NEWSLETTER EUROPEAN UNION REFERENCE LABORATORY FOR SALMONELLA. Vol. 17 No. 3 September, 2011 ISSN

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1 NEWSLETTER EUROPEAN UNION REFERENCE LABORATORY FOR SALMONELLA Vol. 17 No. 3 September, 2011 ISSN Continuation of Newsletter Community Reference Laboratory for Salmonella ISSN

2 Produced by European Union Reference Laboratory for Salmonella National Institute of Public Health and the Environment P.O. Box 1, 3720 BA Bilthoven, The Netherlands phone: (Kirsten Mooijman) (Hennie ter Hoeven) fax:

3 CONTENTS Editorial Note 1 Page For Information 2 Contribution of EURL-Salmonella 3 Literature Information 21

4 EDITORIAL NOTE Bilthoven, 7 October 2011 Dear colleagues, Summer in the Netherlands seem to have been this year in spring (April/May) and in fall (September), but unfortunately not in the holiday period of July and August! I hope you have had better conditions in your country and enjoyed your holiday?! As you may have noticed, we are fully back to business after the holiday break, with one interlaboratory comparison study on detection of Salmonella in minced running at the moment and the study on typing of Salmonella planned in November The planning of this latter study is enclosed again in this Newsletter. Immediately after the holiday period we had to prepare the working plan and budget forecast of the activities of 2012, as this had to be sent to DG-Sanco before 1 September. At the same time also the progress report of the activities performed in 2011 (up to August 2011) was sent to DG-Sanco. Copies of the workplan and the progress report are included in this Newsletter. In August two reports of interlaboratory comparison studies were published: Kuijpers A.F.A., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food IV (2010) - Detection of Salmonella in minced meat. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / 2011 Jacobs-Reitsema, W.F., Maas, H.M.E, de Pinna, E. and Mooijman, K.A. Fourteenth CRL-Salmonella interlaboratory comparison study (2009) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: / Also available at: While still busy with the current interlaboratory comparison studies, we also already started with performing some experiments for the next veterinary interlaboratory comparison study, planned early For this we have tested pig faeces with some Salmonella strains to check which serovar could be a good candidate for use in the study. The data will be further explored and soon we hope to make a decision on the set-up of this study. As soon as we have more definitive information you will be informed. We do the outmost to keep our EURL-Salmonella website up to date, but unfortunately we have had some capacity problems during the year. This is the reason why the information on the site is not always as actually as it should be. For instance, we still use the old name (CRL-Salmonella) for the website and for our logo. However, this is something we are reviewing now together with the review of the full lay-out of the site. The website of the institute (RIVM) is changing, meaning that we also need to change our site as well. For the time being we will use the old link ( As soon as things may have changed you will be informed, but I do not expect this to be the case before the end of Best wishes, Kirsten Mooijman 1

5 FOR INFORMATION Time table of the 16 th interlaboratory comparison study (2011) on serotyping and phage typing of Salmonella spp. Week (2011) Dates Subject October Mailing of the protocol and test report to the NRLs November Mailing of parcels to the NRLs as Biological Substance Cat. B (UN3373) by door-to-door courier service November Starting with the identification of the strains 49 9 December Deadline for sending the electronically completed test report by to the EURL-Salmonella 2

6 CONTRIBUTION OF EURL-Salmonella EURL-Salmonella work-programme 2012 Introduction The working programme of EURL-Salmonella consists of the following activities (the duration of the activities are indicated between brackets): 1. Organisation of interlaboratory comparison studies (yearly); 2. Organisation of a workshop with the NRLs-Salmonella (yearly); 3. Performance of research (depending on the subject: yearly or for a limited period); 4. Giving assistance to the Commission and ad hoc activities (yearly); 5. Communication (every 3 months and yearly); 6. Training (duration dependent on the subject). 1. Interlaboratory comparison studies For 2012 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 2012 will be planned with the NRLs-Salmonella, but an indication of the probable time period is given below. In 2011 for the first time lenticules were used as reference materials in interlaboratory comparison studies for the detection of Salmonella in veterinary and food samples. In earlier years capsules were used for this purpose. The main advantage of the lenticules is that they are easier to dissolve, so that no special reconstitution procedure is needed. Because of this advantage, it is better possible to mimic the pre-treatment of routine samples in the interlaboratory comparison studies. Furthermore, the production process of the lenticules is more flexible than the production process of the capsules so that it may be possible to introduce other serovars in samples of the interlaboratory comparison studies, in stead of only Salmonella Typhimurium, S. Enteritidis or S. Panama. The lenticules are produced by the Health Protection Agency in the United Kingdom, where the EURL-Salmonella orders the batches needed. The EURL-Salmonella has good contacts with the HPA, so that the possibilities can be discussed about amended contamination levels and new serovars. For the interlaboratory comparison studies on detection of Salmonella to be organised in 2012 it is planned to use again lenticules to artificially contaminate a matrix. The choice of the serovars as well as the contamination levels will be decided per study. Interlaboratory comparison study on bacteriological detection of Salmonella in veterinary samples Like for former studies, a Salmonella-free matrix will be selected which will be artificially contaminated with Salmonella reference materials. At the workshop of 2011, it was agreed that the EURL will explore the possibilities for using cattle faeces or pig faeces with Salmonella serovars most frequently found in these types of matrices, e.g. S. Typhimurium and S. Dublin for cattle faeces and S. Typhimurium and S. Derby for pig faeces. The following is planned for the 2012 veterinary ring trial: Time period: February/March; Matrix: cattle faeces or pig faeces; 3

