WORK PROGRAMMES FOR EU REFERENCE LABORATORIES FOR … · b. Enumeration of total flora at 30°C The CRL (Unit HMPA) will organize an inter-laboratory trial on the TF enumeration at

WORK PROGRAMMES FOR EU REFERENCE LABORATORIES

FOR 2009 **********

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 2009

CRL « Milk » 1/7 07/08/08 2009 Programme of Work

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

EU COMMUNITY REFERENCE LABORATORY FOR MILK AND

MILK PRODUCTS

2009 Programme of Work of the Community Reference Laboratory for Milk & Milk Products

Site de Maisons-Alfort


The AFSSA-LERQAP (Laboratory for Studies & Research on Quality of Foods & on Food Processes) foresees to undertake, as Community Reference Laboratory (CRL) for milk, the following works in 2009 according in particular to (a) the actions planned at the 10th Workshop of the National Reference Laboratories (NRLs) (28&29 June 2007), and (b) the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the CRL for the period 2006-2010. These actions are part of the new role of the CRL, restricted to the control of raw and heat-treated liquid milk (total flora, somatic cells count, phosphatase activity), as well as cheeses for phosphatase, in the frame of the Regulation 853/2004 laying down specific hygiene rules for food of animal origin. The Annex III, Section IX of Regulation 853/2004 is dedicated to raw milk and dairy products:

- Microbiological criteria on total flora at 30°C and on somatic cells count are fixed: 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 CRL foresees in particular to provide a support to the NRLs for the implementation of:

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

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

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

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

NB: In brackets under each item, the scheduled duration of the action is indicated: either annual (limited to 2009) , 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 ISO 4833, and the reference method for somatic cells count, Standard ISO 13366-1, as well as conditions for the use of alternative methods.

1.1 Inter-laboratory proficiency testing for the NRLs

(annual)

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

a. Study of sample types used for inter-laboratory trials on total flora in raw milk Up to now, the CRL has relied upon CECALAIT to prepare and dispatch raw milk samples for inter-laboratory trials on total flora (TF) enumeration, since CECALAIT controls sufficiently the TF stability of raw milk during transportation. The CRL (Unit HMPA) will conduct in 2009 an investigation study (stability and homogeneity) to find a way, such as the addition of a chemical agent, to stabilize sufficiently the TF contamination of raw cow’s milk, in order to prepare and dispatch itself the samples used for TF enumeration. It is intended to select a formula adapted to TF; formula which would allow the bacteria to grow on plates after the dilution steps.

b. Enumeration of total flora at 30°C The CRL (Unit HMPA) will organize an inter-laboratory trial on the TF enumeration at 30°C of raw cow’s milk by the reference method, the Standard EN ISO 4833 (total plate count method). 1.2 Analytical development

(multi-annual)

a. Determination of total flora at 30°C and somatic cells in raw milk by an instrumental method

The CRL (Unit HMPA) intends to complete for raw cow’s milk its experimental study 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 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 CRL will launch an experimental study using the Bactocount on raw milk of other species than cow. To go on the collaboration on this topic with the D-NRL (Kiel), a mission to Kiel is scheduled in 2009.


b. Determination of total flora at 30°C in cow’s colostrum

Frame: DG SANCO intends to add hygienic requirements for colostrum in Regulation 853/2004. The CRL and the NRLs have considered that data on acceptable levels of total flora (TF) in colostrum were not readily available, thus a need of investigation from the CRL.

In 2009, the CRL (Unit HMPA) will complete a bibliographical study on the topic of TF at 30 °C in colostrum and will design an experimental study on this determination in colostrum, including the way to get appropriate samples. Moreover, the CRL will launch an experimental study using the reference method EN ISO 4833 and the Bactocount, as an alternative method to the bacterial count, in order to investigate the questions linked to the enumeration of TF in colostrum. For that purpose, samples of colostrum’s batches will be analysed in parallel by the reference method and by the Bactocount. 1.3 Coordination of the NRLs on determination of total flora

(multi-annual) Since the Standard ISO 21187 on the conversion factors between the routine method and the reference method for TF determination has been recently published, the CRL will go on to supervise the NRLs on how they coordinate the implementation of the Standard by the network of laboratories in charge of routine control of raw milk. In particular, all conversion factors should be recalculated according to the Standard in each Member State and it is intended to have only one conversion factor per country. In 2009, the CRL will in particular envisage what follow-up to give to the outcome of the updated enquiry on national practices, re-circulated in 2008. 1.4 Standardization on validation of routine methods for total flora in raw milk

(multi-annual) IDF/ISO has launched 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 CRL will follow this standardization work and will ensure a liaison with the works undertaken as CRL with the NRLs network. 1.5 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 then 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 calibration of instrumental methods. The CRL will take part to the IDF/ISO working group which will develop this reference system as to envisage how it could be beneficial to its 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 ISO 11816-1, the legal limit for negativity of the test for alkaline phosphatase (AP) activity (350 mU/l for cow’s milk) and conditions to use alternative methods.


(annual)

Following the 2007 PT trial on AP, the CRL will organize in 2009 another PT trial on the use of the reference method. The samples to be tested will be discussed with NRLs during the coming workshop dedicated to AP (9&10 October 2008). They could either be goat’s and ewe’s milk or cow’s milk with high AP levels. The statistical model applied to the evaluation of the PT, following the conclusions of the working group on statistics, will also be assessed.

2.2 Analytical development (multi-annual)

In 2009, the CRL intends to conduct the following activities. It should be stressed that the following work program is scheduled on the basis of a normal yearly activity, assuming that the renovation of the laboratory where AP works are conducted, to start in mid-autumn 2008, will not present important constrains to the execution of the work in 2009. The company manufacturing the Fluorophos instruments (Advance Instruments) will provide in situ a training course for the CRL staff so as to enable them to ensure themselves part of the maintenance and revision of the Fluorophos instruments of the laboratory.

a. Determination of the phosphatase activity in other than cow’s milk The CRL will continue the study on fixation of AP limits for species other than cow (hopefully limits for goat’s milk will be finalised during the NRL Workshop in October 2008). If agreed by NRLs at the workshop, the following experiments will relate to ewe’s milk, aiming to support DG SANCO in prescribing legal limits of AP activity in ewe’s pasteurised milk. Ewe milk The CRL will study thermal inactivation of AP in ewe's milk, with input from collaborating NRLs. The CRL will establish the kinetics of AP inactivation in parallel with decrease of the microbial load of the milk samples in order to propose a legal limit for correctly pasteurised ewe's milk. Camel milk To characterize the heat-treatment of camel milk, the CRL intends to launch a study in collaboration with the Central Veterinary Research Laboratory (CVRL) of Dubai. Preliminary results of an informal collaboration with CVRL in 2008 tend to show that AP is not a pertinent indicator of pasteurisation of camel’s milk.


All costs of the entire study are and will be covered by a contract with CVRL.

b. Determination of alkaline phosphatase in cheeses In 2008, the CRL has introduced a revised draft Standard related to the determination of AP activity in cheese, in the framework of the relevant IDF/ISO Joint Action Team. In 2009, the CRL will give a follow-up to comments received and will produce a new draft integrating the remarks forwarded by the experts, if and when acceptable. The CRL will continue the study on the residual AP activity in cheese. Depending on the discussions during the 2008 NRLs Workshop, the next study could deal with hard cheeses. The expected end-product is the fixation of a AP limit allowing for the distinction between cheeses made from pasteurised milk and cheeses made with non pasteurised milk.

c. Comparison of the chemiluminescent/fluorimetric methods The comparison performed in 2008 between the chemiluminescent method (Novalum) and the current regulatory reference method, the fluorimetric method (Fluorophos) showed an improvement but there is still a non-negligible bias between the two methods. In 2009, the CRL intends to apply a new approach based on a conversion factor. This new approach will be detailed for NRLs during the 2008 Workshop on AP and work will be continued only if the NRLs approve this option.

d. Reactivated phosphatase

The CRL believes it is important to study both from a theoretical and an experimental point of view the mechanism of the AP reactivation in milk products with high fat content. This is a unique phenomenon, not encountered with any other enzyme, and which is certainly worthwhile to investigate. The need and content of such a study will be discussed at the NRL Workshop on AP.


3 Assistance to the NRLs

3.1 Training courses Upon requests of NRLs, the CRL may receive NRL staff for individual training on specific topics. 4 NRLs Workshop The CRL will organise in 2009 the 12th NRLs Workshop, of general scope. In particular, this workshop will enable: - to make a point of works undertaken by the CRL since the last general Workshop of

2007, in particular further to the 2008 Workshop dedicated to the phosphatase activity; - to envisage the work programme for the following years. 5 Technical and scientific assistance to the European Commission

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

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

• The 2009 IDF/ISO Analytical Week and the meeting of the groups dealing with the topics mentioned above.

EU REFERENCE LABORATORY FOR SALMONELLA

WORK PROGRAMME 2009

Work-programme CRL-S 2009 22-08-2008 Page 1 of 7

CRL-Salmonella work-programme 2009 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. Quality assurance of serotyping of isolates from (baseline) surveys (related to the

surveys of 2008 and 2009) 5. Performance of ad hoc activities (yearly); 6. Communication (every 3 months and yearly); 7. Missions (duration dependent on the subject); 8. Training (duration dependent on the subject). 1. Interlaboratory comparison studies For 2009 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 2009 will be planned with the NRLs-Salmonella, but an indication of the probable time period is given below. The set-up of the CRL-Salmonella interlaboratory comparison studies on the bacteriological detection of Salmonella has remained the same for many years. In these studies a Salmonella negative matrix is artificially contaminated with Salmonella reference materials containing Salmonella Typhimurium or Salmonella Enteritidis at different contamination levels. The differences over the years have been the choice of the matrix, the number of reference materials to be tested, the contamination level of these reference materials and the choice of the methods. Although it may be more realistic to test naturally contaminated samples, this has only occasionally been used as test matrix, because: a) It is complicated to obtain a sufficient amount of a naturally contaminated matrix; b) The Salmonella contamination in the naturally contaminated sample is in general not homogeneously distributed over the sample; c) The stability of the naturally contaminated samples may not be sufficient for the cause of the study; d) The contamination level and the Salmonella serovar present in the naturally contaminated sample can not be adapted; e) Different interlaboratory comparison studies are hard to compare to each other. Interlaboratory comparison study on bacteriological detection of Salmonella in veterinary samples In the 2008 study, the contamination levels of the reference materials to contaminate the matrix were lowered. The levels were chosen so that the low level materials would be


close to the detection limit of the methods used and the high level materials were approximately 10 times above the detection limit. This resulted in the use of the following reference materials: Salmonella Typhimurium at a contamination level of approximately 5 cfu and 50 cfu per capsule (STM5 and STM50) and Salmonella Enteritidis at a contamination level of approximately 10 cfu and 100 cfu per capsule (SE10 and SE100). In earlier studies STM10, STM100, SE100 and SE500 were used for this purpose. From the 2008 study it could be concluded that these lower levels gave more information on the performance of the laboratories as well as on the performance of the methods. However, it was also shown that the low level materials containing Salmonella Enteritidis were hard to test positive and might contain a too low concentration of Salmonella for the cause of the study. The following is planned for the 2009 veterinary ring trial: - Time period: February/March; - Matrix: animal faeces or environmental sample (to be decided after consultation of

DG-Sanco); - Same reference materials as used in the study of 2008, with the exception of SE10,

which will be replaced by SE20; - Methods: Annex D of ISO 6579 (‘Detection of Salmonella in animal faeces and in

