and Diphyllobotrium Adult, larval and egg stages of zoonotic cestodes will be collected from infected hosts, both humans and animals. Genomic DNA will be extracted and stored. The DNA will be also identified by an available molecular test and the obtained sequences will be compared with those present in GeneBank.

1.5 Opisthorchidae trematodes

1.5.1 To establish a genetic bank of zoonotic Opisthorchidae trematodes

Adult, larval and egg stages of trematodes of the family Opisthorchidae will be collected from infected hosts, both humans and animals. Genomic DNA will be



page 7 of 12

extracted and stored. The DNA will be also identified by an available molecular test and the obtained sequences will be compared with those present in GeneBank.

1.5.2 To establish a serum bank of Opisthorchis-infected humans

Serum samples from Opisthorchis spp. infected humans with a confirmed diagnosis will be collected, aliquoted and stored at -80°C.

1.6 Cryptosporidium 1.6.1 To establish a genetic bank of protozoa of the genus Cryptosporidium

Cryptosporidium spp. oocysts will be collected from domestic and wild animals, humans and environmental samples. Nucleic acids will be extracted and stored at -20°C until their identification by molecular tools.

1.7 Giardia

1.7.1 To establish a genetic bank of protozoa of the genus Giardia

Giardia spp. cysts will be collected from domestic and wild animals, humans and environmental samples. Nucleic acids will be extracted and stored at -20°C until their identification by molecular tools.

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

domestic animals and foodstuffs The use of short DNA sequences as a barcode to differentiate taxa and to discover new species, is becoming a popular technique in the scientific community. There are many possible applications of DNA barcoding, from biodiversity studies to food tracking. Our task will be the identification of specific DNA regions that could be used for the identification at the species, genus or family level and the evaluation of their potential for a large scale application. In the field of food-borne parasites, we would like to focus our attention on: 1) the liver flukes circulating in freshwater fish in Europe. Opisthorchis felineus is the main zoonotic species, but other species can be occasionally zoonotic such as Metorchis bilis and Echinostoma sp.; their larval stage (the metacercaria) present in fish and the eggs shed by the definitive hosts, cannot be easily distinguished from the metacercariae of non-zoonotic liver flukes; and 2) nematode larvae resembling Trichinella that are often collected during the digestion of muscle samples will be also identified at the family, genus or species level.

2.2 Identification of Toxoplasma gondii proteins specific for the oocyst stage The first objective will consist in the validation of the immunomagnetic separation method for the capture of T. gondii oocysts from water samples. To this aim, various anti-TgOWP monoclonal antibodies – including those already described and others that will be presumably obtained – will be used and tested under various experimental conditions. The second objective of the work plan will be the molecular characterization of three tyrosine-rich proteins of T. gondii (TgTRPs), predicted in the ToxoDB database, which likely represent a novel class of oocyst wall constituents. This hypothesis is supported by the sequence similarity of TgTRPs with the proteins gam56, gam82 and gam230, which have been localized to the oocyst wall of Eimeria



page 8 of 12

spp. These molecules may represent novel targets for the development of specific antibodies to be used in various assays for molecular epidemiological studies.

2.3 Identification of polymorphic microsatellites in Trichinella spiralis and

Trichinella britovi The large collection of nucleotide sequences obtained by the Trichinella spiralis whole genome sequencing project represents a new useful tool to identify polymorphic microsatellites in T. spiralis and T. britovi. Previous results showed a peculiar population structure of T. spiralis, which includes the presence of alleles restricted to isolates circulating in specific geographical areas. These peculiar alleles, also known as “private” alleles, are useful to track the parasite transmission. It is planned to increase the number of “private” alleles of T. spiralis and T. britovi, to facilitate the identification of macro and/or local geographic areas where the parasites (and their hosts) are circulating. The program will include: 1. the genotypic analysis of parasites collected both from wild and domestic hosts; 2. the comparison of the alleles circulating in the wild and domestic habitats; 3. the assessment of the transmission rate of the infection from the wild to the domestic habitat and vice versa. For epidemiological studies, it is extremely important to know how much T. spiralis, the so called domestic species, mainly infecting domestic and wild pigs, can be transmitted from the wild to the domestic habitat and vice versa. A similar information is important also for T. britovi, the so called sylvatic species, infecting prevalently wild carnivores.