7 Number of samples and level of reference materials to be decided later (depending on the outcome of the pre-tests); Methods: Annex D of ISO 6579 ( Detection of Salmonella in animal faeces and in environmental samples from the primary production stage ), implying modified semisolid 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 EURL or visiting an NRL by members of the EURL-Salmonella staff). As agreed at the workshop of 2011, also moderate performance of participating laboratories will be further taken into consideration. At the workshop of 2011 the following action for moderate performing laboratories was proposed: if moderate performance is seen in three consecutive studies, the NRL will be contacted by the EURL to discuss a proper follow-up. The type of follow-up will be considered on a case by case basis depending on the nature of the moderate performance. A visit of staff member(s) of the EURL-Salmonella to the NRL can be considered as a possible follow-up. Also in the case of repeated moderate performance DG-Sanco will be informed. Since 2008, also reference laboratories of two third countries (from outside Europe) participated in the veterinary studies, being: Tunisia and Israel. These countries participated on request of DG-Sanco. However, since 2011, Tunesia does not longer participate, as the EC did not agree on their monitoring plan. Therefore it is foreseen that the only third, non-european, country in the veterinary study of 2012 will be Israel. The EURL-Salmonella also offers a limited number of laboratories of EU candidate countries and of EFTA countries to participate in the interlaboratory comparison studies for their own costs. The results of all third countries will be analysed separately from the results of the NRLs of the European Member States. The justification for participation of the third countries (from outside Europe) was given in the work-programme of 2008 and is repeated below: Salmonella control programmes in live poultry are introduced in the European Member States by Regulation (EC) No 2160/2003. The control programmes in breeding hens include the monitoring of Salmonella by the testing of faecal materials in accordance with the provisions in Regulation (EC) No 1003/2005. Third countries, who want to remain or be added to the list of third countries from which Member States may import breeding hens or hatching eggs, should submit a control programme equivalent to the control programmes of the Member States. In order to evaluate the equivalence of testing in these third countries, they should participate in the ring trials organised by the CRL. Tunesia, Canada, Israel and the United States forwarded their control programme for breeding hens and should therefore be included in the ring trial. Interlaboratory comparison study on bacteriological detection of Salmonella in food or feed samples The choice of the matrix for the food/feed study in 2012 will be discussed with the NRLs at the EURL-Salmonella workshop of The prescribed method will be ISO 6579 (2002: Microbiology of food and animal feeding stuffs Horizontal method for the detection of Salmonella spp.), with selective enrichment in RVS and MKTTn. The additionally requested method will be Annex D of ISO 6579, with selective enrichment on MSRV. The planning of the study is September/October 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, as well as in case of repeated moderate performance (see above). 4

8 Interlaboratory comparison study on typing of Salmonella Up to 2008, EnterNet laboratories (analysing human isolates) had the possibility to participate in the typing studies through a collaboration between the EURL-Salmonella and the Health Protection Agency (HPA), Colindale (UK). In fall 2008, this collaboration was formalised in a contract with the ECDC. This collaboration was also profitable for the NRLs, as it still gave them the opportunity to test their phage typing capacities additional to the serotyping of Salmonella in the interlaboratory comparison study on typing. However, the contract with the ECDC ended in August 2011, so that also the formal collaboration with HPA ended. To give the NRLs still the opportunity to test their phage typing capacities, a sub-contract with HPA will be needed to make sure that the expertise of HPA is still available for the study in Like in former studies the EURL-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. At the workshop of 2011, the NRLs asked to include one or more reptile-serovars as well. As not all NRLs are interested in such a type of serovar, it was agreed to add one reptile-serovar as additional ( twenty-first ) strain in the study of The results found with this serovar will not be taken into account for the evaluation of the performance of the laboratory. It will be discussed at the workshop of 2012 whether such a serovar shall be added to the typing study of 2012 as well. The strains will be blindly coded and send to the NRLs for serotyping, one week before the performance of the study. The HPA will select twenty Salmonella strains for phage typing (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 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 The same criteria will be used for evaluating the results of the typing study of 2012, unless agreed otherwise with the NRLs and/or DG-Sanco. Subcontract in relation to activity 1 A subcontract with the Health Protection Agency (HPA), London, United Kingdom will be needed to hire the expertise of the HPA for phage typing of Salmonella in the interlaboratory comparison study on typing. For this, HPA will select and test strains to be used in the typing study and will send the strains to the EURL-Salmonella. Furthermore, HPA will contribute to the analyses and reporting of the phage typing results as found by the NRLs. Missions in relation to activity 1 If necessary a visit to a poor performing NRL by two staff members of the EURL- Salmonella will be made. Time needed: approximately 2 days, country unknown. 5