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

The results of each NRL will be evaluated and compared with the pre-set definition of ‘good performance’. In case of unexplainable ‘poor performance’, the follow-up will be discussed with the relevant NRL (e.g. sending extra samples which need to be tested according to a prescribed protocol, training at the CRL or visiting a NRL by a member of the CRL-Salmonella staff). In the veterinary study of 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 2009. Additionally, two more third countries, Canada and the United States of America, may participate in the 2009 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 In 2008 the first interlaboratory comparison study on the detection of Salmonella in animal feed was organised. It will be discussed with DG-Sanco and with the NRLs for Salmonella, whether the 2009 study will again contain animal feed as matrix, or whether a food matrix should again be the matrix of choice. In both cases, the set-up of the study will be similar to earlier studies and it is planned to use the same reference materials as which will be used for the veterinary study of 2009. The prescribed method will be ISO 6579 (2002: Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp.), with selective enrichment in RVS and MKTTn. The requested method will be Annex D of ISO 6579, with selective enrichment on MSRV. The planning of the study is September/October 2009. 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 In former typing studies, 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). It is foreseen that this collaboration will be formalised in a contract with the ECDC in fall 2008. 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 2009. 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 tool will be used to evaluate the results of the study of 2008. If necessary, the tool will be discussed with the NRLs at the annual workshop in 2009 and amended, when needed, for the evaluation of the results of the 2009 study. 2. Workshop The annual workshop is planned to be organised in Bilthoven in May/June 2009 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 2008 and 2009; - Experiences, problems, results in relation to the (baseline/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; - Working plan of CRL-Salmonella for end 2009, early 2010. 3. Research Activities concerning ISO and CEN The CRL-Salmonella (Kirsten Mooijman) is involved (as project leader or as member of working groups) in several activities of ISO and CEN. More specific in: - ISO/TC34/SC9: International Standardisation Organisation, Technical Committee 34

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

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

Planned activities in which CRL-Salmonella will be involved in 2009: Enumeration of Salmonella (CRL-Salmonella project leader): The CRL-Salmonella

prepared in 2007 a draft protocol on an MPN based method performed in microwell (12 wells) plates, called ‘mini-MSRV’ for the enumeration of Salmonella. It was agreed at the annual ISO meeting in 2008 that Kirsten Mooijman will introduce the comments on this document after which it will be launched for a New Working Item Proposal to become published as an ISO Technical Specification (ISO/TS). In case the voting would result in many (technical) comments, this will be further discussed at the SC9 meeting in 2009. It is planned to perform some further testing of the method for its application to different matrices, at the laboratory of the CRL. It is under discussion (EFSA and DG-Sanco) to introduce the method in the baseline study on broiler meat at retail in 2009. When needed, the CRL-Salmonella will help the NRLs introducing the method in their laboratories and/or give advice (e.g. by training) in case of questions.

Periodical (5 year) review of ISO 6579 (CRL-Salmonella project leader): In 2008, ISO has sent an enquiry to the members for the possible need for revision of ISO 6579. Specific questions were posed in this enquiry and it is planned that an ad hoc group will consider the outcome of the enquiry, convened by Kirsten Mooijman. The CRL will also collect information for answering the questions of the enquiry, by searching literature and by summarising the results of the interlaboratory comparison studies on detection of Salmonella in minced beef and in animal feed (to show the good performance of MSRV for these types of samples as well).

Guide for serotyping Salmonella spp. (CRL-Salmonella project leader): At the annual ISO meeting in 2008 it was agreed that the CRL-Salmonella will prepare a draft proposal for a guide on serotyping of Salmonella. This draft proposal will be sent to


the members of ISO and the need for publication as an ISO document will be further enquired.

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. In 2008 some additional information to the proposal was requested by CEN and the CRL sent this to CEN in June 2008. It is not clear when a contract for the performance of the study can be expected. As soon as this contract comes available, a further planning of the activities can be made, which will involve expertise of the CRL as well as of some NRLs for Salmonella.

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

Methods Several activities and experiments will be carried out in relation to the ISO/CEN activities (see above). In 2007 some artificially contaminated samples (chicken faeces, pig faeces, minced meat) were tested for Salmonella using a Real-Time Polymerase Chain Reaction technique. All samples gave similar results with the RT-PCR and with the bacteriological detection method. In 2008 several naturally contaminated samples were obtained, with different contamination levels. These samples were frozen at -20 °C and are planned to be tested later with the PCR technique. In 2008, Kirsten Mooijman has been asked to revise the chapter on media for isolation of Salmonella of the ‘Handbook of culture media for food microbiology’. Information has been gathered from literature and from media suppliers on ‘new’ culture media. It is also planned to test some of these ‘new’ media at the laboratory of the CRL, so that experience is gained with these media. This can be useful for the activities in ISO as well as for giving advises to NRLs concerning ‘new’ media. Matrices Research activities may be necessary for testing another relevant matrix for the interlaboratory comparison study on the detection of Salmonella in food or in animal feed. The matrix will be tested with and without the addition of Salmonella reference materials. Also the amount of background flora will be analysed.


Participation in Working Groups of DG-Sanco and EFSA A staff member of the CRL-Salmonella participates in the working groups of DG-Sanco and of EFSA for e.g. drafting proposals for (baseline) surveys and monitoring schemes in accordance with EU Directive and Regulations. 4. Quality assurance of serotyping of isolates from (baseline) surveys In December 2008 the baseline studies on the prevalence of Salmonella in broiler carcasses and in herds of breeding pigs will finish. It is foreseen that the last strains for quality assurance, isolated by the NRLs during these studies, will be sent to the CRL up to the first quarter of 2009. The reports of the quality assurance of the serotyping of the two baseline studies will be prepared after receipt of the last isolates of these studies. In 2009, the following survey is expected to take place: • Monitoring programme for Salmonella and Campylobacter in broiler meats at retail

in the EU. In the draft report of EFSA on proposed technical specifications of the survey the following is proposed for the Salmonella analyses: - Quantitative analyses of Salmonella following the mini-MPN method (as drafted for

ISO); - Qualitative analyses of Salmonella, using MSRV as selective enrichment medium; - Serotyping of at least one isolate from each positive sample (following the

Kaufmann-White scheme) and sending a maximum of 16 non-typable strains to CRL-Salmonella for quality assurance.

As the mini-MPN method is relatively new for the majority of the NRLs it is possible that the CRL will be consulted for (technical) advices concerning the method and/or to give training. If all NRLs-Salmonella send in the maximum number of non-typable isolates for quality assurance, the CRL will have to serotype up to 432 strains in 2009 (to be finalised in 2010). For this study a protocol and submission forms will be prepared. By the end of the study the results of the quality assurance will be reported to DG-Sanco and EFSA. 5. 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.


6. Communication A staff member of the CRL-Salmonella has been trained to keep the CRL-Salmonella website up to date (www.rivm.nl/crlsalmonella). 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 via the CRL-Salmonella website. Summaries of several intercomparison studies and related research will be published (if possible) in the scientific literature. 7. Missions Summarising the information as given in the chapters above, the following missions may be needed in 2009. Per mission one (occasionally two) staff member of the CRL-Salmonella will participate. - Visit to a poor performing NRL (if necessary, see information under chapter

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

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

- Meetings of several working groups of ISO/TC34/SC9 (see information under chapter ‘Research’), approximately 2 meetings per working group. The meetings are not yet planned, but will be scheduled as soon as considered needed.

8. Training On request of an NRL, the CRL can give a training for a specific need of an NRL. It is also possible that the CRL will advice an NRL to follow a training at the CRL, especially in case of (repeated) poor performance of the NRL in interlaboratory comparison studies. Mrs. drs. K.A. Mooijman Head CRL-Salmonella

EU REFERENCE LABORATORY FOR MARINE BIOTOXINS

WORK PROGRAMME 2009

CRLMB-Work program-2009-1September2008 1 of 6

WORK PROGRAMME FOR THE COMMUNITY REFERENCE LABORATORY

FOR MARINE BIOTOXINS

January-December 2009

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

COMMUNITY REFERENCE LABORATORY FOR MARINE BIOTOXINS


Work Programme for the Community Reference Laboratory for Marine Biotoxins Objectives for the period January-December 2009 LEGAL FUNCTIONS AND DUTIES The functions and duties of the Community Reference Laboratories for feed and food are described in Article 32(1) of Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules. 1. Coordination of activities of NRL network and

provision of technical assistance and training

1.1. Organization of the XII Workshop of the European NRL network of marine biotoxins

Date: to be determined (expected to be in October-November) Place: to be agreed during the XI Workshop to be held in Slovenia (23-24 October 2008). Duration: 1,5-2 days

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

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

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

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

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

COMMUNITY REFERENCE LABORATORY

FOR MARINE BIOTOXINS


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

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

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

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

See point 5.2.

1.7. Working group on EFSA Opinion on Marine Biotoxins. Activities focused on possibilities of the EU-NRLs network on the potential/practical implementation of the Opinion on marine biotoxins in shellfish provided by the EFSA Scientific Panel on Contaminants in the Food Chain (CONTAM).

NOTE: If it is required after the next XI Workshop of EU-NRLs (October 2008, Slovenia) the ongoing working groups could be modified (eliminated, modified, or new ones created).

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

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

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

Duration: ongoing

2.3. Participation in the expert working group to asses EFSA Scientific Panel on Contaminants in the Food Chain (CONTAM) to provide Opinion on marine biotoxins in shellfish. 3. Interlaboratory Proficiency Testing The interlaboratory proficiency testing trials organised by the CRLMB for the NRLs are aimed at evaluating the ability of the NRLs to apply satisfactorily the recognised testing methods for marine biotoxins for the purpose of Regulations (EC) Nos. 853/2004 and 854/2004 stated in Annex III of Commission Regulation (EC) Nº 2074/2005. At the same time, the equivalence of the different methods applied is


studied. The CRLMB will organise one trial for each marine biotoxins group legislated in the EU:

3.1. CRLMB 2009 Proficiency Testing for PSP toxins determination (CRLMB-2009-PT-PSP). 3.2. CRLMB 2009 Proficiency Testing for LIPOPHILIC toxins determination (CRLMB-2009-PT-LIPO). 3.3. CRLMB 2009 Proficiency Testing for ASP toxins determination (CRLMB-2009-PT-ASP). Schedule for the Proficiency Testing trials (participants registration, samples dispatch, results submission and PT report) will be agreed by the EU-NRLs network and published in the “EU CRLMB/NRLs timetable of activities for 2009”, which will be circulated to the network in the first quarter of 2009. NRLs will receive in advance the “Application Form” to be registered at each trial together with information about the foreseen dates for samples dispatch and results submission. Samples for the trial will be accompanied with the identity code for each participant. At the same time, participants will receive by email the “Protocol” for the exercise, a “Sample Arrival Form” to be sent to the CRLMB just after samples reception and the “Reporting Sheet” for results submission. Preliminary results on CRLMB-2009-PT exercises will be presented by the CRLMB during the XII Workshop of the European NRL network of marine biotoxins and discussed by the group. A final and detailed report (with all information on participants, aim of the exercise, schedule and instructions, materials preparation, methods used by participants, evaluation of results, proficiency results,..) will be elaborated and edited by the CRLMB for each PT, circulated to participants in the trial and published on the CRLMB website. A follow-up report on the management in cases of underperformance in the Proficiency Testing by NRLs will be submitted to DG SANCO together with the CRLMB technical report for 2009 (planned for March 2010). 4. Missions 4.1. Visit to NRLs when necessary, in order to solve problems with results after ring trials or validation studies (see points 3 and 5).

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

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

4.4. Assist third countries on technical issues on request.


5. Development of analytical and detection methods 5.1. Study on further refinement of the “Standard Operating Procedure for lipophilic toxins detection by Mouse Bioassay” (based on harmonised MBA-SOP version 4).

5.2. Development and validation of single toxins group methods, starting with a method for OA-group toxins, for LC-MS analysis of the lipophilic toxins legislated in the EU.

5.3. If the performance problems detected during the validation study carried out in 2007-2008 are solved, continuation with the formal validation study of the method for the determination of Okadaic acid and dinophysistoxins in molluscs by a Fluorimetric Phosphatase Assay through the TOXILINE-DSP test. 5.4. Validation and accreditation of a multitoxin method by LC-MS for the determination of Okadaic acid-group, Azaspiracid-group for the confirmative determination at the CRLMB of these toxins. 5.5. Implementation of a LC-MS method for the confirmative determination of ASP toxins at the CRLMB. 5.6. Pending on the reactivation of the activities related to phycotoxins through CEN/TC 275/WG5-Biotoxins, finalization of necessary requirements to get the validated method for “the determination of Domoic acid in shellfish and finfish by RP-HPLC using UV detection” as a CEN standard. 5.7. Pending on the reactivation of the activities related to phycotoxins through CEN/TC 275/WG5-Biotoxins, collaboration in the elaboration of a document about “Criteria of analytical methods for marine biotoxins” 5.8. Identification of alternative methods within scientific reach and feasibility to initiate validation studies of those methods. For the year 2009, the CRLMB will implement and study possibilities for validation of two alternative analytical methods for PSP toxins detection:

HPLC-Fluorescence method with post-column derivatization LC-MS method

5.9. Identification of methodological options to changes in regulatory levels for marine toxins (see point 1.7).

5.10. Identification within current scientific knowledge the conversion factor for each group of toxins.


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

6.1. Underperformance (i.e failure in proficiency test). See Point 3. The CRLMB will carry out a follow-up of those NRLs that fails in the CRLMB-2009-Proficiency Testing for PSP, lipophilic and ASP toxins determination in order to identify the origin of the bad results. 6.2. Lack of collaboration by the NRLs with the CRLMB. According to DG SANCO protocol, the CRLMB will contact those NRLs for which a lack of collaboration with the CRL is detected. This lack of collaboration can be due to lack of collaboration in working groups’ activities or to a lack of participation in the CRLMB proficiency testing exercises.