2.4 Identification of Trichinella-specific antigens most frequently recognised by human sera by Western blot A main problem in the serological diagnosis of parasitic infections in humans is the cross-reactivity with parasitic and non-parasitic antigens. Crude and excretory/ secretory antigens (ESA) from muscle larvae are widely used for diagnosis, but, since these antigens are complex mixtures of molecules, cross-reactivity to other antigenically related parasites may occur. In fact, the presence of shared antigens of Trichinella sp. has been largely reported in other parasites and pathogens and, even if several serological techniques have been developed and attempts have been made to increase the specificity of the available tests, there is still a high percentage of false-positive reactions. In a previous study, it was shown that the specificity of ELISA is greatly influenced by the panel of sera tested, increasing the percentage of false positive-reaction when considering persons with other parasitic and non-parasitic infections or with non-infective pathologies. The aim of this study is to determine in detail the distinctive pattern of reactivity of the Trichinella positive human sera with ESA by Western blot to discriminate the non specific-cross reactive bands from those corresponding to Trichinella-specific antigens. To this end, we will test human sera from Trichinella infected persons and from persons with other parasitic or non parasitic infections, or persons coming from highly endemic areas for parasitic infections. This study will allow the establishment of a model of sera reactivity with Trichinella ESA by Western blot which will be used to confirm ELISA positive sera.

2.5 Development of a molecular test to identify Alaria spp. mesocercariae

The number of reports of Alaria spp. trematodes in wild boars meat samples tested for Trichinella is strongly increasing and resulting in the condemnation of the infected carcasses; consequently, there is an urgent need to acquire more



page 9 of 12

information on the epidemiology of these trematodes and the related human risk. For this purpose, a PCR-based method will be developed to identify the mesocercariae at the species level for molecular epidemiological studies. PCR primers will be designed on the ITS2 ribosomal DNA.

2.6 Study of the genetic polymorphisms of Echinococcus granulosus sensu stricto

The population genetic structure of Echinococcus granulosus sensu stricto in Europe will be evaluated by the DNA sequencing analysis of mitochondrial gene cytochrome c oxidase 1 (CO1) of hydatid cysts. The conserved primers COI_F (forward) 5'-TTTTTTGGCCA TCCTGAGGTTTAT-3' and COI_R (reverse) 5'-TAACGACATAACATAATGAAAATG-3' will be used to amplify the CO1 gene. The presence of PCR products will be confirmed by capillary electrophoresis using the QIAxcel apparatus. Purified products will be Dye labelled. The Dye labelled products will be subsequently purified for a second time. Forward and reverse sequences will be detected. Consensus sequence derived from the forward and the reverse sequences will be aligned and compared to the reference sequences. Amino acid sequences will be inferred from the nucleotide sequences by the echinoderm mitochondrial genetic code and the standard genetic code. The percentage divergence values of nucleotide sequences will be determined using the Kimura’s two parameter model. The molecular results joined with the epidemiological information, will enable us to develop more appropriate control strategies.