9 Output in relation to activity 1 Type interlaboratory comparison study Detection of Salmonella in veterinary samples Detection of Salmonella in food/feed samples Typing of salmonella Planning study Planning interim summary report Planning final draft full report (including the possible follow-up study) 1 Feb./March 2012 May 2012 September 2012 Sept./Oct December 2012 May 2013 Nov./Dec February 2013 July : The full reports will be published as RIVM-reports. The publication of these reports takes some time consuming administrative steps which can not be fully controlled by the author(s). Therefore, the planning of the (final) draft report is indicated in stead of the planning of the publication of the final report. 2. Workshop The annual workshop is planned to be organised in the Netherlands in May/June 2012 and will last 1,5-2 days. The exact place of the workshop will be decided later. The programme may contain the following items: - Introductory presentations (e.g., by EU representative and EURL-Salmonella); - Zoonoses in Europe (EFSA, DG-Sanco); - Results of research activities of EURL-Salmonella; - Results of interlaboratory comparison studies of 2011 and 2012; - Experiences, problems, results in relation to monitoring surveys for Salmonella; - Plans and results of research activities of the NRLs-Salmonella; - Discussion on methods (e.g. typing, molecular, serological); - Activities in ISO and CEN; - Future working plan of EURL-Salmonella; - Information on research in relation to Salmonella by one or more guest speakers. According to the draft regulation concerning the financial aid to the EU reference laboratories (SANCO/10536/2011 Rev.1), it will be possible to invite up to 3 invited speakers and up to 10 representatives of third countries additional to up to 32 representatives of NRLs of EU Member States. Concerning the third countries, the EURL- Salmonella will (most likely) at least invite representatives of the following countries: Bosnia and Herzegovina, Croatia, Iceland, Former Yugoslav Republic of Macedonia, Norway, Serbia, Switzerland and Turkey. Output in relation to activity 2 1. Publication of the presentations of the workshop at the EURL-Salmonella website ( within a few weeks after the workshop. 2. Draft report of the workshop, including a summary of the discussion performed per item at the workshop: within 2 months after the workshop (together with the cost statement). 6

10 3. Research Activities concerning ISO and CEN The EURL-Salmonella (Kirsten Mooijman) is involved (as project leader or as member of working groups) in several activities of ISO and CEN. More specific in: - ISO/TC34/SC9: International Standardisation Organisation, Technical Committee 34 on Food products, Subcommittee 9 Microbiology. - CEN/TC275/WG6: European Committee for Standardisation, Technical Committee 275 for Food analysis Horizontal methods, Working Group 6 for Microbial contaminants. Kirsten Mooijman of the EURL-Salmonella is convenor of three groups in CEN/ISO dealing with methods for Salmonella. The activities for these groups will be continued in 2012: Detection of Salmonella (EN ISO ): In fall 2011, most likely, the first working draft of the EN/ISO document will be sent to the members of CEN/TC275/WG6 and ISO/TC34/SC9 for comments and for voting. It may be necessary to discuss the outcome of this voting in the CEN Technical Advisory Group (TAG) on Salmonella detection, in spring As a follow up, the document will need to be amended (action by the convenor of the group Kirsten Mooijman), before it can be sent around for a new voting round. The progress of the group with the document needs to be presented at the plenary meeting of CEN and ISO (spring 2012) by the convenor (Kirsten) of the TAG group. Enumeration of Salmonella (ISO ): Due to administrative problems in CEN, the launching of the final vote of the document is delayed. It is hoped that the document can be finished in Before the final publication, it may be necessary to introduce some last (editorial) amendments to the document (by Kirsten Mooijman). Guide for serotyping Salmonella spp. (ISO ): In fall 2011, most likely, the first working draft of the EN/ISO document will be sent to the members of CEN/TC275/WG6 and ISO/TC34/SC9 for comments and for voting. It may be necessary to discuss the outcome of this voting in the ISO ad hoc group in spring As a follow up, the document will need to be amended (action by the convenor of the group Kirsten Mooijman), before it can be sent around for a new voting round. The progress of the ad hoc group with the document needs to be presented at the plenary meeting of CEN and ISO (spring 2012) by the convenor (Kirsten) of the ad hoc group. Other activities related to CEN CEN mandate on validation of microbiological methods: In 2007 the EURL-Salmonella was assigned as project leader for the validation of Annex D of ISO By then the EURL- Salmonella also submitted a work plan and budget forecast. By the end of 2010 the contract was signed between CEN and the EC. Next, subcontracts needed to be made between CEN (Afnor, France) and each project leader. This will hopefully be arranged before the end of 2011, so that the validation study for Annex D of ISO 6579 can be performed in The main part of the validation study is the organisation of an interlaboratory comparison study in which at least ten laboratories should produce acceptable results. To be sure that sufficient data will be available, it is the intention to ask at least 15 laboratories to participate. The EURL-Salmonella will invite NRLs for Salmonella to participate in the study. Although the work for this CEN mandate will be performed under a separate contract from the EURL-Salmonella, it is still the expertise of the EURL which is needed to perform the study. Therefore, information on this study is given here as well. Methods If necessary, literature research and experiments may be done in relation to the ISO/CEN activities in the field of Salmonella (see above). The need and the nature for this will depend on the comments on the CEN/ISO documents which may be given during the voting rounds. 7