EU REFERENCE LABORATORY FOR BACTERIOLOGICAL AND VIRAL CONTAMINANTS OF MOLLUSCS

WORK PROGRAMME 2009

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

1. Scientific advice and support

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

10 days

1.2. Participate in relevant EU and International scientific committees (ISO/CEN, WHO/FAO, ICMSS etc). In 2009 the CRL will:

• Continue to chair and co-ordinate the activities of the CEN/TC 275/WG6/TAG4 developing a CEN standard for detection of norovirus and hepatitis A in foodstuffs, including bivalve molluscs (see resolution 23, 7th workshop of NRLs).

• Lead and co-ordinate the activities of CEN/TC 275/WG6/TAG3 in the elaboration of molecular based enumeration methods for pathogenic marine vibrios in bivalve shellfish (see resolution 28, 7th workshop of NRLs).

• Participate in ISO/TC34/SC9/WG3 working group on validation of methods (revision of EN ISO 16140) to include the elaboration of ISO technical report on recommendations for establishing/revising reference methods.

• Participate in working group CEN/TC 275/WG6/TAG6 on sampling methods to include recommendations on sampling bivalve molluscan shellfish.

35 days




2

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

1.4. Support the industry initiative SUMO through technical oversight of

the work programme.

5 days 5 days

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

4 days

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

• During 2009 missions are foreseen in relation to the annual meetings of ISO and CEN (up to 2 missions); the CEN/TAG4 working group on viruses in food (2 missions); CEN/TAG3 working group on vibrios (2 missions); ISO/WG3 working group on validation of methods (2 missions) and up to 3 missions in support of NRLs and DG Sanco activities.

30 days

1.7. Participation in relevant international scientific conferences, e.g. International Conference on Molluscan Shellfish Safety, France, June 2009, and the IWA Health-Related Water Microbiology Conference, Greece, September 2009, Interstate Shellfish Sanitation Conference, US, August 2009.

1.8. Participate as a member of the steering committee in the ICMSS’

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

10 days 3 days

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

technical assistance and training

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

5 days

2.2 Organise, host, and participate in the eighth annual NRL workshop, produce resolutions and other workshop outputs (May 2009, CRL Weymouth). To include CRL administrative assistance.

30 days

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

Up to 100 days

2.4 Organise specialist, targeted, practical training for NRLs, MS competent authorities and the FVO on sanitary surveys - in

8 days




3

accordance with the requirements of 854/2004 on official controls. 2.5 Complete follow-up activities associated with the joint EU/US FDA

workshop on implementation and approaches to sanitary surveys.

5 days

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

5 days

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

10 days

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

7 days

3 Ring trials, comparative testing and quality assurance

3.1 Organise comparative (proficiency) testing for NRLs for E.coli and Salmonella spp. in bivalve molluscs via the CRL/HPA shellfish EQA scheme (see resolution 14, 7th workshop of NRLs). Analyse results, produce report, advice and recommendations (by May 09).

40 days

3.2 Organise Norovirus and hepatitis A ring trials (see resolution 21, 7th workshop of NRLs). Analyse results, produce report and recommendations (by May 09).

50 days

3.3 Undertake Vibrio parahaemolyticus ring trials appropriate for methods enabling enumeration of pathogenicity principles (thermostable direct and thermostable direct related haemolysins) (see resolution 27, 7th workshop of NRLs). Analyse results, produce report and recommendations (by May 09).

50 days

3.4 Provision of EQA material, methods of analysis for FRNA bacteriophage (see resolution 20, 7th workshop of NRLs)

10 days




4

3.5 To challenge aspects of the E. coli and Salmonella spp. methods not covered by the standard shellfish EQA scheme organise a whole animal ring trial (see resolution 17, 7th workshop of NRLs)) for NRLs, the scheme will be extended to selected Official Control Laboratories. Analyse results, produce report, advice and recommendations (by May 09).

30 days

3.6 Prepare stable reference material using biological carriers for norovirus and Hepatitis A (see resolution 22, 7th workshop of NRLs). Perform homogeneity and stability analyses. Distribute data and LENTICULES to NRLs for use as control material on request.

10 days

4 Confirmatory testing

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

Up to 50 days

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

50 days

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

10 days

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

Included in above

5 Development of analytical methods (undertaken at CRL)

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

15 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 - both nucleic acid hybridisation and real time PCR approaches.

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

3 specified in Commission Regulation (EC) No 2073/2005 is published as a technical specification with an expiry of December

20 days Up to 20 days




5

2008. It is proposed that the CRL request that ISO SC9 grant an extension for the TS for a further 2 years. In addition, in collaboration with the NRL network, work to generate data enabling adoption as a full horizontal standard should be undertaken. Thus preventing the withdrawal the E. coli reference method.

5.4 It is proposed that limited research to assist in the clarification of

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

Note. The target date for a fully validated horizontal standard method for the detection of norovirus in bivalve shellfish is 2012 (see 5.1). Thus previous restrictions on the implementation of norovirus testing within a European legislative framework, such as the absence of a standard reference method, will no longer apply. Norovirus cannot be routinely cultured in the laboratory and all existing assays in shellfish are based upon detection of a small fragment of viral genome. Application of these assays has shown that a high percentage of shellfish are contaminated with norovirus. Thus the implications of implementation of a European standard for norovirus in BMS may be very significant. It is however unclear whether detection of viral genome always correlates with presence of whole infectious virus and thus a health risk. Limited studies to correlate RT-QPCR data with capsid based assays and the use of analogous detection systems using the closely related but cultivable murine norovirus will assist in understanding the relationship between norovirus positive data generated by the proposed reference method and the presence of infectious virus in shellfish. It is proposed that a financial contribution of up to a maximum of £8000 is provided to partially support a studentship to develop this work area.

10 days

Rachel Rangdale CRL Co-ordinator August 2008




6

WORK PROGRAMME FOR THE COMMUNITY REFERENCE LABORATORY FOR BACTERIOLOGICAL AND VIRAL CONTAMINANTS OF MOLLUSCS, 2007 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 2006 Item Baseline Activity Resources

required (£) 7% overheads

TOTAL budget requested

1 Scientific advice and support

£21,343.30 £1,494.03 £22,837.33

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

£32,014.95 £2,241.04 £34,255.99

3 Ring trials, comparative testing and quality assurance

£103,285.48

£7,229.99

£110,515.47

4 Confirmatory testing

5 Development of analytical methods (undertaken at CRL)

£67,065.21 £8,000.00

£4,694.57 £0.00

£79,759.78

Total Baseline Costs

£231,708.94 £15,659.63 £247,368.57

Workshop

£27,691.07 £1938.37 £29,629.44

EU REFERENCE LABORATORY FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

WORK PROGRAMME 2009

CRL Work Plan 2009

2009 workplan.doc Page 1 of 8 31/05/1115/09/0826/08/08

Ref Function

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

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

1.2 Data on the strengths and weaknesses of existing, as well as new/emerging antibodies and protocols used in confirmatory diagnostic tests will be maintained. This, and other information relating to assessment of antibody performance, will be made available through the technical IHC QA round (see section 2.2), and at the annual NRL meetings. Summary data will ultimately be accessible on the website (see section 3.3).

1.3

Collection and storage of infected tissues from suspects in the UK will continue in order to maintain (as far as possible) a supply of reference materials for National Laboratories (or at least sufficient to enable appropriate characterisation of internal reference material). This collection of tissues is managed by the VLA TSE Archive, and all release of tissues from this collection to the CRL (or any other user) is subject to the approval of Defra’s Independent Archive Advisory Group (IAAG) and charges may be made (http://www.defra.gov.uk/corporate/vla/science/science-tse-arc-intro.htm). Standardised reference materials will also be prepared to facilitate batch testing activities (see section 6.1). Some reference materials, specifically for CRL use, will be generated through experimental challenge of animals (see sections 1.5, 1.6 and 1.7).

1.4

Positive and negative material necessary for annual QA purposes (see section 2 below) will be requested ‘en bloc’ from the TSE Archive, and reserved for CRL purposes. (This will require the continued maintenance of dedicated freezers. It was agreed in 2003 that the Commission would pay 1/5 depreciation costs each year.) We will request material in 2009, to prepare samples for EQA use in 2010.

1.5

As discriminatory tests become more widely used, it will be necessary for the CRL to maintain stocks of ovine BSE for the provision of QA and QC material. To obviate the need for repeat requests to the UK Archive Group (with no guarantee of continued successful application), we challenged four ARQ/ARQ sheep with bovine BSE in 2005, five in 2006, ten in 2007 and five in 2008. This will provide material in the medium term against which other potential alternative supplies (such as the Tg mouse pool being created by IRMM) can be appropriately validated. It also provides material for test evaluation and sensitivity exercises, and QA panels. To date, five sheep have succumbed to disease. The surviving 17 sheep will incur maintenance costs in 2009 if they do not succumb to disease within the next 6 months. It is proposed to challenge a further five sheep in 2009.

1.6

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

CRL Work Plan 2009


An experimental challenge of animals with ‘atypical’ scrapie was started in 2006, with a further five challenged in 2007, to provide a bank of such material exclusively for EU QA purposes. To date, one animal has developed disease, and one is clinically suspect. There was one loss due to intercurrent disease. A further five sheep were challenged in 2008. The 12 animals that are currently healthy are predicted to incur maintenance costs into 2009. It is proposed to challenge a further five sheep in 2009.

1.7

The identification and characterisation of H and L type BSE raises the need for reference material from such cases. Global supplies are currently very limited, and reserved for research. We have challenged (Autumn 2008) two cattle with H-type BSE and two with L-type BSE to generate a small bank of reference material for statutory testing purposes. These animals will incur maintenance charges in 2009.

2.0 Proficiency testing (see appended timetable)

2.1

The CRL will organise two proficiency tests for the interpretation of histopathology and immunohistochemistry (IHC) for BSE in bovines, and scrapie in sheep. The first of these will be in April 2009 and the second in October 2009. The format of these QA distributions will change this year. The existing glass slide distribution system required the preparation of six sets of matching slides from 50 cases, which then provided material for multiple distributions which took five years to complete. We are currently trialling 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 have a number of advantages –greatly reduced postal costs, 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 before, but the web images will be hosted by ‘Slidepath’, an external company which specialises in web-based imaging. This system was demonstrated at the CRL workshop in 2008 and will be run as a pilot in parallel with the final round of the slide distribution system in the Autumn of 2008 to ensure parity of outcome before the change-over.

2.2

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

2.3

A proficiency test panel of ovine blood samples will be provided for the QA of laboratories undertaking genotyping for statutory purposes. Information will be requested about the methods used in each country. Reporting on 4 codons (136, 141, 154 and 171) of the ovine PrP gene will be required from all labs. It is not intended to make provision for caprine testing this year.

CRL Work Plan 2009


2.4

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

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

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

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

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

those NRLs which are operating such methods. Following successful transmission of ovine BSE (see 1.5), it is proposed that this round in 2009 will include an ovine BSE sample in addition to the bovine BSE, to ensure the adequacy of the bovine BSE sample for routine use. However, please note that such ovine BSE samples are precious and will not necessarily be included in subsequent years, unless challenged animals continue to succumb.

• One proficiency test round for cervid rapid tests can be issued if this is still

deemed necessary by the Commission. We understand that the surveillance requirements have been extended to include the 2008 hunting season (which extends into 2009), but the timing of this round would fall beyond the anticipated completion of the surveillance programme.