2.7 Development of analytical methods for the speciation of parasites of the

genus Cryptosporidium Parasites of the genus Cryptosporidium have similar morphological features and their identification to the level of species can only be achieved by molecular characterization. In Europe, Cryptosporidium parvum and C. hominis are responsible for the majority of human infections, but an increasing number of species are detected in people at risk (immunocompromised individuals, children) and can also be associated with outbreaks. Due to the lack of morphological differences, the species and genotypes present in faecal and environmental samples can only be identified using molecular techniques. In the course of the activities of the EURLP, we intend to develop an assay based on the CpA135 gene, which codes for a multi-modular protein with characteristic domains (such as ricin B, discoidin, fibrinogen-like and LCCL domain), and is expressed specifically by the sporozoite. Primers will be designed to bind conserved regions as deduced from a multiple alignment of the full-length gene of C. parvum, C. hominis and C. muris. Isolates of Cryptosporidium parvum, C. hominis, C. meleagridis, C. felis, C. ubiquitum, C. canis, C. andersoni, and C. muris are available in our laboratory and will be tested by PCR. Amplification products will be sequenced and the presence of genetic polymorphisms will be assessed. If possible, a PCR-RFLP (restriction fragment length polymorphism) method will be adapted to allow for a rapid and cheap identification of these species.

3 Interlaboratory comparison studies

According to the requests of NRLs expressed in the course of the fifth NRL workshop, held in Rome from 3 to 4 June, 2010, three proficiency tests (PTs) will be organised by the EURLP in the course of 2011.



page 10 of 12

3.1 Proficiency test (PT) to detect Trichinella larvae in meat samples The fifth PT on the detection of Trichinella larvae in meat samples, will be organised among NRLs to evaluate the sensitivity of the magnetic stirred method as reported in the EU legislation 2075/2005 on Trichinella. Test samples (100 or 35 g meatballs made with diaphragm tissue from pigs, horses and/or wild boars) will be spiked with a known number of T. spiralis larvae obtained from experimentally infected mice. Each NRL will receive nine samples containing three different numbers of Trichinella larvae, plus a negative control sample. 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 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, compared to those of the previous years, and possible critical points will be identified and corrected.

3.2 Proficiency test to identify Trichinella larvae at the species level by a PCR-

derived method The PT will be organised among NRLs to evaluate their skill to properly identify Trichinella larvae at the species level. Trichinella larvae from reference strains representing the species circulating in Europe and those which have been sometimes imported from non-EU countries into Europe, will be collected from infected mice by artificial digestion and stored in a 0.5 ml conical vials with ethyl alcohol. Vials will be coded and forwarded to participating labs for molecular identification by a PCR derived method according to the PCR- method used in each laboratory. Participant laboratories will be invited to identify single larvae in stead of a pool of larvae.

3.3 Proficiency test to identify Echinococcus sp. adult worms in the intestinal

content of a definitive host For the third time, this PT will be organised among NRLs to detect adult worms or their portions of Echinococcus sp. spiked in the natural matrix (intestinal content). Each NRL will receive five samples containing three similar amounts of worms, plus two negative controls. Samples will be packed and sent as bio-hazardous material in cool freeze containers to ensure a stable temperature. Every participating partner in the PT 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, compared to those of the previous years, and possible critical points will be identified and corrected.

4 Workshop

In the first half of 2011, a two day-workshop will be held at the Istituto Superiore di Sanità of Rome, or in another MS, to present and discuss the results of the PTs 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 these pathogens. A dedicated session of the workshop will focus on the sample preparation according to the ISO 17043:2010 standard and to the PT organisation to reach an agreement among NRLs to allow the comparison of the PT results obtained in each member state.



page 11 of 12

5 Visit to NRLs

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

6 Training for the personnel of NRL and developing countries

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

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

To improve the usefulness of the EURLP web site, a section for the running of proficiency tests will be created, in order to provide a quick and real-time tool for NRLs participating to the PTs. In the PT section the participant laboratories, after the login to get their individual code, will find the detailed instructions on the PT procedure, will submit their results and receive the final report, including an evaluation of their performance and the results of all the other participants in an anonymous form.

8 Production of videos on diagnostic methods The availability of various media tools is of invaluable importance for training, therefore the EURLP will start to develop, through the collaboration of specialized companies, training material such as pictures and videos. Pictures showing morphologic features (both macro- and microscopic) of foodborne parasites, will be uploaded onto the “forum” section of the web site, thus starting the creation of an e-archive open to the contribution of NRLs and international experts. Moreover, videos showing the correct implementation of diagnostic methods, with special emphasis on critical points, will be developed and make available onto the EURLP web site and/or by other media (ex: by DVD).