11 In 2011 a large study design was set-up to test the influence of pooling of poultry meat samples on the sensitivity of the Salmonella detection method. In the study design many combinations of Salmonella serovars, different ways of stressing the strains, type of matrices, different ways of pooling and different detection methods are tested: - Of each of the following three Salmonella serovars two strains are tested: Enteritidis, Typhimurium and 1,4,[5],12:i:-; - The strains will be subjected to different types of stress: cold stress, freeze stress and heat stress; - Four types of poultry meat will be tested: chicken and turkey meat with and without skin. At least five different samples of each type of meat will be tested; - Dry pooling (pooling of sample units) as well as and wet pooling (pooling of preenriched cultures) will be tested; - For the analyses ISO 6579 (2002) and Annex D of ISO 6579 (2007) are followed. Due to the large study design, not all combinations may already be fully tested in Therefore, it may be necessary to continue the study in Reference materials and matrices Per interlaboratory comparison study the serovar(s) and contamination levels in the reference materials will be chosen. Before ordering lenticules with a new Salmonella serovar, it may be necessary to test the interference of the matrix with the new serovar. If the results are promising, the batch of lenticules will be ordered and the tests will be repeated in an interlaboratory comparison set-up with the relevant lenticules. Research activities may be necessary for testing relevant matrices for the interlaboratory comparison study on the detection of Salmonella. The matrix will be tested with and without the addition of Salmonella reference materials. Also the amount of background flora will be analysed. Missions in relation to activity 3 - Participation of one staff member of the EURL-Salmonella in the annual meetings of ISO/TC34/SC9 and CEN/TC275/WG6. Duration of the meetings: approximately 5 days. Period of the year: spring/early summer Location: Belgium. - Meetings of several working groups or ad hoc groups of ISO/TC34/SC9 and CEN/TC275/WG6. Approximately 2 meetings per working group, with 1 meeting, for most of the groups, in conjunction with the meeting of ISO/TC34/SC9 and CEN/TC275/WG6. The meetings are not yet planned, but will be scheduled as soon as considered necessary. Output in relation to activity 3 ISO and CEN New draft version of EN ISO : fall 2012 Publication of ISO end 2012 New draft version of EN ISO : fall 2012 Report of relevant items in relation to standardisation as discussed at the plenary meetings of ISO/TC34/SC9 and CEN/TC275/WG6: summer 2012 Note: For the progress of the work with the EN ISO documents, the EURL-Salmonella is very much dependent on the cooperation and on the speed of the administrative processes in CEN and ISO. Methods Summary of the results of the pooling experiment in a draft manuscript: end

12 Reference materials and matrices Results of research performed to test optimal matrix and reference material combinations, will be published in the reports related to the interlaboratory comparison studies (see Activity 1). 4. Giving assistance to the Commission and ad hoc activities The EURL-Salmonella is regularly 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 EURL-Salmonella will in principle always react positively and will try to include the ad hoc work required in the working plan although it is difficult to plan the time needed to answer the different questions. Participation in Working Groups of DG-Sanco and EFSA When requested and when possible, one or two staff members of the EURL-Salmonella participate in the working groups of DG-Sanco and of EFSA for, among others, to give technical support in drafting EU legislation, for preparation of technical specifications of monitoring and control programmes, for drafting (EFSA) opinions for certain items. Missions in relation to activity 4 Participation in working groups of DG-Sanco and EFSA will be funded by DG-Sanco and EFSA and will not be charged on the EURL-Salmonella budget. For ad hoc activities, no missions are foreseen. Output in relation to activity 4 Input in EFSA working groups are published by EFSA in for example EFSA opinions. Input in working groups of DG-Sanco will be used by DG-Sanco to prepare/amend specific documents (e.g. EU legislation). In case a question needs substantial input of the EURL-Salmonella, it will be summarised in more detail in the annual technical report of the EURL-Salmonella over the year under review (2012). March Communication A staff member of the EURL-Salmonella has been trained to keep the EURL-Salmonella website up to date ( In 2011, the RIVM website has been amended. Therefore, it is necessary to amend the EURL-Salmonella website as well. However, in August 2011, no start was yet made with the amendment of the English version of the RIVM website. It is therefore expected that the amendment of the EURL-Salmonella website will continue in With this amendment of the site, it will also be investigated whether it is possible to change the name in the url (from CRLSalmonella to EURLSalmonella ). Furthermore, also the logo of the EURL-Salmonella will be reviewed. The newsletter of EURL-Salmonella is published every quarter with information from the EURL-Salmonella relevant for the NRLs-Salmonella or from NRLs-Salmonella relevant for the other NRLs-Salmonella. Also, a literature search is included in each newsletter covering the previous 3-month period. Results on the interlaboratory comparison studies, the workshop and relevant research will be published in RIVM reports. The reports will be distributed to the EC and to the NRLs and other interested bodies. Furthermore they will also become available at the EURL-Salmonella website. Summaries of several interlaboratory comparison studies and related research will be published (if possible) in the scientific literature. By comparing several studies over the years it is possible to determine the existence of trend analyses in the studies. An overview of 7 years of interlaboratory comparison studies on detection 9

13 of Salmonella in veterinary and food samples was published in It will be investigated whether a publication of a similar summary for the typing studies is possible in Output in relation to activity 5 Website Keeping the EURL-Salmonella website up to date: continuously Amending the EURL-Salmonella website to the new RIVM demands, including investigation to the possibility of a new url: end 2012 Investigation for a new logo of the EURL-Salmonella: end 2012 Newsletter Publication of 4 newsletters through the website: ; ; ; Trend analyses interlaboratory comparison studies Draft review of several interlaboratory comparison studies on (sero)typing of Salmonella: end Training On request of an NRL, the EURL can give a training for a specific need of an NRL. It is also possible that the EURL will advice an NRL to follow a training at the EURL or that staff members of the EURL give a training at the laboratory of the NRL, especially in case of (repeated) poor performance of the NRL in interlaboratory comparison studies. Output in relation to activity 6 Short summary on the number and type of trainings performed in 2012, in the annual technical report of the EURL-Salmonella. March 2013 Mrs. Drs. K.A. Mooijman Head EURL-Salmonella Bitlhoven, 29 August