2.5

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

CRL Work Plan 2009


2.6

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

3.0 Provision of diagnostic and confirmatory testing and advice

3.1

The demand for diagnostic testing will depend on individual countries. Most Member States have adequate arrangements and do not require significant help with routine diagnostic testing. However, confirmation of results may be an important task for CRL, which does not anticipate having to conduct large numbers of confirmatory tests but the service will be available on an ad hoc basis for difficult or perplexing cases (see also section 5). These tests will include HE sections, IHC sections and Western Blotting on unfixed material. The CRL will continue to attempt to collect data on cases which are in some way ‘unusual’, to enable comprehensive cross-referencing and collation of information on such cases for the Commission. For sheep, there is a standardised format for basic data collection and presentation on all positive cases (i.e. not pre-categorised by individual MS), so that more standardised interpretation of results as ‘usual’ or ‘unusual’ (to be undertaken by the CRL Strain Typing Expert Group) is possible across the whole of the EU27. Full instructions have been issued as part of the manual on Discriminatory Testing. The success of any parallel system for cattle data will be dependent on the willingness of MS to comply with such a request if our diagnostic opinion is not sought initially. Additionally, as part of the approval for the heat treatment protocol for the IDEXX test, NRLs must send any samples which are initially reactive on the IDEXX tests, but where the signal is ablated by the heat treatment protocol, to the CRL. Depending upon the amount of such samples which arrive, investigations into the nature of these samples will be conducted by the CRL. No costs have been explicitly planned for this purpose on the assumption that the submission rate will be very low. However, if significant numbers of referrals are made, a separate plan of work will be proposed for this group of samples.

3.2

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

3.3

All relevant information will be published on the CRL website, when appropriate. Discussion fora will be possible on the password protected TSE-LAB-NET system due to go live in 2008.

4.0 Provision of training

4.1 A workshop for National experts will be arranged in the first half of 2009. This workshop will cover all aspects of NRL functions, with the exception of 4.2 below. Feedback will be provided and training needs identified following the outcome of the QA assessments outlined in 2.0.

4.2

Assistance and guidance will be provided to those laboratories experiencing difficulties. Specialist input to Commission fora on an ad hoc basis. Provision has been made in 2009 for three laboratory visits to assist with technical issues should the need arise.

CRL Work Plan 2009


4.3

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

5.0 Strain typing

5.1

The CRL has established a working group of experts in the field of strain differentiation. It will continue to be responsible for the evaluation of any unusual results arising from TSE testing within Europe, and will agree the criteria on which strains will be classified ‘BSE-like’ (and what that means). Advice will be provided on appropriate further investigation and interpretation, to enable the submitting NRL to appropriately and competently brief the relevant National authorities. The panel is drawn partly from experts within the CRL and NRLs, and partly from other sources. [A self-financing representative from SEAC is also on this group, with the Commission’s approval]. This group plans to meet twice in 2009. Discussion will continue to focus on the validation/interpretation of the increasing range of Tg bioassay methodologies. The group will also agree criteria for the classification of bovine isolates. This group will coordinate the provision of material (see also 2.4 and 7.2) for the ring trial of any new potential discriminatory method not presented with sufficient supporting data to be approved by the group without further assessment.

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

5.3 Any isolate still considered BSE-like following ring trial (see section 7.4) will be forwarded for bioassay in mice. At present, only conventional mice are sufficiently evaluated and defined for this purpose, and interpretation is based on a full panel (i.e. RIII, VM and C57Bl6). [The choice of mouse strains is under active discussion (see section 5.1) including the use of Tg strains. There are fewer bioassay data regarding Tg lines compared to the wild type mice. This is because wild type mice have been used for bioassay for over forty years while data from Tg lines have been available only during the last decade, and much of it has no comparative data from conventional lines. Also data from the Tg lines are not directly comparable as a large number of Tg lines are produced and used independently in the originating laboratories. However, there are Tg lines which are used (and published) widely and have also been adopted by the CRL for bioassay of demanding referred samples. These include the ovinised Tg338 line and the bovinised Tg110 mouse line. A cervid Tg line (Tg(CerPrP)1536+/-) has also been adopted for bioassay of TSE cases in deer should they emerge in the EU (see section 7.3). ] These estimates are based on the assumption that a maximum of 1 isolate will

CRL Work Plan 2009


require further characterisation by bioassay.

6.0 Rapid diagnostic methods

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

6.2

The CRL will continue investigations into a sustainable standardised source of positive control material. Currently, this is based upon a macerate or homogenate of bovine brain material to provide all NRLs with the same material, which could be used to compare test performance. However, attempts will also be made to assess more sustainable source materials for the long term. (This issue is partially addressed under sections 1.5 –1.7). There are also ongoing discussions with IRMM about the potential of using transgenic mouse material for this purpose. Homogenates have been prepared in 2008 for QA testing in 2009. There will be requirement to prepare further supplies of homogenate in 2009 for use in 2010. The CRL will ask the COM to write to MS regarding their plans for the continuation of independent NRL status following the EFSA recommendations on changes to cattle testing. Until there is a clear indication of how many NRLs will continue to require full EQA panels, accurate predictions of future requirements cannot be made.

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

6.4

Batch testing of approved rapid tests for the detection of BSE in bovine samples has been introduced in 2008. Nominated NRLs are responsible for testing to an

CRL Work Plan 2009


agreed protocol and the CRL approves batches for release and communicates this information to NRLs for cascade to testing labs throughout the EU. Currently (August 2008) this is via email, but we expect to move to a web-based system on the password protected web-site later in 2008, when passwords have been issued to participants.

6.5 Instigate a batch testing system for small ruminant TSE rapid tests based upon classical scrapie samples, which is run along the same lines as the estabished system for BSE kits. This is dependent upon EU- NRL co-operation.

7.0 Discriminatory testing

7.1 The Discriminatory Testing Handbook detailing precise methods for discriminatory blotting was produced in 2005, and updated in 2007. 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 is being drafted (based on a proposal from J Langeveld, as presented at the STEG meeting in April 2008) for the discrimination of H and L type BSE from C type. If this protocol is made available on line, it should be noted that the material does not yet exist (see section 1.7) to perform adequate - or indeed any -QA or QC and any testing will, by definition, be ‘out of control’. (A referral system will be recommended, but cannot be enforced).

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

7.3 The CRL will continue the evaluation of cervinised mice (developed by Glenn Telling, University of Kentucky) for susceptibility to BSE, and experimental cervid passaged BSE, to establish the suitability of this model for investigation of strain should any European cervid be identified through the surveillance programme. The experimental challenge of these mice was started mid – 2008 and the mice will incur costs into 2009 unless incubation period is less than 150 days. Maintenance of the Tg line at the CRL - either by minimal breeding, or the cryopreservation of the line, should further challenges not be required in the short to medium term, will incur some cost.

7.4 Any positive isolate which presents a discriminatory blot result which is BSE-like should be sent to the CRL. Such cases will be referred to the STEG (see section 5.1) and material distributed around ring-trial laboratories. Cost estimates have been based on the very low level (8 so far) of referrals to date, and assume that a maximum of 1 ring trial will be required.

CRL Work Plan 2009


Appendix 1

PROVISIONAL TIMETABLE FOR TSE CRL QA EXERCISES IN 2009

Intended Start

Date i QA activity

February 2009

Ovine genotyping

March 2009 Immuno-histochemical technique

April 2009 Histopathology and immunohistochemistry interpretation (round 1)

October 2009 Histopathology and immunohistochemistry interpretation (round 2)

October 2009 Bovine rapid testing incorporating confirmatory blotting if appropriate

November 2009 Ovine rapid testing, incorporating confirmatory blotting if appropriate

September 2009 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 2009

CRL « L. monocytogenes » 1/8 23/07/08 2009 Programme of Work


EU COMMUNITY REFERENCE LABORATORY FOR

LISTERIA MONOCYTOGENES

2009 Programme of Work of the Community Reference Laboratory for

Listeria monocytogenes



Introduction In May 2006, the laboratory LERQAP (Laboratory for Studies & Research on Quality of Foods & on Food Processes) of AFSSA (French Agency for Food Safety) has been nominated Community Reference Laboratory (CRL) for Listeria monocytogenes (see Regulation 776/2006). The CRL foresees to undertake the following actions in 2009, according to the actions planned at the 2nd Workshop of the National Reference Laboratories (NRLs) (10&11 April 2008), as well as to the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the CRL for the period 2006-2010. Most of these activities aim at implementing, from an analytical point of view, the EC Regulation 2073/2005 on microbiological criteria for foodstuffs, which includes in particular 4 food safety criteria on L. monocytogenes (Annex I, Chapter 1):

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

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

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

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



1. Detection and enumeration of L. monocytogenes in food

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

a. Study of sample types for inter-laboratory trials The CRL (Unit HMPA) will conduct a study to develop the sample types to be used for the PT trial to be organized in 2009, using as matrix cold-smoked salmon, to which will be added a competitive flora possibly interfering with L. monocytogenes. In particular, the stability of this type of samples will be studied in different transportation conditions to the NRLs.

b. Enumeration of L. monocytogenes The CRL (Unit HMPA) will organize in 2009 a PT trial for the NRLs on the enumeration of L. monocytogenes by the reference method EN ISO 11290-1, using cold-smoked salmon as matrix (1.1.a). A competitive microflora will be added to the samples.

1.2 Analytical development (multi-annual)

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

a. Enumeration method using a membrane filtration

The Standard horizontal method EN ISO 11290-2 for enumeration of L. monocytogenes in food is characterized by a theoretical limit of enumeration of 10-100 cfu/g or ml. Meanwhile, it has been shown that the precision of this Standard method is quite poor, especially a low levels. Even if the Standard has been amended with a more precise method (an enumeration agar, ALOA, now more specific to L. monocytogenes), the method still lacks of enough sensitivity to control precisely a limit at 100 cfu/g or ml. The CRL (Unit HMPA) has developed and validated a more sensitive enumeration method, including a concentration step based on membrane filtration followed by transfer of the filter to a selective medium, for the enumeration of L. monocytogenes in cold-smoked fish. In 2008, the CRL would have tested the applicability of this membrane filtration method to various foods. In 2009, the CRL will further study performances of this method for some particular matrices, defined in agreement with the NRLs (see the enquiry launched in 2008).


b. Environnemental sampling techniques After having reviewed the ISO 18593 Standard, the CRL (Unit HMPA) would have initiated in 2008 a bibliographic study in order to check whether the ISO Standard fully cover the case of L. monocytogenes control in the environment of food production and food handling, or whether there would be a need to add to the ISO Standard specific guidance on sampling techniques for L. monocytogenes control. The CRL will continue this study in 2009.

1.3 Coordination of the NRL network (multi-annual)

a. Measurement uncertainty

The CRL will give the agreed follow-up to the NRL training session on estimation and use of measurement uncertainty (9 April 2008), in the form of recommendations or enquiry to NRLs.

1.4 Training of the NRLs (annual) The CRL (Unit HMPA) will organize a training session for the NRLs on the membrane filtration method previously developed by the CRL, for the enumeration of L. monocytogenes at low contamination levels in cold-smoked salmon. This session will be organized in conjunction with the annual 2009 NRL Workshop.


2. Predictive microbiology

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

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

2.1 Classification of products (with or without L. monocytogenes growth) (multi-annual)

In the guide on durability studies and challenge tests prepared by the CRL (Unit MQER) in 2007 and 2008, the question of the food classification as regards to the potential of L. monocytogenes growth was particularly considered through challenge tests demonstrating or not a significantly positive growth potential. In 2009, the CRL (Unit MQER) will consider this question of classification on the basis of chemical parameters, in particular organic acids, in addition to the pH and aw factors already mentioned in Regulation (EC) 2073/2005. The role of competing flora will be studied as well.

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

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

2.3 Training package (multi-annual)

The CRL (Unit MQER) has initiated the development of an English-language training package on shelf-life of foods related to L. monocytogenes. This package targets food business operators and/or official control services, and the CRL intends to propose to the NRLs to use this presentation for training at national level. In 2008, a PowerPoint presentation would have been prepared by the CRL for the 2008 training session for the NRLs. In 2009, the CRL will prepare an instruction booklet to complement the PP presentation.