9 Standardization of methods for the detection of parasites in food

A EURLP representative will participate to the next ISO/TC 34/SC 9 and CEN/TC 275/WG 6 joint meetings to be held in United Kingdom in the second half of April, 2011, in order to follow as project leader of TAG 7 the standardization process on Trichinella. After the call for experts will be launched, a meeting will be organized with experts designated by the national standardization bodies. Moreover, a EURLP researcher will participate as expert to the working group of the ISO/TC34 SC9 WG work on standardization of methods for the detection of Cryptosporidium and Giardia in food.



page 12 of 12

10 Development of reference materials for diagnostic methods to detect Trichinella infections The EURLP hopefully will establish a collaboration with the Institute for Reference Material and Measurements, Joint Research Centre of the European Commission (Belgium), to start working on the development of international reference materials for Trichinella detection methods. The potential reference material, already available at the EURLP, needs to be further characterized for one or several parameters of relevance, since if the reference material is used as an arbitrary standard it must be ensured that its production is reproducible, i.e. the parameters to be reproducible and to be characterized must be known.

11 Validation of apparatuses for the detection of Trichinella larvae in meat

samples According to the Guidelines for the validation of apparatuses for the detection of Trichinella larvae in meat samples by digestion, the EURLP will organize the validation process involving four National Reference Laboratories for Parasites. At the present time, the EURLP has received the request from one company to validate its apparatus.

12 Quality assurance system

The continuous improvement of the EURLP Quality Assurance System, is a key factor to assure to the NRLs the highest level of reliability of EURLP services. For this aim, the EURLP will apply for an additional accreditation according to the ISO 17043:2010 standard as a Proficiency Test provider. Probably, the accreditation will be reached in the 2011, as soon as the Italian accreditation body, ACCREDIA, will be able to grant such accreditation.

Rome, 19th August, 2010 The Director of EURL for Parasites

Dr. Edoardo Pozio

EU REFERENCE LABORATORY FOR ANTIMICROBIAL RESISTANCE

WORK PROGRAMME 2011

Work plan for 2011

The main purpose of the European Union Reference Laboratory on Antimicrobial Resistance (EURL-AMR) is to ensure the quality of antimicrobial susceptibility testing in the Member States and to harmonise the procedures and methodologies used. Thus, most of the activities aim at implementing, from an analytical point of view, the provisions of monitoring of antimicrobial resistance set down in Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents. In addition the EURL-AR provides assistance to the member States and the Commission on other relevant aspects of antimicrobial resistance. Furthermore, the EURL-AR should work in an international context and ensure that EU influences and follows global standards and guidelines.

Scientific advice and support to the Commission During 2011 the EURL will provide advice as stated under the general terms. The EURL will participate in workshops and working groups on antimicrobial resistance initiated by EFSA, EMA, Codex, FAO/WHO/OIE and when relevant other organisations. The WHO has established an Advisory Group in Surveillance of Antimicrobial Resistance (AGIS AR), which has as the aim to develop global standards for monitoring of antimicrobial resistance. The EURL-AR is obliged to actively support this initiative that also involves EFSA.

Co-ordination of National Reference Laboratories and provision of technical support

Workshops In 2011, the EURL will host a workshop held in Kgs. Lyngby, Denmark with the following tentative agenda:

Introduction and presentation - Update of the functions of the EURL-AR - Update on other EURL's - Update from EFSA, EMA, the Commission and other parties - Updates on ongoing projects e.g. streptomycin, ESC, and qnr.

Break points and cut-off values, updates and new projects MRSA isolation, detection and epidemiology Results of the ring trials performed in 2010 Presentation and discussions on national programmes on susceptibility testing.