14 Progress report EURL-Salmonella 2011 (up to August 2011) Introduction General In 2011 the following activities were carried out or are planned for late 2011: 1. Organisation of three interlaboratory comparison studies 2. Organisation of a workshop with the NRLs-Salmonella 3. Performance of research 4. Giving assistance to the Commission and ad hoc activities 5. Communication 6. Training 7. Missions Since January 2011 the Food department of the Laboratory for Zoonoses and Environmental Microbiology (LZO) of the Dutch National Institute for Public Health and the Environment (RIVM) (where the EURL-Salmonella is situated), works with a pool of laboratory technicians. Meaning that not only one or two technicians are exclusively working for the EURL, but that also other technicians of LZO may perform some tasks for the EURL, depending on the workload in the laboratory. Less experienced technicians will work under supervision of an experienced technician to make sure that the work is performed in a proper way. In June this was communicated to DG-Sanco and it was agreed that in the budget planning the technicians will not longer be indicated by name but more anonymous as technicians. Only, in the financial report the names will be indicated. Due to the move in tasks of the technicians at LZO it was also agreed that the management of the EURL-Salmonella website will be done by the secretariat of the laboratory from May For this, support is given by Hennie ter Hoeven. For the organisation of the EURL-Salmonella workshop, the secretariat of LZO is also very important. This year Noël Peters and Jeanette van Essen of the secretariat helped with the organisation. The change in names (addition of Hennie ter Hoeven and input of Noël Peters in stead of Loes van Dijk) was also communicated to DG-Sanco in June Deliverables RIVM-Reports In 2011 (up to August 2011) the following RIVM-reports were published: Mooijman, K.A. The fifteenth CRL-Salmonella workshop 27 June 2010, Saint Malo, France. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / 2010 (published January 2011). Kuijpers A.F.A., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food IV (2010) - Detection of Salmonella in minced meat. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / 2011 (published August 2011). Jacobs-Reitsema, W.F., Maas, H.M.E, de Pinna, E. and Mooijman, K.A. Fourteenth CRL- Salmonella interlaboratory comparison study (2009) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: / 2011 (published August 2011). The following RIVM-reports are under preparation: Kuijpers A.F.A. and Mooijman, K.A. EU Interlaboratory comparison study Veterinary-XIV (2011) - Detection of Salmonella in chicken faeces. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: /

15 The interim summary of this interlaboratory comparison study was published in April The final report is under preparation. Mooijman, K.A. The sixteenth EURL-Salmonella workshop 19 and 20 May 2011, Zandvoort, the Netherlands. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / The draft report was sent to DG-Sanco on 19 July The presentations were published on the EURL- Salmonella website on 10 June 2011: The final report is under preparation. Pol-Hofstad, I, Jacobs-Reitsema, W.F., Maas, H.M.E, de Pinna, E. and Mooijman, K.A. Fifteenth EURL-Salmonella interlaboratory comparison study (2010) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: / The interim summary of this interlaboratory comparison study was published in April The final report is under preparation. Publication in a journal Kuijpers, A.F.A and Mooijman, K.A. Detection of Salmonella in food, feed and veterinary sample by EU laboratories. Food Reaearch International, in press since May ISO and CEN A consolidated report of 8 EURLs on the meetings of ISO/TC34/SC9 and CEN/TC275/WG6 held in Bournemouth, United Kingdom on June 2011, was sent to DG-Sanco on 18 July ISO6579-1: Detection of Salmonella A second working draft document was prepared by Kirsten Mooijman and sent to ISOworking group (WG)9 for further comments in May ISO : Enumeration of Salmonella The document was amended by Kirsten Mooijman and sent to the secretariat of ISO in February The publication for final vote as Technical Specification (TS) has been delayed due to administrative problems at CEN and ISO. ISO : Serotyping of Salmonella A second draft guidance document was prepared by Kirsten Mooijman and was sent for further comments to the ISO ad hoc group on serotyping in April Symposium An oral presentation was given by Kirsten Mooijman of the EURL-Salmonella at the Dutch symposium Goed of slecht nieuws van oude vrienden - even bijpraten over voedselpathogenen. Organised by the foundation on food industry micro methods (FiMM) in Wageningen on 17 March 2011.The presentations were published in the proceedings of the symposium, by FiMM, Wageningen, Mooijman, Kirsten. Salmonella: nooit weggeweest! 1. Interlaboratory comparison studies In the interlaboratory comparison studies of the EURL-Salmonella, matrices artificially contaminated with Salmonella reference materials are used for many years. The contamination levels has varied during the years, but always low level and high level materials with one or two Salmonella serovars have been used. The low level is chosen as close to the detection limit as possible. The high level samples are approximately 5-10 times higher than the low level materials. It is expected that when samples with a contamination level close to the detection limit are tested, approximately 50 % of the tested samples will be negative. 12