2.4 Training of the NRLs (annual)

Further to the 1st session organised in 2008, the CRL (Unit MQER) will organize for the NRLs a 2nd session of a 3-day theoretical and practical training on:

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


2.5 Effect of the inoculum size on growth of L. monocytogenes

(multi-annual) Obtaining quantitative data concerning the relative impact of various factors that may influence bacterial growth is of great importance for microbial risk assessment and predictive microbiology, in particular to predict whether foodstuffs which can permit L. monocytogenes growth will respect the limit of 100 cfu/g-ml at the end of shelf-life. The CRL (Unit HMPA), in collaboration with the Unit MQER and other AFSSA units, will conduct a study concerning the impact of initial contamination level on maximum population attained by L. monocytogenes in food. In 2009, data will be collected from predictive microbiology softwares and databases, and initial assays will be performed on the impact of initial contamination level on L. monocytogenes growth in food within various conditions.


3. Characterization and typing of strains, epidemiosurveillance

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


(annual) This inter-laboratory proficiency testing (PT) trial organised by the CRL (Unit CEB) will aim at evaluating the ability of the NRLs to perform satisfactorily the PFGE (pulsed field gel electrophoresis) method for sub-typing L. monocytogenes strains.

3.2 Dispatch of strains (multi-annual)

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

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

The CRL (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. These new techniques may be used in complement of the PFGE in the future. The CRL will use MLVA or other new molecular sub-typing methods in addition to PFGE for some investigations of listeriosis cases.

3.4 Training sessions of the NRLs (multi-annual)

Further to the 2008 session, the CRL (Unit CEB) will organize in 2009 a 2nd theoretical and practical training session for the NRLs on L. monocytogenes serotyping by conventional and molecular methods. The CRL (Unit CEB) will also schedule a second training session on PFGE sub-typing, especially for the NRLs who would have failed the PT trial (see 3.1).


4. Workshop of the NRLs (annual) The CRL will organise the 3rd Workshop of the NRLs in 2009, of general scope : - to make a progress report on works undertaken by the CRL since the 2nd 2008 Workshop; - to envisage the work programme for 2010 and further.

5. Technical and scientific assistance to the European Commission

5.1 DG SANCO activities (multi-annual)

Upon request of the services of DG SANCO in charge of food hygiene: • Participation to the revision of Regulation 2073/2005 on microbiological criteria related

to L. monocytogenes of: - the CRL coordinator, for the analytical aspects; - 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 and Codex activities (multi-annual)

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

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

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

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

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

EU REFERENCE LABORATORY FOR COAGULASE POSITIVE STAPHYLOCOCCI

WORK PROGRAMME 2009

CRL “CPS” 1/7 23/07/08 2009 Programme of Work


EU COMMUNITY REFERENCE LABORATORY FOR

COAGULASE POSITIVE STAPHYLOCOCCI

2009 Programme of Work of the Community Reference Laboratory for

Coagulase Positive Staphylococci (including Staphylococcus aureus and their toxins)



Introduction In May 2006, the laboratory LERQAP (Laboratory for Studies & Research on Quality of Foods & on Food Processes) of AFSSA (French Agency for Food Safety) has been nominated Community Reference Laboratory (CRL) for Coagulase Positive Staphylococci (CPS), including Staphylococcus aureus and their toxins (see Regulation 776/2006). The CRL foresees to undertake the following actions in 2009, according to the actions planned at the 2nd Workshop of the National Reference Laboratories (NRLs) (26&27 June 2008), as well as to the work programme defined in Annex I of the Framework Partnership Agreement between EC/DG SANCO and the CRL for the period 2006-2010. Most of these activities aim at implementing, from an analytical point of view, the EC Regulation 2073/2005 on microbiological criteria for foodstuffs, 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.



1. Detection/enumeration of coagulase positive staphylococci in food

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

1.1 Study of sample types used for inter-laboratory trials

(annual) As to be able to organize an inter-laboratory proficiency testing (PT) trial on a new matrix compared to the former PT trials, the CRL (Unit HMPA) will conduct in 2009 an investigation study (stability and homogeneity) of freeze drying to prepare an artificially contaminated milk powder which could be used to prepare and dispatch samples for quantitative determinations.

1.2 Inter-laboratory PT trial for the NRLs (annual) The inter-laboratory PT trials organised by the CRL for the NRLs aim at evaluating the ability of the NRLs to apply satisfactory the methods for the analyses performed in the frame of controls prescribed by Regulation 2073/2005. The CRL (Unit HMPA) will organize in 2009 an inter-laboratory trial on the CPS enumeration by one of the reference methods EN ISO 6888-1 & 2, using as matrix fresh cheese, investigated in 2008.

1.3 Optimisation study of the reference method EN ISO 6888-1 (confirmation step) (multi-annual)

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


2. Characterization and typing of strains, epidemiosurveillance

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

2.1 Dispatch of strains

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

2.2 Optimisation of molecular typing by PFGE (multi-annual) The CRL (Unit CEB) will screen its pulsotype database and go on with PFGE optimisation. Once optimised, the CRL will dispatch the PFGE typing method to the NRLs and, upon request of the NRLs, organize a training session.

2.3 Development of SE genes detection by multiplex PCR (multi-annual) The CRL (Unit CEB) has launched in 2008 the development of two multiplex PCR techniques to detect 11 SE genes in the isolated strains. One multiplex technique detects sea-see + ser genes, the other one detects seg-sej + sep. In 2009, the two multiplex PCR techniques will be validated on a collection of strains already tested for these genes using simplex PCR. Once validated, the method will be dispatched to the NRLs.

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

The CRL (Unit CEB) has launched in 2008 a study for the evaluation of recent molecular typing techniques, in comparison with PFGE sub-typing. The CRL has already used spa-typing for some investigations. In 2009, the spa-typing technique will be applied to a collection of food strains. Additionally, a few strains, one per spa-type detected, will be typed using Multi-Locus Sequence Typing (MLST). The CRL will pay attention to any other typing method of interest.

2.5 Training of the NRLs on PFGE typing (annual)

If the optimised PFGE method is found satisfactory (see 2.2), the CRL (Unit CEB) would organize a training session for the NRLs.


3. Detection of staphylococcal enterotoxins in food

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

In order to be able to transfer to the NRLs a confirmatory method, the CRL (Team TOP-BAC) has launched the evaluation of commercially available antibodies to perform a quantitative and specific ELISA test to detect SE type A (SEA) to SEH in milk-based products. In 2008, the antibodies against SEA and SED would have been tested. If the first results obtained in 2008 are satisfactory, the CRL (Team TOP-BAC) would go on this study on antibodies against SEB, SEC and SEH. Once the characterization study conducted, the CRL would provide the NRLs with the instructions for use.

3.2 Feasibility study to develop an internal reference material other than

cheese (annual)

In order to be able to organize a future PT trial on another matrix than cheese, the CRL (Team TOP-BAC) will conduct in 2009 a feasibility study to develop an internal reference material. The aim of this study is to develop and to test a protocol to prepare frozen or freeze-dried samples of SEs spiked food matrices, such as meat products (ex: sausage or minced beef). The stability and homogeneity of such matrices will be studied.

3.3 Development of certified reference materials (CRM) (multi-annual)

The need of (certified) reference materials for SE in food has been highlighted at several opportunities both by the CRL and the NRLs, at the NRL workshops in particular (also in the former frame of CRL/NRLs Milk, including SEs in milk products). Up to now, the CRL (Team TOP-BAC) has organized inter-laboratory trials for the NRLs on SEs detection with freeze-dried SE-spiked cheese matrices. These are the only “reference materials” available. In 2009, the CRL together with DG SANCO intends to identify the possibilities to have developed certified reference materials, the possible collaborators and to contact them, in particular the EC/JRC in Geel. A visit of the latter is scheduled.

3.4 Use of mass spectrometry for SEs characterisation and quantification in food (multi-annual)

To date, 20 SEs and/or SE-like types (SEA to SEIV) have been identified. Among these toxins, only a few of them can be detected using immunological tools, whereas others have an emetic action which can lead to food poisoning. In addition, the currently available immunological detection methods have several drawbacks, including false positive and false negative results. In these conditions, an ELISA-based method is not a satisfactory solution to be the reference method to confirm positive results obtained with the European Screening


Method (prescribed in Regulation 2073/2005 modified). In this context, the LERQAP laboratory has launched a project in collaboration to investigate other tools such as mass spectrometry (MS) to confirm SEs in food matrices. In order to implement this new quantitative method for SEs, the CRL (Team TOP-BAC) will launch the development of a MS Specific Protein Standard Absolute Quantification (SPSAQ) for SE types involved or suspected in staphylococcal food poisoning outbreaks, such as SEH, SEG and SEI.

3.5 Development 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 European Screening Method-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 CRL (Unit HMPA and Team TOP-BAC in collaboration) intends to at first develop and apply this technique to detect and quantify SEA and SED in food:

• The Team TOP-BAC will focus on the evaluation of commercially available antibodies against SEs and the development of quantitative ELISA steps (coating, antigen immobilization).

• The Unit HMPA will focus on the combination of immuno-complex signal and PCR detection steps.

3.6 Training of the NRLs

(multi-annual) The CRL (Team TOP-BAC) will organize and conduct one (or two, depending on the needs) training session(s) on the detection of SEs for the benefit of staff from NRLs.

3.7 Technical and scientific assistance (multi-annual)

The CRL (Team TOP-BAC) will collaborate and provide scientific and technical assistance to DG SANCO and to the NRLs, especially to confirm in some cases positive results obtained by the NRLs with the screening method ESM, in the frame of official controls performed according to the Regulation 2073/2005 modified.


4. Workshop of the NRLs (annual) The CRL will organise the 3rd Workshop of the NRLs in 2009, of general scope : - to make a progress report on works undertaken by the CRL since the 2008 Workshop; - to envisage the work programme for 2010 and further.

5. Technical and scientific assistance to the European Commission

5.1 DG SANCO activities (multi-annual)

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

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

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

On behalf of the CRL and as EC representative, follow-up by the CRL coordinator of the activities of ISO/TC 34/SC 91 & CEN/TC 275/WG 62 for aspects related to the standardization of reference methods for CPS and SETs (1 jointed plenary meeting –budget CRL L. monocytogenes). In particular, participation to the works of 2 working groups of ISO/TC 34/SC 9 of specific interest for the CRL activities and for DG SANCO: - WG 3 “Method Validation” (2 meetings); - WG 4 “Proficiency Testing” (1 meeting).

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

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

(VTEC)

WORK PROGRAMME 2009

Page 1 of 7

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

Unit of Foodborne Zoonoses and Veterinary Epidemiology Istituto Superiore di Sanità

Community Reference laboratory (CRL)

for Escherichia coli,

including Verotoxigenic E. coli (VTEC)

Work Programme

1st January - 31st December, 2009

Page 2 of 7

Introduction

The work programme of CRL for VTEC (CRL-VTEC) for the year 2009 will consist of

the following activities:

1. Consolidating the CRL structures

1.1. Staff 1.2. Accreditation of the laboratory

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

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

2.4. Characterization of VTEC strains isolated by the NRLs

3. Implementation of the CRL-VTEC web site 4. Co-operation with other European Community structures

4.1. The European Food Safety Authority (EFSA) 4.2. The European Committee for Standardization (CEN)

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

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

5.2. Validation study of the method EN/ISO 16654 for E.coli O157 6. Research on VTEC accessory virulence factors 7. Missions

The duration of each action is indicated, and will be either limited to 2008 or multi-

annual (ongoing program).

Page 3 of 7

1. Consolidating the CRL structures

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

activities. Other dedicated staff, hired with CRL funds, will work full time at the CRL-

related activities. The administrative procedures to change the administrative status of

this staff from “temporary research collaborators” to the more stable status of “temporary

staff employees” have been activated in 2008 and should be completed within 2009.

(Duration: ongoing)

1.2. Accreditation of the laboratory

The CRL-VTEC will maintain the formal accreditation UNI EN/ISO 17025 of its

quality assurance system (obtained from the Italian body for accreditation, SINAL,

accreditation N. 0779) and of the methods for detection and typing of VTEC related

with CRL’s tasks and activities. The possibility to submit additional methods for

accreditation will be evaluated.

(Duration: ongoing)

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

training 2.1. Annual workshop with NRLs The 4th annual workshop will be held in the second half of 2009 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 2009 inter-laboratory studies will be

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

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

NRLs needs.

2.2. Assistance to NRLs

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

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

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

standard operating procedures for detection of other pathogenic Escherichia coli in

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

be developed and discussed with the NRLs.