- Presentation of activities at the NRL's Experiences from other EURL's

Meetings on standardization of monitoring of antimicrobial resistance The most important problem in relation to ring trails and monitoring of resistance is the lack of common interpretive criteria. This is a global problem and not only related to EU. The EU is the world's largest exporter and importer of food products and European citizen travel with an increasing frequency outside the EU. Thus, the development and harmonisation of global standards is a high priority. The EURL will in 2011 continue the work with WHO (AGISAR) and other important stakeholders such as EUCAST, CLSI, OIE, FAO and Global Foodbome Infections Network (GFN) in order to promote a common international standard. This is also essential for the ongoing work in Codex Alimentarius.

Maintaining the network of NRL's The EURL will during 2011 maintain and continuously update a full list of contact persons from all NRL's. In addition, the EURL will attempt to identify expected members from applicant countries to include in the network. This list will also be maintained during the following years.

Dissemination of knowledge and information The EURL will maintain the EURL web page (www.eurl-ar.eu) where relevant information is posted. In addition, the EURL will distribute if any; updates, highlights or other relevant information through newsletters to the NRL's as pdf-files. Specifically for 2011 the EURL will provide updates on the current situation in Europe and suggested methods used for isolation and identification of extended spectrum beta-lactamase producing and quinolone resistant bacteria, as well as methicillin-resistant Staphylococcus aureus and other issues considered critical important.

The EURL will provide updated lists of suggested breakpoints based on the work done by EUCAST (www.eucast.org) and other international standardization committees. Specially the EURL will host and participate in meetings with the aim to get standardised breakpoints and cut-off values globally as mentioned above. In addition, knowledge on which antibiotics to test for and ranges to use as well as other problems encountered will also be disseminated between the NRL's.

Specific assistance to individual laboratories, site visits or individual training courses Some NRL's might have a need for special assistance. The EURL will to the extent possible within the financial limits provide specific assistance to individual laboratories based on individual needs.

Molecular methods are becoming increasingly important for verification of results obtained using conventional methods, but also as a substitute for conventional testing in the routine laboratory. In agreement with the decisions taken by the NRL's at the annual work shop in 2010, the EURL will in 2011 in collaboration with one or more of the NRLs organise a one-week training course on genotypie characterisation of relevant Gram positive and negative bacteria for a maximum of 30 participants.

E-leaming The continuous changing of staff at the different NRL's make it difficult to ensure sufficient training though individual and larger training courses. The EURL-AR will continue testing the possibilities to provide e-leaming on the web-site showing the most basic principles.

Ring trials, comparative testing and quality assurance External quality control is an important part of ensuring and maintaining the analytic quality of laboratory tests performed. The EURL will in the spring and autumn 2011 organize the following ring trials on antimicrobial susceptibility testing for the NRL's:

1. Salmonella 2. Campylobacter 3. Escherichia coli 4. Enterococci 5. Staphylococci 6. Detection of MRSA 7. Genotypie characterization of Gram positive and Gram negative bacteria (optional)

http://www.eurl-ar.eu

http://www.eucast.org

The organization and evaluation of the results are given under the general terms.

Confirmatory testing The EURL will provide confirmatory testing for NRL's on bacterial isolates of particular relevance or on request by the Commission. Specifically, the EURL will provide reference testing of putative Salmonella and E. coli isolates producing beta-lactamases with extended spectrum, and carbapenemases. Additionally, isolates resistant to fluoroquinolone or harbouring transferable fluoroquinolone resistance mechanisms, and confirmation of MRS A.

Evaluation and development of analytic methods

Reference strains Reference strains for use in quality control or other analyses are an important part of the internal quality control and validation of on-going analyses. The EURL will extend it's already available strain collection and make the strains available for NRL's on request.

