NEWSLETTER EUROPEAN UNION REFERENCE LABORATORY FOR SALMONELLA

Transcription

1 NEWSLETTER EUROPEAN UNION REFERENCE LABORATORY FOR SALMONELLA Vol. 17 No. 1 April 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) fax:

3 CONTENTS Editorial Note 1 Page Contribution of CRL-Salmonella 3 Literature Information 19

4 EDITORIAL NOTE Bilthoven, 8 April 2010 Dear colleagues, One year ago I informed you about the fact that according to the Lisbon Treaty of December 2009, the term Community had to be replaced by European Union. However, it took until March 2011 until it was officially arranged, by the publication of Commission Regulation 208/2011, that the name of the Community Reference Laboratory has been changed into European Union Reference Laboratory. For the official abbreviation to be used no rules are set yet, meaning that we can choose anything like EURL, EU-RL, ERL. For the moment we will use EU-RL, unless DG-Sanco will inform us to do differently. Due to this change of name, also the newsletter has become a new name and because of that also a new International Standard Serial Number (ISSN). However, the content of the newsletter will remain the same, containing information for you as NRLs for Salmonella. Our activities performed in 2010 were recently reported to DG-Sanco. For your information this technical report is also included in this newsletter. In November 2010 the fifteenth interlaboratory comparison study on typing was organised. The results have been analysed and recently you should have received the interim summary report of this study. In February 2011 the fourteenth interlaboratory comparison study on the detection of Salmonella in chicken faeces was organised. In this study for the first time lenticules were used as reference materials. I hope you were satisfied with the use of these new reference materials? The results of the study look good. Only a few NRLs were not able to reach the level of good performance. Also of this study you should recently have received your own results and the interim summary report showing the results of all laboratories. As you may know we are also very busy with the preparation of our annual workshop (19 and 20 May 2011). I hope you will like the new location: Zandvoort aan Zee (translated: Zandvoort at the sea). We hope for nice weather so that you can take a swim in the sea! Our secretariat (Jeanette) has been very busy with booking of the flights and on short term (if not already received) you should receive your e-ticket(s). The programme is almost final. At the moment it undergoes a final check by the speakers. I hope to be able to send you the final programme and information on the location, hotel and how to reach the meeting venue one month before the workshop. I look forward to meet you all in Zandvoort! Best wishes, Kirsten Mooijman 1

5 CONTRIBUTION OF CRL-Salmonella Technical report of 2010 Introduction The work plan of CRL-Salmonella for the year under review, 2010, was submitted to the European Commission in August This report details consequential activities of the CRL according to the agreed work plan. General In 2010 the following activities were carried out: 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 1 January 2010 the microbiological department of RIKILT Institute for Food Safety has been moved to the RIVM. By this move, 7 additional persons became staff members of the Laboratory for Zoonoses and Environmental Microbiology (LZO), the laboratory at RIVM where the CRL-Salmonella is located. After the introduction of the new staff members it was agreed to reshuffle the input of staff members on different projects, to obtain an optimal exchange of know how in all projects. By this, in total 3 new staff members give input to the CRL-Salmonella: Wilma Jacobs-Reitsema (who was already subcontracted since 1 September 2009), Irene Pol-Hofstad and Wendy van Overbeek. The new staff members took over some planned hours of other staff members, so that the original planned budget for 2010 remained the same. The change in input was communicated to DG-Sanco in January Deliverables RIVM-Reports In 2010 the following RIVM-reports were published: Mooijman, K.A. The fourteenth CRL-Salmonella workshop 25 and 26 May 2009, Bilthoven, the Netherlands. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / Kuijpers A.F.A., Veenman C., van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food III (2009) - Bacteriological detection of Salmonella in minced chicken meat. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / Berk, P.A., Maas, H.M.E, de Pinna, E. and Mooijman, K.A. Thirteenth CRL-Salmonella interlaboratory comparison study (2008) on typing of Salmonella spp. National Institute for Public Health and the Environment, Bilthoven, the Netherlands. RIVM Report no.: /