16 Up to the end of 2010 reference materials were used which were prepared by the EURL- Salmonella and existed of gelatine capsules filled with milk powder artificially contaminated with either one of the following Salmonella serovars: Typhimurium, Enteritidis or Panama. However, as the reconstitution of the capsules required a specific procedure, it was not possible to mimic very well the routine procedure for pre-treatment of samples in the interlaboratory studies. In an attempt to improve this, it was decided to replace the capsules by lenticules for the studies organised in 2011 and later. The lenticule reference materials are produced by the Health Protection Agency (HPA) in the United Kingdom. The HPA is able to produce lenticules with different serovars at various contamination levels. This also offers some flexibility in the set-up of the interlaboratory comparison studies over the years. In 2010, several studies on lenticules (performed by the EURL-Salmonella) showed that this type of reference materials could be a good replacement for the capsules, especially as no special reconstitution procedure was needed (see technical report of 2010). To be able to compare the results of old interlaboratory comparisons studies, in which capsules were used, with the results of new studies, in which lenticules are used, the set-up of the studies with lenticules is initially kept similar to earlier studies. In September 2010 the fourth interlaboratory comparison study on the bacteriological detection of Salmonella spp. in a food sample was organised. As matrix a mixture of minced pork and minced beef was used. Portions of 25 g of this matrix had to be mixed with low and high level reference materials and with blank reference materials. In this study, still capsules were used. It was decided to use only capsules containing Salmonella Typhimurium, as this serovar is often found in meat products from pigs and cattle. In the study, 31 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States and 3 NRLs from member countries of the European Free Trade Association. The prescribed method for analyses was ISO 6579:2002 (with selective enrichment in RVS and MKTTn). Additional it was requested to also use Annex D of ISO 6579:2007 (with selective enrichment on MSRV). In November 2010 an interim summary report of the study was sent to the participants. The results of two NRLs did not fulfil the criteria of good performance (false positive blank results). A follow up study was organised for these laboratories in January 2011, in which they both scored good performance. The results were presented at the EURL-Salmonella workshop in May The full report of this study was published in August 2011 (see Introduction). In November 2010 the 15 th interlaboratory comparison study on typing of Salmonella spp. was organised for the NRLs-Salmonella. In parallel a third typing study for the laboratories of the Food- and Waterborne Diseases and Zoonoses (FWD) surveillance network Europe, under contract with the ECDC, was organised. The typing study was organised in collaboration with the Health Protection Agency (HPA, London, United Kingdom), for phage typing. In total 33 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 2 NRLs from member countries of the European Free Trade Association and 3 NRLs from EU candidate Member States. Each NRL performed the serotyping of 20 different Salmonella serovars and 7 NRLs performed the phage typing of 10 different strains of Salmonella Typhimurium and 10 different strains of Salmonella Enteritidis. The results were sent to the EURL before the end of December The analysis of the results was performed in January 2011 after which the NRLs received their own results. Four laboratories did not fulfil the criteria for good performance for the serotyping, In March 2011 a follow-up study was organised in which three of the four poor performing laboratories participated. The fourth laboratory was situated in an EU candidate Member State and did not have the resources to participate in a follow-up study. The three participating laboratories showed good performance in the follow-up study. The interim summary report was published in April 2011 and the results were presented at the EURL-Salmonella workshop in May The final report is under preparation (see Introduction). In February/March 2011 the 14 th interlaboratory comparison study on the detection of Salmonella spp. in a veterinary matrix (chicken faeces) was organised. In this study, 32 13

17 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 3 NRLs from member countries of the European Free Trade Association and, on request of DG-Sanco, one NRL from a third country (non-europe). In this study for the first time lenticule reference materials were used. The following lenticules, to artificially contaminate portions of 25 g chicken faeces, were used: Salmonella Typhimurium at a contamination level of approximately 6 cfu and 60 cfu per lenticule (STM6 and STM60) and Salmonella Enteritidis at a contamination level of approximately 6 cfu and 60 cfu per lenticule (SE6 and SE60). Furthermore, also blanc lenticules were used, as well as some control lenticules (which were not added to a matrix). The prescribed method for analyses was Annex D of ISO 6579 (2007), with selective enrichment on Modified semi-solid Rappaport Vassiliadis (MSRV) agar. The results were sent to the EURL-Salmonella before mid March 2011, after which the analyses of the results were performed. The results of three laboratories did not fulfil the criteria of good performance. One laboratory reported an electricity breakdown for many hours which could have affected the quality of the stored samples. A follow-up study for this laboratory was not considered necessary, but the results were indicated as moderate. The other two laboratories participated in a follow-up study in June 2011 and both scored a good performance. In April 2011 an interim summary report of the study was sent to the participants and the results were also presented at the EURL-Salmonella workshop in May The final report is under preparation (see Introduction). In September 2011 an interlaboratory comparison studies on the detection of Salmonella spp. in a food sample will be organised. This will be the first EURL-Salmonella food study in which lenticules will be used. Lenticules with low and high level Salmonella serovars, as well as blanc lenticules will be used to artificially contaminate portions of 25 g matrix. The matrix will concern a mixture of minced beef and minced pork. The planning of the study was published in the EURL-Salmonella newsletter of June 2011 and was also sent to the NRLs in July Early September the protocol and Standard Operating Procedure will be sent to the NRLs, followed by the samples two weeks later. The study is planned to be performed in the last week of September In November 2011 the 16 th interlaboratory comparison study on typing of Salmonella spp. is planned for the NRLs-Salmonella. There will be no study organised in parallel for the laboratories of the Food- and Waterborne Diseases and Zoonoses (FWD) surveillance network Europe, as the contract with the ECDC was not prolonged after August The typing study will be organised in collaboration with the Health Protection Agency (HPA, London, United Kingdom), for phage typing. The planning of the study was sent to the NRLs for Salmonella in August At the workshop in May 2011, the NRLs asked to add a reptile-serovar to the study. As not all NRLs are interested in such a type of serovar, it was agreed to add one reptile-serovar as additional ( twenty-first ) strain. The results found with this serovar will not be taken into account for the evaluation of the performance of the laboratory. 2. Workshop On 19 and 20 May 2011, the annual EURL-Salmonella workshop was organised in Zandvoort, the Netherlands. This location was chosen as it was easier to reach by public transport than Bilthoven/De Bilt, where the workshop was organised before. On both days representatives of the National Reference Laboratories for Salmonella (NRLs-Salmonella) were present from 25 EU Member States, from one EU candidate country (for own costs) and from two member countries of the European Free Trade Association (for own costs). From the EU Member States excuses were received from the NRLs of Spain and Cyprus, due to health problems of the representatives of both countries. Furthermore, representatives of the European Commission, Directorate- General for Health and Consumer Protection (DG-Sanco), of the European Food Safety Authority (EFSA) and several guest speakers were present. A total of 47 participants were present at the workshop which lasted one and a half day. 14