Interpretative criteria The EURL will if needed perform studies on the susceptibility of food borne pathogens to various antimicrobial agents in order to provide data for the establishment of interpretative criteria for categorizing isolates as susceptible or resistant. On the annual work shop between all NRL's the most urgent needs and problems were discussed. Thus, especially for 2011 the EU RL will initiate the following projects:

Detection of synacid resistance and interpretative criteria in Enterococcus Establishment epidemiological cut off values for florfenicol in E. coli

- Evaluation the current interpretatrive criteria for Colistin resistance in Salmonella.

MRSA detection The emergence of MRSA in food animal production is a matter of increasing concern. The EURL will together with other institutions continue to evaluate different methods for the optimal detection of MRSA from animal sources and if possible food of animal origin.

Transferable quinolone resistance Transferable quinolone resistance has recently emerged. The EURL will continue and expect to finalise the 2009 project into 2011.

Extended spectrum beta-lactamases The emergence of ESC producing isolates poses a major problem for human health. The EURL will together with the NRL's collect information on the occunences of ESC resistance in Europe and if necessary initiate further studies into this.

EU REFERENCE LABORATORY FOR ANIMAL PROTEINS IN FEEDINGSTUFFS

WORK PROGRAMME 2011

EURL-Animal Proteins

CRA-W

Work programme 2011

EURL-AP Work programme 2011 Ver 00, Fnal

1

PROPOSED 2011 WORK PROGRAMME FOR THE EUROPEAN UNION

REFERENCE LABORATORY “Detection of animal proteins in feedingstuffs”

EURL-AP

Walloon Agricultural Research Centre – CRA-W (Belgium)

1 Scientific advice and support to the European Commission (34 p/m)

1.1 Provide scientific and technical assistance to the European Commission in

relation to the development of EC feed legislation. (2 p/m)

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

EURL, participate in 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 EURL-AP activities.

(1 p/m)

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

EURL, participate in meetings for the standardisation of analytical methods

relating to the detection of animal proteins in feedstuffs and their

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

missions/year are foreseen in support to DG Sanco and/or EURL-AP activities.

(2 p/m)

Among the possible activities in 2011 there might be the starting of a CEN

working group (within CEN TC327) defining guidelines for PCR in detection of

PAP in feed.

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

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

animal proteins. (3 p/m)


CRA-W

Work programme 2011


2

1.5 To keep at EURL-AP the highest standard possible of technical skill, scientific

awareness and quality management under accreditation (ISO17025) 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 EURL-AP lab. (14 p/m)

1.5.1 To maintain scientific awareness in general about techniques that

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

1.5.2 To maintain the accreditation scope

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

of the new recruits

1.5.4 Extend the accreditation scope of the EURL-AP by including

additional animal targets DNA in the scope for PCR analyses

1.5.5 Submission of the dossier for the accreditation of the organisation of

interlaboratory studies

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

with disputed results. (12 p/m)

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

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

Albania, Bosnia and Herzegovina, Montenegro, Serbia) (0 p/m)

No activity is forecasted under this task for 2011


CRA-W

Work programme 2011


3

2 Coordination of activities of NRL network (11 p/m)

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

share information with NRLs and others stake holders. (5 p/m)

2.1.1 Information collection and validation

2.1.2 Maintenance of the website

2.1.3 Update and test of the information system and validation

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

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

2.3.1 Organisation of the 5th annual EURL-AP workshop

2.3.2 Preparation of the agenda

2.3.3 Invitation of the attendees

2.3.4 Realisation of the workshop

2.3.5 Minutes of the annual workshop

Note : For 2011 it is proposed to organise the annual workshop in Vienna, Austria

The Austrian NRL agrees to host this meeting.