6 Kuijpers A.F.A., Veenman C. and Mooijman, K.A. EU Interlaboratory comparison study Veterinary-XIII (2010) - Bacteriological detection of Salmonella in chicken faeces. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / Mooijman, K.A. The fifteenth CRL-Salmonella workshop 27 June, Saint Malo, France. National Institute for Public Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: / The draft report was sent to DG-Sanco on 22 July The presentations were published on the CRL-Salmonella website on 6 July 2010: The final report was published January The following RIVM-reports are under preparation: 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.: / The interim summary of this interlaboratory comparison study was published in May The final report is under preparation. Kuijpers A.F.A, van de Kassteele, J. and Mooijman, K.A. EU Interlaboratory comparison study food IV (2010) - Bacteriological detection of Salmonella in minced meat. 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 November The final report is under preparation. Reports on trainings In January 2010, two staff members of CRL-Salmonella visited an NRL-Salmonella who showed poor performance in several interlaboratory comparison studies on detection of Salmonella. The report of this visit was sent to the NRL and to DG-Sanco in February In April 2010, two staff members of CRL-Salmonella visited an NRL-Salmonella who showed poor performance in several interlaboratory comparison studies on serotyping of Salmonella. The report of this visit was sent to the NRL and to DG-Sanco in May Presentations on symposium A poster was presented and an oral presentation was given by two staff members of CRL- Salmonella at the International Symposium on Salmonella and Salmonellosis (I3S), St. Malo, France on June 2010 ( Furthermore, Kirsten Mooijman was asked to co-chair a session of the symposium. Both presentations were published in the proceedings of the 2010 symposium, by the symposium secretariat ISPAIA, Ploufragan: Mooijman, K.A. Salmonella and its standard methods (oral presentation). Kuijpers, A.F.A and Mooijman, K.A. Detection of Salmonella in food, feed and veterinary sample by different EU laboratories (poster presentation). ISO and CEN A consolidated report of 7 CRLs on the meetings of ISO/TC34/SC9 and CEN/TC275/WG6 held in Buenos Aires, Argentina on 31 May-4 June 2010, was sent to DG-Sanco on 22 July ISO6579-1: Detection of Salmonella A first draft document was prepared by Kirsten Mooijman and sent to ISO-working group (WG)9 in May After receiving comments and additional contributions, a second draft document was prepared in October

7 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 The draft guidance document was amended by Kirsten Mooijman and sent to the ISO ad hoc group on serotyping in May Interlaboratory comparison studies In the interlaboratory comparison studies of the CRL-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 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. The reference materials are prepared by the CRL and exist of gelatine capsules containing milk powder with Salmonella serovars at different contamination levels. The milk powders are prepared by spray drying milk, artificially contaminated with a Salmonella serovar. The obtained highly contaminated milk powder is mixed with sterile milk powder to obtain the desired contamination level. Next gelatine capsules are filled with the mixed powder to obtain the final reference materials and these materials are stored at -20 C. The following reference materials (capsules), to artificially contaminate a matrix, have been used in the bacteriological detection studies since fall 2008: 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 20 cfu and 100 cfu per capsule (SE20 and SE100). In October 2009 an interlaboratory comparison studies on the bacteriological detection of Salmonella spp. in minced chicken meat was organised. In this study, 32 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 3 NRLs from member countries of the European Free Trade Association and 1 NRL from a EU candidate member state. The prescribed method for analyses was ISO 6579:2002 (with selective enrichment in Rappaport Vassiliadis broth with Soya (RVS) and in Muller Kauffmann Tetrathionatenovobiocin broth (MKTTn)). Additional it was requested to also use Annex D of ISO 6579:2007 (with selective enrichment on Modified semi-solid Rappaport Vassiliadis (MSRV) agar). In November 2009 an interim summary report of the study was sent to the participants. The results of only one NRL did not fulfil the criteria of good performance (one false positive blank control sample and a too low number of positive results with the artificially contaminated chicken meat sample with low level Salmonella Enteritidis). It was observed that this NRL has had similar problems in two earlier interlaboratory comparison studies. After this last study, the NRL had taken several (extra) actions in trying to improve their performance: cleaning and disinfection to prevent cross contamination, change from plastic bags to jars for pre-enrichment in BPW, extra controls to check the performance of the media, etc. To check the implementation of these measures and to see whether extra advices could be given, a follow-up in combination with a visit of two staff members of the CRL was organised in January The results of the follow up study fulfilled the criteria of good performance, albeit at the lower end for the sensitivity results. The NRL agreed to take additional actions to try to further improve their performance. The report of the visit and the results of the follow-up study have been reported to the relevant NRL and to DG-Sanco in February The conclusions of this follow-up were included in the full report of the study, which was published in September 2010 (see Introduction). 4