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Duration: ongoing (as required)

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

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

training on specific topics related with detection and typing methods.

Duration: ongoing (as required) 2.4. Characterization of VTEC strains isolated by the NRLs After the introduction of phage-typing in the panel of typing methods available at

CRL-VTEC (foreseen by the end of 2008), the NRLs will be asked to send

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

different sources for phage-typing and characterization by molecular techniques,

such as pulse-field gel electrophoresis (PFGE). The shipment of the strains from

each NRLs will be made periodically to contain the delivery costs. The data obtained

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

from animals, harmonized with the Enter-net database for human isolates,

maintained by the ECDC in the framework of the recently established Food- and

Waterborne Diseases (FWD) Network. Such a project could allow the comparison of

the phage-types of VTEC O157 isolated from non-human sources with those of the

strains causing human infections throughout Europe.

Duration: ongoing

3. Maintaining and implementing the CRL-VTEC web site

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

section, to allow NRLs to directly upload their data.

Duration: ongoing

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

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

4.1. The European Food Safety Authority (EFSA) Dr. Caprioli will participate in the Working Group on monitoring of VTEC in animals

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and food, established by the EFSA Task Force on Zoonoses Data Collection. The

task of the WG is the preparation of a harmonized monitoring scheme for VTEC in

animals and/or foods, which should take place in the framework of the Zoonoses

Directive 2003/99/EC. More generally, the CRL-VTEC will contribute as reference

laboratory (elaboration of methods, pahge-typing of VTEC O157, etc.) to any program

on VTEC Monitoring that will be implemented by EFSA.

4.2 The European Committee for Standardization (CEN), Technical Committee 275 – Food analysis – Horizontal methods, WG 6 – Microbial contamination. 4.2.1. Validation study of the method EN/ISO 16654 for E.coli O157

The CRL-VTEC has presented, as project leader, a proposal for the validation and

revision study of the method EN/ISO 16654 for E.coli O157 in foodstuffs. The study,

which will involve 13 NRLs for E.coli, had been scheduled for 2008 but, due to delay

in the approval of the project by CEN, will probably take place in 2009. In any case it

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

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

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

edited a draft Technical Specification “Horizontal method for the detection of Shiga

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

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

Method”, recently submitted to CEN for final approval. Upon adoption, such a

Technical Specification will undergo a 3-years period of evaluation and the CRL will

organize ad hoc evaluation studies.

4.3. The European Centre for Disease Control (ECDC) Food- and Waterborne Diseases (FWD) Network (formerly Enter-net) The coordination of the Enter-net network has been taken over by the ECDC, who

has created the Food- and Waterborne Diseases (FWD) Network, constituted by the

reference laboratories for human infections in EU Member States. The ECDC is in the

process of selecting a reference laboratory for VTEC infections, which will be in

charge of the external quality assurance activities for the network. The CRL-VTEC will

establish connections and liaisons with the FWD reference laboratory for VTEC, with

the objective of harmonizing the identification and typing schemes and making the

respective monitoring programs and databases compatible for comparison of human

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and non-human data.

4.3 MED-VET-NET, the European Network of Excellence for research on the prevention and control of zoonoses. The collaboration with several institutes on the virulotyping of VTEC will continue

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

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

the deputy coordinator.

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

VTEC will contribute to the preparation of the technical booklets on VTEC

epidemiology and ecology.

Duration: ongoing

5. Inter-laboratory comparison studies 5.1. 3rd inter-laboratory study with the NRLs The 3rd inter-laboratory study will presumably still be focused on VTEC identification

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

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

discussed with the NRLs during the 2008 Annual Workshop and harmonized with the

ECDC-FWD reference laboratory for VTEC.

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

The CRL-VTEC is project leader of the validation and revision study of the method

EN/ISO 16654 for E.coli O157 in foodstuffs (see point 4.2.1). The study will begin as

soon as the financial support from CEN will be available and will involve 13 NRLs,

which had been selected on the basis of a call for expressions of interest. The

validation study will also function as external quality assurance test for the 13

participating NRLs with respect to the EN/ISO 16654 method. It will be possible to

prepare additional samples at CRL own costs to be sent to the other NRLs, in order

to extend the external quality assurance test for the EN/ISO 16654 method to all the

available NRLs.

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6. Research on VTEC accessory virulence factors

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

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

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

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

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

Duration: ongoing

7. Missions

The following missions may be needed in 2008:

• Participation of a scientist at VTEC 2009, the 7th International Symposium on

Shiga Toxin (Verocytotoxin)-producing Escherichia coli infections that will be

held in Buenos Aires10-13 May, 2009.

• Participation of a scientist at the 2009 CEN/TC275 WG6 annual plenary

meeting. • Participation of a scientist at the 2009 WG6 TAG3 general meeting. • A visit to one NRL can be planned for 2009, upon agreement with the EC and the

interested countries.

August 20th, 2008

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

EU REFERENCE LABORATORY FOR CAMPYLOBACTER

WORK PROGRAMME 2009

CRL- CAMPYLOBACTER WORK PROGRAMME FOR 1st OF JANUARY 2009 TO 31st OF DECEMBER 2009 Introduction The activities in the working programme for 2009 for the Community Reference Laboratory (CRL) for Campylobacter will follow EU legislation on CRLs functions, duties and designation (Regulation (EC) No 882/2004 and Commission Regulation (EC) 776/2006). The work programme for 2009 will consist of the following key activities, repeated annually:

1. Ring test/s (interlaboratory comparison study/ies) 2. Workshop 3. Research 4. Scientific and technical assistance to the European Commission and to the NRLs-

Campylobacter, including ad hoc activities 5. Missions 6. Communication 1. Organisation of a ring test in 2009 In May 2008, a ring test for detection and enumeration of Campylobacter in broiler skin was organised. The test material and protocol for analysis were designed to be similar to the EU survey of the prevalence of Campylobacterin broiler flocks and broiler carcasses (Commission Decision of 19 July 2007, 2007/516/EC) that is carried out in 2008. A second, voluntary ring test is organised for identification of Campylobacter species by three PCR-based assays. Results from all ring tests organised by the CRL so far have shown that confirmation and species identification of Campylobacter by molecular methods (PCR-based assays) is more reliable compared to phenotypic identification. The majority of NRLs are using molecular techniques, but not all of them. At the workshop in 2007, it was agreed that the CRL would organise this voluntary PCR ring test which will be submitted to the NRLs in September 2008. The reason is to provide a solid basis for giving recommendations about useful and simple PCRs for NRLs that want to set up this type of assays. For the ring test in 2009, the CRL will follow on from the activities in 2008, and prepare a test for detection and enumeration of Campylobacter in chicken retail meat. As for the 2008 ring test, the standardised protocols of ISO 10272 part 1 and 2: 2006 “Microbiology of food and animal feeding stuffs – Horizontal method for detection and enumeration of Campylobacter spp”, will be applied. The choice of culture media for detection and enumeration of Campylobacter is always discussed, also at ISO standardisation meetings, and the ring test will therefore include use of one or two new media that differ from the media indicated in the ISO 10272 standard. An EU survey on Campylobacter and Salmonella in broiler meats at retail is being proposed, and the planned ring test in 2009 will be a good way to evaluate the

performance of the Member States´ NRLs for this type of matrix and analyses. It will also give the opportunity to evaluate new and improved media for Campylobacter analyses. From the preparations of the 2008 ring test, the CRL obtained useful experience about using live cultures for inoculation of samples. However, for the 2009 ring test, the plan is to use freeze dried cultures with Campylobacter. For more information about the preparation of test material, please see point 3 (Research). The ring test is planned to be distributed by courier in the spring 2009. 2. Organisation of a workshop The workshop will be organised in the fall (September or October) in 2009. Representatives from the Member States´ NRLs for Campylobacter will be invited. The EU Candidate Countries (financed by TAIEX) and Iceland, Norway and Switzerland (on their own budgets) will also be invited to participate. Further, experts from EC, the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) will be invited. The agenda will include presentations and discussions on:

- Campylobacter activities in the EU. Results of zoonosis monitoring (EFSA), the EU surveys in broilers (EC), and monitoring and control of Campylobacter in humans (ECDC).

- Results of ring tests - Molecular methods for identification and characterization of Campylobacter - Campylobacter activities in MS, including national monitoring and research

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

matters of common interest

3. Research 3.1. Studies on bacterial and matrix reference materials for the ring tests. For the 2009 ring test, the CRL plan to purchase freeze dried reference material consisting of different Campylobacter species of varying and specified levels. The CRL will test batches of the freeze dried reference material with matrices of broiler retail meat. In addition to plating on mCCD agar (which is indicated in the standard ISO 10272- 2:2006), two other selective agar plates are planned to be tested for the enumeration of Campylobacter. Different types of broiler meat, included in the proposed next baseline survey on Campylobacter in broiler meat at retail will be tested to ascertain the stability of the test. The test will be delivered by courier and should be delivered within 48 hours. However, the aim is to have samples that are stable for at least 5 days. If there are problems with the freeze dried material, the CRL will use live bacterial cultures applying the technique that was developed for the ring test in 2008.

3.2. DNA-based (molecular) and alternative methods for detection, species identification and strain characterization of Campylobacter. The traditional methods for detection and identification of Campylobacter include culture in selective media, followed by phenotypic methods for genus and species identification. Although molecular or DNA based methods are considered more reliable, there are no ‘golden standard’ methods for detection, species identification or strain characterization (subtyping/fingerprinting). Among the most commonly used fingerprinting or subtyping methods are macrorestriction profiling of genomic DNA by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and others, such as short variable region (SVR) sequencing of the flaA gene. Although they are useful for different purposes and in research settings, there is still a need to agree on methods and interpretation of results. The CRL will continue to review and test new and alternative methods for analysis of Campylobacter. 3.2.1 As a follow-up to the voluntary PCR ring test in 2008, other molecular methods (i.e. other PCRs) for detection and identification of Campylobacter will be reviewed and discussed with the NRLs for future interlaboratory comparative testing. A questionnaire will be prepared in 2009 to collect information about subtyping/fingerprinting methods that are used at the NRLs for Campylobacter. The obtained information and possibility of future ring tests including subtyping will be discussed at the workshop in 2009. 3.2.2. Mass spectrometry for detection and strain characterization The CRL will continue to investigate the usefulness of mass spectrometry technology for identification of Campylobacter species and also for strain characterization. Obtained mass spectra indicate that mass spectrometry could be used for subtyping and would provide a very rapid and automated method for analysis of large number of samples. 3.2.3. Detection of Campylobacter in Acanthamoeba polyphaga culture Broiler carcass samples from the baseline study in 2008 (Commission Decision of 19 July 2007, 2007/516/EC) are, in addition to being tested according to the Technical specifications for the baseline study, co-cultured in amoebas. The intention is to evaluate the capacity of the amoebas for enrichment of Campylobacter spp. The results from parallel testing with amoebas will be summarised and reported in 2009. 3.3. Database and analyses of Campylobacter isolates from EU monitoring survey/s The EU monitoring of Campylobacter in broilers in 2008 specifies that MS shall submit a maximum of 16 isolates/MS to the CRL for quality control. This means that a maximum of 432 isolates can be submitted to the CRL. The CRL has prepared instructions for submission of isolates and protocols for the analysis and how the documentation shall be done at the CRL. The CRL will set up a database for the strain collection from 2008 including test results of conventional phenotyping and PCR assays. A summary of test results will be prepared and reported to the Commission in 2009. In addition, the CRL will subtype a selection of the isolates by PFGE in 2009 and possibly perform other genotyping. This will provide valuable information about what type of strains are prevalent and also show strain diversity among Campylobacter in broilers in Europe.

If the new, proposed EU survey on Campylobacter and Salmonella in broiler meats at retail is performed in 2009, isolates of Campylobacter will be submitted to the CRL for quality control also in 2009. The CRL will apply the same routines for the isolates of that survey. 3.4. Participation in international research networks, research projects and international conferences In 2009, CRL staff members will participate in:

- EU funded network of excellence Med-Vet-Net Workpackages: WP 24 “Development of a European consensus frame work on risk assessment of Campylobacter in broiler meat”, WP 30 “Towards a combined microbiological and epidemiological approach for investigation of host-microbe interactions of C. jejuni” (Campynet III), WP 33 “Early host responses to Salmonella and Campylobacter” and WP 34 “Control and prevention of Campylobacter in poultry”

- ISO/CEN standardisation activities: Participation in 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. A study regarding Campylobacter analysis of samples from the primary production was initiated in 2008 to gain more knowledge for the development of an ISO 10272- Part 4. The study will be summarized and reported at ISO/CEN meetings in 2009

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

campylobacters in poultry farm environments and suggested control measures”.