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

laboratories, detection, quantification and identification of animal proteins in

feed ingredients and feedingstuffs. (0 p/m)

This task has been merged with task 3.3

2.5 Participate in the annual EURL Directors co-ordination meeting. (0 p/m)

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

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


CRA-W

Work programme 2011


4

3 Interlaboratory studies and quality assurance (38 p/m)

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

national reference laboratories (NRL) of samples containing animal proteins

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

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

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

(12 p/m)

3.1.1 Definition of the needs

3.1.2 Collection of the raw materials

3.1.3 Control of the raw materials

3.1.4 Production of the samples

3.1.5 Test of the homogeneity of the samples produced

3.1.6 Report on the produced samples

3.1.7 Distribution of the samples

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

classical microscopy. (9 p/m)

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

Autumn 2010

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

of the ring trial to perform at the end of 2011

3.2.3 Preparation of the interlaboratory study 2011.

3.2.4 Invitation of the NRLs to participate.

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

3.2.6 Collection of the results.

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

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

missions/year are foreseen in support to DG SANCO and/or EURL-AP

activities

(1 p/m)


CRA-W

Work programme 2011


5

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. (8 p/m) (Note : This task has been

merged with task 2.4.)

3.3.1 Definition of the needs of the NRLs

3.3.2 Provide the requested help to the NRLs

3.3.3 Preparation of syllabi including all the information needed for an

appropriate detection of processed animal proteins in feedingstuffs

3.4 Organization of an interlaboratory study for GTH (with the collaboration of

JRC-IRMM) (8 p/m)

Note: This task is proposed by EURL-AP but has not been yet discussed with

DG-Sanco.

4 Development of analytical methods and tools (42 p/m)

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

existing methods of analysis. (14p/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 EURL-AP to these initiatives

4.1.3 Establishment of the needs in the development of methods

4.1.4 Evaluation of immunoassays proposed for the detection of PAP (4 p/m)

4.1.5 Harmonisation of analytical methods for determining insoluble

impurities in rendered animal fat (‘tallow’) (Methods ISO 663 and AOCS Ca

3a-46) (7 p/m)

Note : This task refer to the request made by DG-Sanco in the letter sent on

28/07/2010 (Ref SANCO/D1/MK/(2010)/D)


CRA-W

Work programme 2011


6

4.2 Contribute to the development of complementary analytical methods necessary

to assure the correct implementation of official methods and explorative or

alternative methods. (3 p/m)

4.2.1 Test of methods for the detection of blood products

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. (18 p/m)

4.3.1 Organisation of interlaboratory studies based on alternative tests

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

4.3.2 On the basis of former interlaboratory studies regarding PCR

methods, define the strategy for the optimum implementation in the NRL

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

implementation of the PCR methods

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

network – training courses and manuals will be prepared during 2011

4.3.5 Production and verification of PCR kits for the NRLs

4.4 Performing EURL-AP available methods or adapting them on outbreak material

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

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

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

well as production of microscopic image representative of the particles making

up the samples collected and selected to be included in the EURL-AP samples

bank. (5 p/m)

4.5.1 Establishment of the specification for the EURL-AP samples bank

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

samples bank

4.5.3 Maintenance of informatics tools for the appropriate management of

the samples

4.5.4 Collection/production of samples of animal origin


CRA-W

Work programme 2011


7

4.5.5 Test of the samples collected

4.5.6 Storing of the samples and maintenance of the samples bank

5 Workshops/trainings (8 p/m)

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

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

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

development or regulation related to the detection, identification and

quantification of animal proteins in feed. (8 p/m)

5.1.1 Light microscopy training

5.1.2 PCR training

On the basis of the former interlaboratory studies regarding PCR methods, to

decide of the opportunity to organise a specific workshop or training sessions

in 2011 for the implementation of the PCR method.

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

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

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

identification and quantification of animal proteins in feed. (0 p/m)

No specific activity is foreseen in 2011.

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

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

skill of laboratories performing classical microscopy or other validated method

(0 p/m).

No specific activity is foreseen in 2011.

Documents

WORK PROGRAMMES FOR EU REFERENCE LABORATORIES … · WORK PROGRAMMES FOR EU REFERENCE LABORATORIES ... MMP , formerly CRL ... determination of alkaline phosphatase activity in cheese