8 In December 2009 the 14 th interlaboratory comparison study on typing of Salmonella spp. was organised for the NRLs-Salmonella. In parallel a 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 31 NRLs for Salmonella participated: 28 NRLs from the (27) EU Member States, 2 NRLs from member countries of the European Free Trade Association and 1 NRL from an EU candidate member state. Each NRL performed the serotyping of 20 different Salmonella serovars and 5 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 CRL by the end of December 2009/early January The analysis of the results was performed in January/February Eight laboratories did not fulfil the criteria for good performance for the serotyping and performed a follow-up study in April For one of these laboratories this was a repeated poor performance. Therefore it was decided to visit this laboratory to give a training at the spot during the follow-up study. After the follow-up study all participating laboratories fulfilled the criteria of good performance. For the first time also an interim summary report was prepared for this interlaboratory comparison study on typing, which was published in May The results were presented at the CRL-Salmonella workshop in June The final report of the study is still under preparation (see Introduction). In March 2010 the 13 th interlaboratory comparison study on the detection of Salmonella spp. in a veterinary matrix (chicken faeces) was organised. In this study, 33 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, 2 NRLs from third countries (non-europe). The prescribed method for analyses was Annex D of ISO 6579 (2007), with selective enrichment on Modified semi-solid Rappaport Vassiliadis (MSRV) agar. In April 2010 an interim summary report of the study was sent to the participants and the results were presented at the CRL-Salmonella workshop in June In the study additional control samples were introduced. Chicken faeces mixed with an antibiotic (gentamicin) for which Salmonella Enteritidis (SE) was sensitive had to be tested in the presence of SE20 capsules. The laboratories were not aware of this deviating matrix. In total four of these control samples had to be tested. Tests performed at the laboratory of the CRL showed that occasionally a sample could still be tested positive for Salmonella but finding more than 2/4 samples positive in one laboratory was quite unlikely and might indicate that no faeces was added to the SE capsules. One NRL found 4/4 of the antibiotic control samples positive for the prescribed procedure. The NRL performed several additional tests to try to find an explanation for these results. They indicated to have followed the protocol. If the laboratory indeed followed the protocol correctly, the only explanation could be that they added the faeces without antibiotics to the specific SE control samples. It was not considered necessary to organise a follow-up study for this laboratory, although their results were indicated as moderate. Another participant had problems with reconstitution of some capsules so that they missed some positive samples. This result was also indicated as moderate and no follow-up was considered necessary as all other samples were analysed correctly. Furthermore the laboratory has always shown correct results during earlier studies. The final report of the study was published in December 2010 (see Introduction). In September 2010 the fourth interlaboratory comparison studies on the bacteriological detection of Salmonella spp. in a food sample was organised. At the workshop it was discussed that the NRLs as well as the CRL prefer to introduce samples in the interlaboratory comparison studies of which treatment is more closely related to real life samples. For the current samples the reference materials (capsules) first need to be reconstituted at 37 C for 45 min, after which the matrix can be added. This procedure enhances the risk of cross contamination. Furthermore, for routine analyses of food samples it is common practice to mix the food sample with BPW by placing the sample in a stomacher or pulsifier for 1 min. However, with the current samples of the 5