- 15th International Workshop on Campylobacter, Helicobacter and Related

Organisms (CHRO). CHRO meetings are held every other year and are the most important scientific meetings for researchers and other people active in the area of diagnostics, epidemiology and prevention and control of Campylobacter. The CRL will participate and plans to present the organisation of the European CRL-NRL-network for Campylobacter and its activities.

- Participate in other relevant national and international seminars and research

meetings in order to assure competence and knowledge on recent advancement within the Campylobacter area

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

The CRL will provide scientific and technical assistance to the Commission and the NRLs on issues regarding Campylobacter. The ongoing EU monitoring surveys of Campylobacter in broilers and in broiler meat generate many questions on technical aspects of the analyses and related issues. It is expected that these types of requests for assistance, including ad hoc activities, will also be made in 2009.

Request from the NRLs and the Commission for support and advice will be handled by the CRL scientific staff as soon as possible. Assistance to the Commission and EFSA services will have priority. The proposed monitoring of Campylobacter in broiler meat at retail also indicates that isolates be sent to the CRL- Campylobacter for quality control. The CRL will analyse and keep the isolates in the same way as for the monitoring survey performed in 2008. 5. Missions

The following missions are planned to be made (or may be necessary) in 2009:

- The 28th meeting of ISO/TC34/SC9 and the 16th meeting of

CEN/TC275/WG6, which will be in Spain in the second half of April 2009. 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.

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

- The 15th International Workshop on Campylobacter, Helicobacter and

Related Organisms (CHRO) in Niigata, Japan, 2- 5 September 2009

- Approximately 2 meetings with Med-Vet-Net workpackages, financed by Med-Vet-Net.

6. Communication

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

EU REFERENCE LABORATORY FOR PARASITES (IN PARTICULAR TRICHINELLA,

ECHINOCOCCUS AND ANISAKIS)

WORK PROGRAMME 2009

Community Reference Laboratory for Parasites

Istituto Superiore di Sanità

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FOR

PARASITES

WORK PROGRAMME

2009



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CRL for Parasites work programme 2009

Introduction The 2009 working programme of CRL for Parasites (CRLP) consists of the following activities: 1. Ad hoc activities 1.1 Trichinella

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

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

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

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

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

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

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

1.2 Anisakidae worms

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

o Diagnostic activity

1.3 Echinococcus

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

1.4 Cryptosporidium

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

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

animals and foodstuffs (multi-years)

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

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

2.4 Identification and development of analytical methods for the speciation of parasites of the genus Cryptosporidium (one-year)

2.5 Identification and development of analytical methods for the speciation of liverflukes (multi-years)



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2.6 Identification and development of analytical methods for identification of microsatellites in Echinococcus multilocularis (multi-years)

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

3 An interlaboratory comparison study

4 Workshop

5 Visit to NRLs

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



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

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

1.1.2 To establish an international reference standard serum for the serological

surveillance of Trichinella infection in swine Four freeze-dried preparations of pooled swine sera containing specific immunoglobulin G (IgG) to Trichinella spp, are now available and ready to use in quantitative assays to determine the anti-Trichinella IgG in swine. The material has been proposed to be considered for the establishment of an international standard for the diagnosis of swine trichinellosis. During the last NRLs Workshop held in Rome, this proposal was unanimously accepted. A collaborative study with five laboratories will be organized with the aim to determine the potency of the preparations and to give them arbitrary units of their biological activity. Further, other EU laboratories and three laboratories from Canada, USA and Switzerland will be invited to joint the project to evaluate the reference standard/s. Since the National Institute for Biological Standards and Control (NIBSC) maintains a central role in the development of the scientific basis for control and standardization of biologicals, its assistance will be asked for.

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

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

1.1.4 To produce reference Trichinella antigens for serology

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



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1.1.5 Maintenance of reference strains of Trichinella in vivo

Reference strains for each species or genotype of Trichinella identified so far, will be maintained in laboratory animals. Fresh mouse carcasses infested with Trichinella species/genotypes will be provided to laboratories for training of personnel and for typing of wild isolates. Trichinella larvae from reference strains will be stored in ethyl alcohol and forwarded to laboratories as reference material for typing of wild isolates. To increase the quality and the reliability of the maintenance of these reference strains in vivo, the identity of each infected mouse will be monitored by a microchip which will be inserted under the skin.

1.1.6 Diagnostic activity Diagnostic samples provided from NRLs will be tested with the following accredited tests:

i. Identification of parasites of the genus Trichinella by a multiplex-PCR analysis

ii. Identification of anti-Trichinella IgG antibodies in swine sera iii. Identification of anti-Trichinella IgG antibodies in human sera iv. Detection of Trichinella larvae in meat samples

1.1.7 Collection of serum and/or meat juice samples from wild boars and foxes

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

1.2 Anisakidae worms

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

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

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



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1.4 Cryptosporidium

1.4.1 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.

1.5 Diagnostic activity A number of diagnostic tests are also available at the CRLP for the diagnosis of other foodborne parasitic infections in food, animals and humans.

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 increasingly taking hold inside the scientific community. The are many possible applications of the DNA barcoding from biodiversity studies to food tracking. In the field of the food-borne parasite control, we would like to focus our attention on the liver flukes circulating in freshwater fish of Europe, because only one species, Opisthorchis felineus, is zoonotic, and the larval stage present in fish (the metacercaria) and the eggs shed by the definitive hosts cannot easily been distinguished from the metacercariae of non-zoonotic liver flukes. Recently, O. felineus has been identified as the etiological agent of 4 human outbreaks which occurred in the Mediterranean basin. Little information is available on the spread of zoonotic and non-zoonotic liver flukes in Europe. Hence the need for a molecular based identification system allowing a reliable an quick response and that can be used also by non-specialist staff. Our task will be the identification of specific DNA regions that could be used for the identification at the species level and the evaluation of their potential for a large scale application. Nevertheless, the application of the DNA barcoding to the identification of other parasites detected in Europe will be also implemented as, for example, the identification of nematode larvae resembling Trichinella that frequently are collected during the digestion of muscle samples.

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

course of 2009, our work is going to be focused on two main objectives. The first will be the production of monoclonal antibodies (mAbs) specific for at least one of the validated oocyst wall proteins (OWPs) of T. gondii. Mice will be immunized with recombinant fragments of selected TgOWPs and cell lines producing monospecific antibodies will be obtained following the standard hybridoma technology. Hybridoma supernatants will be screened by ELISA against the recombinant protein used as immunogen or by immunofluorescence on intact T. gondii oocysts. The production of mAbs specifically reacting with the outer layer of the oocyst wall will be exploited to develop a rapid immunofluorescence assay for the coprodiagnosis of toxoplasmosis in cats. The availability of the above mentioned mAbs will also open the possibility to set up an immuno-magnetic capture assay for the detection of T. gondii oocysts in environmental



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samples. The second objective of our study will be a more accurate definition of the TgOWP family through the cloning of the cDNAs encoding 27m, 41m, 50m and 76m and the immunolocalization of each protein using specific antisera.

2.3 Identification of intraspecific genetic variability of Trichinella spiralis and T. britovi.

We will continue the nucleotide characterization of allele sequences detected at the heterozygote status. For sequence analysis, alleles will be isolated in single colonies by amplification, cloning and sequencing. The results obtained so far have shown the presence of unique alleles in single isolates. Since single isolates from specific areas have been investigated so far, the number of isolates from each area will be increased in order to understand if the allele is shared with other isolates circulating in the area, allowing to use the allele as geographical marker. Twelve loci of at least five isolates for each geographical area will be screened. At least 20 single larvae will be analysed for each isolates. On the basis of the allele frequencies from a large number of isolates, the genetic relationships among the isolates will be analysed to draw a map of dispersion of the alleles over the European continent. Preliminary results show that the panel of primer pairs studied for the Trichinella spiralis genome can be used to amplify DNA from Trichinella britovi. A program similar to that used in past for T. spiralis, will be used to analyse the population genetic of T. britovi isolates. The DNA extracted from isolates present in the collection of the International Trichinella Reference Centre, will be used.

2.4 Evaluation of the use of serology to monitor Trichinella infections in wild boars

and/or red foxes. Sera and/or meat juices collected from hunted wild boars and/or red foxes will be tested by an ELISA. Sera and/or meat juices from wild boars and/or red foxes originating from Trichinella-free regions (e.g. Great Britain, Denmark) will be used as negative controls to establish the ELISA cut off and the ELISA index “IE”. Since the Western blot analysis allows to distinguish specific Trichinella antigens from cross-reactive antigens, ELISA positive samples will be tested by Western blot using excretory/secretory antigens.

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

of the genus Cryptosporidium. Cryptosporidium is an enteric parasite, which has a global impact on the health, survival and economic development of millions of people and animals world-wide for which there is currently no effective chemotherapy. Currently 19 species of Cryptosporidium are regarded as valid and over 40 genotypes are recognised, with new genotypes continually being identified. However, only nine Cryptosporidium species/genotypes have been identified in humans, including C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris, C. suis, the cervine genotype and the skunk genotype. Traditionally, the detection of Cryptosporidium oocysts in environmental, water, food and faecal samples has primarily relied on examination by microscopy. However, morphological characters for identifying Cryptosporidium are few, and identification based on light microscopy alone is unreliable and relatively time consuming. Since the development of the polymerase chain reaction (PCR), PCR-based techniques have permitted specific and sensitive detection of Cryptosporidium spp. for clinical diagnosis and environmental monitoring. There is no standard locus recommended for detecting Cryptosporidium, and a wide variety of loci have been utilized. The purposes of our activity are (i) to compare the available methods



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in terms of their specificity and sensitivity, and (ii) to develop new assays with better characteristics of specificity and sensitivity to be used to identify these parasites in environmental samples, vegetables, water and foodstuff.

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

of Echinococcus granulosus s.l. Parasites belonging to E. granulosus s.l. have been separated in ten genotypes (G1-G10) grouped in four species (G1-G3, E. granulosus s.s.; G4, E. equinus; G5, E. ortleppi; and G6-G10, E. canadensis) showing different epidemiological patterns (e.g., intermediate hosts), geographical distribution and different pathogenicity to humans. Consequently, there is an urgent need to map the distribution and circulation of these genotypes in the MS. Literature data and sequence information available in the Genebanks, will be used to prepare specific primer sets to develop a standard protocol for the identification of the different parasite stages (eggs in faecal samples of definitive hosts; protoscolices in cysts of intermediate hosts) at the species or genotype level by PCR, PCR-RFLP and sequencing.

2.7 Collection of epidemiological data on the prevalence of the trematode worm Alaria alata in wild boar populations of MS. In the course of the workshop, which was held in Rome from 29 to 30 May, 2008, several participants expressed their concern on the increased detection of Alaria alata infestations in wild boars. Alaria alata is an intestinal trematode of carnivores (e.g. dogs, cats, foxes, wolves) which can reach other mammalian hosts such as the wild boar, which acts as paratenic host. This trematode can infect the human being, even if the number of documented infestations in humans is very limited. In collaboration with NRLs, the presence of A. alata in the wild boar population of MS, and the prevalence in endemic regions will be monitored to map the risk for this parasite in the EU and in neighbouring countries from which the infection can spread into EU.

3 Interlaboratory comparison studies 3.2 Trichinella. According to the request of NRLs, expressed in the course of the Workshop

which was held in Rome from 29 to 30 May, 2008, a ring trial will be organised among NRLs for the third time to evaluate the sensitivity of the magnetic stirred method as reported in the EU legislation 2075/2005 on Trichinella. Test samples (100gr meatballs made with diaphragm tissue from pigs) will be spiked with a known number of T. spiralis larvae obtained from experimentally infected mice. Each NRL will receive 9 samples containing three different number 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 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.