18 Presentations were given on the following subjects: Results of the interlaboratory comparison studies as organised by the EURL- Salmonella since the former workshop (June 2010); The European summary report on Zoonoses of 2009 as published by EFSA; Policy issues at EC level in relation to Salmonella; Technical (method) issues concerning Salmonella; Validation of a protocol for MLVA (Multi-Locus Variable number of tandem repeats Analysis) typing of Salmonella Typhimurium; Salmonella in the pork slaughter chain (as part of EU project Biotracer ); Activities of Five NRLs for Salmonella; Salmonella Typhimurium-like strains: o EFSA opinion on monitoring and assessment of the public health risk of this strain; o a molecular (PCR) technique to type these strains and o two outbreaks in France caused by this type of strain. Work programme of the EURL-Salmonella for the coming year. The draft report of the workshop was sent to EC DG-Sanco in July All presentations were placed on the EURL-Salmonella website ( on 10 June Research Activities in ISO and CEN EURL-Salmonella 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. Both groups organised its annual meeting in Bournemouth, United Kingdom from 20 to 24 June Kirsten Mooijman of the EURL-Salmonella is convenor of three groups in ISO dealing with methods for Salmonella. She presented the progress of these groups at the plenary meeting of SC9 and of WG6. For Salmonella three procedures are under revision or preparation in CEN and ISO. The existing standard procedure for detection of Salmonella in food and animal feed is described in EN ISO 6579 (published in 2002) and in Annex D of this document for the detection of Salmonella in samples from primary production (published in 2007). After the five years review of EN ISO 6579 in 2007, it was decided to start the revision of this document. At the same time it was also agreed to start working on standard documents for enumeration of Salmonella and for serotyping of Salmonella. Therefore, it was agreed to split EN ISO 6579 into 3 parts to deal with detection (part 1), enumeration (part 2) and serotyping (part 3) of Salmonella spp. under one EN ISO number. The work for the three items is performed in three different working groups or ad hoc groups, of which Kirsten Mooijman is project leader. ISO : Detection of Salmonella Historical overview: 2007, July-December: Five years systematic review of ISO 6579 (2002). Result of voting: 10 members confirmed, 2 members voted confirmation & correction, 4 members voted revision, 0 members voted withdrawal, 2 members abstained. Comments were included. 2008, May: Discussion of outcome systematic review at the meeting of ISO/TC34/SC9 in Helsinki. 2008, July-November: Enquiry launched to ask for specific comments and data. 2008/2009, December/January: an ad hoc group was raised. 2009, 6 April: meeting ad hoc group to review outcome enquiry. Fifteen decisions/proposals were made. 15

19 2009, May: Presentation (by Kirsten Mooijman) of the decisions/proposals at SC9 meeting in Valencia. SC9 agreed that revision of EN ISO 6579 (2002) was considered necessary. The 15 decisions were summarised by SC9 in resolution 395. It was also agreed to split EN ISO 6579 into 3 parts: part 1, detection; part 2, enumeration; part 3, serotyping of Salmonella spp. 2009, June-September: Call for experts to raise new Working Group (WG9), with Kirsten Mooijman (EURL-Salmonella) as convenor. 17 experts from 7 different countries were nominated. 2009, 15 December: First meeting WG9 (Paris). Several items and distribution of tasks agreed. 2010, January: Report of first meeting WG9 sent to the secretariat of SC , May: First working draft of ISO prepared by Kirsten Mooijman and sent to the members of WG , June: Summary of the progress of WG9 presented at the meeting of SC9 in Buenos Aires. At this meeting it was agreed that for this work the lead should be at CEN level because of the fact that the method is part of validation studies under a mandate at CEN level. 2010, October: Second meeting WG9 (Brussels), discussion of outcome SC9 meeting and discussion of first working draft. 2010/2011, October-February: Contributions for update first working draft sent by members of WG9 to Kirsten. 2011, May: Second working draft prepared by Kirsten and sent to the members of WG9 for further comments. The final working draft (including the final comments of WG9) will be sent to the secretariat of ISO/TC34/SC9 and CEN/TC275/WG6 to send it around for a first official voting round (expected approximately September 2011). 2011, June: Summary of the progress of WG9 presented at the meeting of CEN/TC275/WG6 in Bournemouth, UK. The main changes in EN ISO 6579 part 1 compared to the version of 2002 are: Incorporation of ISO 6785 (milk and milk products); Samples for primary production added to the scope; Description of detection of S. Typhi and S. Paratyphi in normative annex: use of Selenite Cystine broth for selective enrichment Selective enrichment media: o First selective enrichment: choose either Rappaport Vassiliadis broth with Soya o (RVS) or Modified Semi-solid Rappaport Vassiliadis (MSRV) agar. Second selective enrichment: Mueller Kauffmann Tetrathionate broth with novobiocin (MKTTn). Primary production samples: only MSRV (like in the current Annex D of EN ISO 6579; Anonymous, 2002); Incubation time of selective enrichment media retained for 24 h, except for some specific samples (e.g. milk powder) and for primary production samples cultured on MSRV (48 h if necessary); In informative notes the possibility to refrigerate pre,- and selective enrichment cultures for a maximum of 72 h is added; Xylose Lysine Deoxycholate (XLD) is retained as mandatory isolation medium; Plating stage has been made less prescriptive (only indicate need to obtain isolated colonies); Tables are added in an annex to give clearer direction for the choice of suitable second plating media; Confirmation on only one suspect colony (in stead of one colony of each medium combination). If negative, 4 more suspect colonies from different media combinations have to be tested; Allowed to perform parallel biochemical testing and purity check; The non-selective medium for purification has been left for choice; Two confirmation tests have become optional: ß-Galactosidase test and indole reaction. One confirmation test has been deleted: Voges-Proskauer reaction; 16

20 Details on serotyping moved to ISO 6579 part 3. In part 1 only serological confirmation (to serogroup level) is described; Performance testing for quality assurance of media is added; Validation data for analysing food samples on MSRV are added to an annex. ISO : Enumeration of Salmonella Historical overview: 2007, April: At plenary meeting ISO/TC34/SC9 agreed to base enumeration standard on publication of Fravalo et al (2003): a Most Probable Number (MPN) technique in 12 well microtitre plates, with selective enrichment on MSRV (also in microtitre plates). 2007, December: First draft protocol for the mini-msrv technique distributed to the members of SC9 for testing. 2008, fall: Information from the experiences of different SC9 members gathered and used to amend the document. 2009, January: Amended document launched for voting: positive outcome with some comments. 2009, May: Comments discussed in an ad hoc group. Finishing final draft document had to wait for a MPN calulation tool from the ISO working group on statistics 2010, Februay: Final draft document sent to the secretariat of SC9 to launch it for final voting. 2011, May: Final vote still not launched due to administrative problems at CEN level. However, it is expected that these administrative problems are solved by June Next the document needs to be translated in French and in German. Hence, the final voting is not expected before September ISO : Serotyping of Salmonella Historical overview: 2008 & 2009: Enquiries were sent to the members of SC9 to ask for their interest in a standard document for serotyping of Salmonella. 2009, May: The outcome of the enquiries was presented at the plenary meeting of SC9 and it was agreed that there was a need for a standard document on serotyping of Salmonella. Next it was agreed to raise an ISO ad hoc group to start the work. 2009, June: A call for experts for raising the ad hoc group was launched. Currently the ad hoc group exists of 9 experts from 7 countries (including WHO reference centre Paris) with EURL-Salmonella (Kirsten) being project leader. 2009, December: First meeting of the ad hoc group in Paris. At this meeting the ad hoc group indicated to prefer publication of the standard as an informative document, meaning an ISO/CEN Technical Report. 2010, March: Members of ad hoc group sent comments/contributions to Kirsten. 2010, May: Kirsten made a first working draft and sent it to the ad hoc members. 2010, June: progress reported at plenary meeting of SC9 (Buenos Aires). 2010, June-fall 2010: Ad hoc group members gave comments/contributions to first working draft. 2011, April: Kirsten made second working draft and sent it to members of ad hoc group for comments. The final working draft (including the final comments of the ad hoc group) will be sent to the secretariat of ISO/TC34/SC9 and CEN/TC275/WG6 to send it around for a first official voting round (expected approximately September 2011). 2011, June: June: Summary of the progress of the ad hoc group presented at the meeting of ISO/TC34/SC9 in Bournemouth, UK. When the draft documents will be sent around for comments and voting in ISO and CEN, the EURL-Salmonella will also send them for comments to the NRLs for Salmonella. CEN mandate on validation of microbiological methods In 2006, the European Commission (DG-Sanco) sent a mandate to CEN/TC275/WG6 for the validation of 15 microbiological methods as mentioned in Regulation EC 2073/2005, on Microbiological criteria. Annex D of EN ISO 6579 (Anonymous, 2007) is one of the methods to become validated. By the end of 2007, the EURL-Salmonella was already 17