3.3 Other parasites. Worms (e.g., Echinococcus, Anisakis) or protozoa (e.g.,

Cryptosporidium) spiked in their natural matrices, will be forwarded to NRLs to evaluate their skill to detect and identify the parasites.



page 9 of 9

4 Workshop

In the first half of 2009, 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 ring trial/s, and other issues including epidemiological problems related to foodborne parasitic zoonoses occurring in the MS. Some experts in the field of foodborne parasitic zoonoses will be invited to present the most recent knowledge on the epidemiology, diagnosis and control of some zoonoses.

5 Visit to NRLs

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

6 Training for Personnel of NRLs and from developing countries

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

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

A discussion forum will be developed on the CRL web site, in order to provide a tool to strengthen the collaboration among NRLs-CRLP. Only authorized users will have access to the forum, while it will be free for reading. It will be possible to upload and download documents and images, and to share every material that could be useful for the network. In addition, the web site will be up dated with epidemiological information (accessible to the NRLs and the public).

Rome, 1st August, 2008 The Director of CRL for Parasites

Dr. Edoardo Pozio

EU REFERENCE LABORATORY FOR ANTIMICROBIAL RESISTANCE

WORK PROGRAMME 2009

Work plan for 2009 The main purpose of the Community Reference Laboratory on Antimicrobial Resistance (CRL-AR) is to ensure the quality of antimicrobial susceptibility testing in the Member States and to harmonise the procedures and methodologies used. Thus, most of the activities aim at implementing, from an analytical point of view, the provisions of monitoring of antimicrobial resistance set down in Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents. In addition the CRL-AR provides assistance to the member States and the Commission on other relevant aspects of antimicrobial resistance. Furthermore, the CRL-AR is requested to work in an international context and ensure that EU influences and follows global standards and guidelines. Scientific advice and support to the Commission During 2009 the CRL will provide advice as stated under the general terms. The CRL will participate in workshops and working groups on antimicrobial resistance initiated by EFSA, Codex and FAO/WHO/OIE. In addition, the CRL-AMR will participate ad hoc in meetings arranged by EMEA. The WHO has recently established an Advisory Group in Surveillance of Antimicrobial Resistance (AGISAR), which has as the aim to develop global standards for monitoring. The CRL-AR has and will actively support this initiative that also involves EFSA. Co-ordination of National Reference Laboratories and provision of technical support Workshops In 2009, the CRL will host a workshop in June with the following tentative agenda:

- Introduction and presentation - Update of the functions of the CRL-AR - Update on other CRL’s - Update from EFSA, the commission and other parties - Updates on ongoing projects (streptomycin, ESBL, qnr) - Break points and cut-off values - MRSA isolation, detection and epidemiology - Results of the ring trial performed in 2008/2009 - Presentation and discussions on national programmes on susceptibility testing, control

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

- Presentation of activities at the NRL’s Meetings on standardization of monitoring of antimicrobial resistance The most important problem in relation to ring trails and monitoring of resistance is the lack of common interpretive criteria. This is a global problem and not only related to EU. The CRL will in 2009 together with WHO (AGISAR) arrange meetings with important stakeholder in order to promote a common international standard. This is also essential for the work planned in Codex Alimentarius. Maintaining the network of NRL’s The CRL will during 2009 maintain and continuously update a full list of contact persons from all NRL’s. In addition, the CRL will attempt to identify expected members from

applicant countries to include in the network. This list will also be maintained during the following years. Dissemination of knowledge and information The CRL will maintain the CRL web page (www.crl-ar.eu) where relevant information is posted. In addition, the CRL will send two newsletters with highlights to the NRL’s as pdf-files. Specifically for 2009 the CRL will provide updates on the current situation in Europe and suggested methods used for isolation and identification of extended spectrum beta-lactamase producing and quinolone resistant bacteria, as well as methicillin-resistant Staphylococcus aureus. The CRL will provide updated lists of suggested break points based on the work done by EUCAST (www.eucast.org) and other international standardization committees. Specially the CRL will host and participate in meetings with the aim to get standardised break points and cut-off values globally as mentioned above. In addition, knowledge on which antibiotics to test for and ranges to use as well as other problems encountered will also be disseminated between the NRL’s. Collection of information on activities at the NRL’s The CRL will through the NRL’s continue to gather information on the activities in relation to antimicrobial resistance from the different member countries. This will include information on:

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

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

Specific assistance to individual laboratories, site visits or individual training courses Some NRL’s might have a need for special assistance. The CRL will to the extent possible within the financial limits provide specific assistance to individual laboratories based on individual needs. Specifically for 2009 the CRL will organise a one-week training course on susceptibility testing of Salmonella and Campylobacter for a maximum of 15 participants. The participants will be selected from the NRL’s where the performance in the ring trails was below the threshold set by the CRL and discussed during the workshop in 2008. Furthermore, the CR_AR will together with the CRL for Staphylococci organise and host a training course on isolation, identification and characterisation of MRSA. Ring trials, comparative testing and quality assurance External quality control is an important part of ensuring and maintaining the analytic quality of laboratory tests performed. The CRL will in the spring and autumn 2009 organize the following ring trials on antimicrobial susceptibility testing for the NRL’s:

http://www.crl-ar.eu/

http://www.eucast.org/

1. Salmonella 2. Campylobacter 3. Escherichia coli 4. Enterococci 5. Staphylococci 6. Detection of MRSA (together with CRL Staphylococci)

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

EU REFERENCE LABORATORY FOR ANIMAL PROTEINS IN FEEDINGSTUFFS

WORK PROGRAMME 2009


for animal proteins in feedingstuffs” CRL-AP

Walloon Agricultural Research Centre – CRA-W (Belgium)

Work programme for 2009

1.Scientific advice and support to the European Commission

1.1 Provide scientific and technical assistance to the European Commission in relation to the

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

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

reference laboratory, participate to international fora/committees relating to the detection of

animal proteins in feedstuffs (EFSA, WHO/FAO, JRC, etc) with eventual presentations to

prepare for it. As up to 2 European or international missions/year are foreseen in support to DG

SANCO and/or CRL activities. (2 p/m)

1.2.1 Preparation and participation in international meeting/fora

1.2.2 Report/minutes following completion of the mission

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

reference laboratory, participate in meetings for the standardisation of analytical methods relating

to the detection of animal proteins in feedstuffs and their implementation (CEMA, ISO/CEN,

OIE, IAG, etc). Up to 3 European missions/year are foreseen in support to DG SANCO and/or

CRL activities. (1 p/m)

1.3.1 Participation in the CEMA, CEN and IAG meetings

1.3.2 Reports/minutes of the meetings

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

context of incidents or crises linked to incorrect use of animal proteins. (3 p/m)

1.4.1 Provide technical and scientific support

1.4.2 Submission of the report on the technical and scientific support provided

1.5 To keep at CRL the highest standard possible of technical skills, scientific awareness and quality

management under accreditation (ISO17025, later on maybe even ISO9001) on analytical

methods for detection, quantification and identification of animal proteins in feed ingredients and

in feedingstuffs. To maintain and extend the accreditation scope of the CRL lab. (12 p/m)

1.5.1 Maintain of the accreditation scope

1.5.2 Extend of the accreditation scope : accreditation of the CRA-W PCR method for the

detection of cattle DNA

1.5.3 Preparation of the file for the organisation of interlaboratory studies This task includes the

maintenance of the competences of the CRL-AP team and the training of the new recruits

1.6 On the request of DG SANCO, to perform analyses on samples with disputed results. (12 p/m)

1.6.1 Perform the requested analyses

1.6.2 Report on the analyses performed

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

or/and candidate countries

1.7.1 Administration and participation of candidate countries (Croatia, Turkey) to the

annual NRLs network workshop.

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

2. Coordination of activities of NRL network (13 p/m)

2.1 Construction, development and maintenance of CRL website (internet/intranet) to

disseminate and share information with NRLs and others stake holders. (3 p/m)

2.1.1 Information collection and validation

2.1.2 Development of the website (internet and intranet)

2.1.3 Development of the management tools of the website

2.1.4 Test of the information system and validation

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

2.2.1 Preparation and sending of three newsletters in 2009

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

2.3.1 Organisation of the 3rd annual CRL-AP workshop

2.3.2 Preparation of the agenda

2.3.3 Invitation of the attendees

2.3.4 Realisation of the workshop

2.3.5 Minutes of the annual workshop

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

quantification and identification of animal proteins in feed ingredients and feedingstuffs.

2.5 Participate to annual CRL Directors co-ordination meeting.

2.5.1 Participation to the CRL directors co-ordination meeting

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

transmitted by DG SANCO.

2.6.1 Prepare and submit the 6-months report (January 2009 – June 2009) and annual report

(January 2009 – December 2009)

3. Interlaboratory studies and quality assurance

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

reference laboratories (NRL) of samples containing animal proteins derived from different

species and in particular from fish, poultry, pigs and ruminants to be used as reference

materials or to carry out comparative testing.

3.1.1 Definition of the needs

3.1.2 Set up of a planning of the samples to produce in the 2009-2010 period

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

3.1.4 Control of the raw materials

3.1.5 Production of the samples

3.1.6 Test of the homogeneity of the samples produced

3.1.7 Report on the produced samples 3.1.8 Distribution of the samples

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

microscopy.

3.2.1 Redaction of the report of the CRL-AP interlaboratory study of Autumn 2008

3.2.2 Definition (with the collaboration of the DG SANCO) of the objectives of the ring trial

to perform at the end of 2009.

3.2.3 Preparation of the interlaboratory study.

3.2.4 Invitation of the NRLs to participate.

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

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

3.2.7 Collection of the data.

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

comparative testing reveals limited experience. Up to 3 European missions/year are foreseen

in support to DG SANCO and/or CRL activities

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

of the audit

3.3.2 Report of the audits

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

quality management under accreditation on analytical methods for detection, quantification

and identification of animal proteins in feed ingredients and in feedingstuffs. (2 p/m)

3.4.1 Definition of the needs of the NRLs

3.4.2 Preparation of the programme, in consultation with the European Commission,

including actions to be undertaken

3.4.3 Provide the requested help to the NRLs

4. Development of analytical methods and tools

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

methods of analysis.

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

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

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

4.1.3 Establishment of the needs in the development of methods

4.1.4 Improvement of the Neogen dipstick method of ruminant PAP detection for the

screening of PAPs by immunological methods

4.2 Contribute to the development of complementary analytical methods necessary to assure the

correct implementation of official methods and explorative or alternative methods.

4.2.1 Specification of the needs

4.2.2 Report of the results of the tests

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

available, methods specifically detecting ruminant, pig or poultry proteins should be

evaluated.

4.3.1 Organization of an interlaboratory study for PCR methods

4.3.2 Organization of a validation study for PCR methods

4.3.3 Transfer of validated PCR methods to the CRL and to the NRLs network

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

available for the NRLs network.

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

network. On the basis of the results of the validation of the PCR and immunological

dipstick methods (see tasks 1.5.3.2. and 1.5.3.3.) preparation of CRL protocol to apply

these methods in the NRLs labs.

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

one single species origin (e.g. fish, poultry, pig, bovine and sheep) from different processes.

Test, packaging and storage of the new samples as well as production of microscopic image

representative of the particles making up the samples collected and selected to be included in

the CRL-AP samples bank.

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

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

4.5.3 Production and validation of informatics tools for the appropriate management

of the samples

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

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

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

4.5.6 Test of the samples collected

4.5.7 Preparation of the samples for the storing

4.5.8 Storing of the samples

4.5.9 Maintenance of the samples bank

5. Workshops/trainings

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

126/2003/EC directive to detect animal proteins in feed (Classical microscopy) and any new

directive linked to the detection, identification and quantification of animal proteins in feed.

5.1.1 Organisation of a workshop for the identification PAP particles (raw and

sediment fraction) using the 126/2003/EC method

5.1.2 Invitation of the attendees

5.1.3 Preparation and submission of the minutes of the workshop

5.2 Provide specific workshop for the benefit of NRLs for the detection, identification and/or

quantification of PAPs in feed according new validated method.

5.3 Provide specific workshop of experts from candidate Member States for the correct

application of the 126/2003/EC directive to detect animal proteins in feed (Classical

microscopy) and any new directive linked to the detection, identification and quantification

of animal proteins in feed.

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

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

laboratories performing classical microscopy or other validated method.

Documents

WORK PROGRAMMES FOR EU REFERENCE LABORATORIES FOR … · b. Enumeration of total flora at 30°C The CRL (Unit HMPA) will organize an inter-laboratory trial on the TF enumeration at