9 interlaboratory comparison studies it is preferred not to do so. Therefore alternative samples have been tested at the laboratory of the CRL (use of lenticules, see 3. Research ). The tests with lenticules were not yet finalised before the interlaboratory comparison study of September, so that for this study still capsules were used as reference materials. The only improvement to mimic better the routine samples, the amount of matrix to be tested was enhanced (from 10 g to 25 g). As matrix a mixture of minced pork and minced beef was used which had to be mixed with low and high level reference materials. In this study only reference materials containing Salmonella Typhimurium were used, as this serovar is more related to meat products from pigs and cattle than Salmonella Enteritidis. The NRLs had to test 24 (blind) capsules with matrix (8 containing STM50, 8 containing STM5 and 8 being blank capsules) and 5 (blind) control samples without matrix (1 capsule containing STM50, 3 capsules containing STM 5 and 1 blank capsule). 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 final report is under preparation. 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 CRL 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. A follow-up is planned to be organised in March It is also planned to prepare an interim summary report in March 2011, containing the results of all laboratories. 2. Workshop On 27 June 2010, the annual CRL-Salmonella workshop was organised in Saint Malo, France. The location was chosen to make it possible to organise the workshop in conjunction with the International Symposium on Salmonella and Salmonellosis (I3S), which was organised in Saint Malo from 28 to 30 June Each NRL was represented by, at least, one participant. Two Member States were not able to participate in the workshop: Cyprus and Malta. The NRL of Cyprus indicated not to be able to come due to the fact that at the time of the workshop their laboratory activities were assessed for accreditation and they could not miss any staff at this audit. Malta indicated personal reasons for not being present at the workshop. From the EFTA countries, Switzerland and Iceland did not participate due to respectively personal and financial reasons. Candidate country Former Yugoslav Republic of Macedonia neither participated as they could not receive a financial contribution for participation in the workshop from TAIEX. In total 38 participants were present at the workshop, of which several for their own costs. The workshop lasted only one day and the presentations were focussed on the results of the CRL interlaboratory comparison studies, in which the NRLs-Salmonella had participated. Plans for the coming year were also presented. Besides the activities of the 6

10 CRL, other subjects included: Community summary report on Zoonoses (EFSA 2008), overview of the baseline studies on Salmonella and information on the standardisation of methods at International (ISO) and European (CEN) level. The draft report of the workshop was sent to EC DG-Sanco in July All presentations were placed on the CRL-Salmonella website ( one week after the workshop. The final report was published in January 2011 (see Introduction). 3. Research Activities in ISO and CEN CRL-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 Buenos Aires, Argentina from 31 May until 4 June Kirsten Mooijman of the CRL-Salmonella is convenor of three groups in ISO dealing with methods for Salmonella. She presented the progress of these groups at the plenary meeting SC9 and WG6. In 2009 it was decided to revise EN ISO 6579:2002 on detection of Salmonella spp. in food and animal feed. By that time it was also 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. ISO : Detection of Salmonella At the ISO/TC34/SC9 meeting in Valencia in May 2009, it was decided to raise a working group (WG9) to deal with the revision of ISO Kirsten Mooijman was appointed as convenor of this working group. At this 2009 meeting, a resolution was taken (no. 395) in which several items for revision of the ISO document for detection of Salmonella has been indicated. The items of Resolution 395 were dealt with item by item during the meeting at Buenos Aires: 1. Description of the detection of S. Typhi and S. Paratyphi in a normative annex of ISO 6579, considering the use of the Selenite Cystine (SC) enrichment broth: A text proposal has been made for this annex and it is suggested by WG9 to include the detection of S. Gallinarum (biovars gallinarum and pullorum) as well. However, it was argued by SC9 that these latter Salmonella strains are not related to human health and are only pathogenic for animals. It was agreed that this will be checked with the WHO reference centre for Salmonella. Additional, the secretariat of SC9 will ask OIE whether they would agree with inclusion of the detection of S. Gallinarum in an annex of the amended ISO The annex will become normative and in the full text of ISO 6579 it should be clearly indicated when this annex will be used. 2. To launch a trial comparing selective enrichment in the BAM/USP formulation of tetrathionate broth and in MKTTn (ISO6579 formulation). Kirsten Mooijman will prepare a protocol and will provide this to the SC9 secretariat. The secretariat will send this protocol to the SC9 members: The protocol was prepared and sent to the members in August In total 5 datasets were received, all showing similar results. In summary MKTTn (ISO formulation) gave the best results. WG9 therefore suggested to retain MKTTn in the amended ISO SC9 agreed. 3. The SC9 secretariat will launch an enquiry for data comparing the use of RVS and MSRV for food analysis. WG9 will also perform a literature review on this subject: In total 7 datasets were received in which MSRV was compared to ISO 6579 (or to only RVS). In one study the comparison was done between MSRV and ISO 6785 (for milk and milk products, selective enrichment in SC and RVS). All studies showed similar 7

11 results: MSRV gave equal or better results than ISO WG9 therefore suggests to allow the choice of subculturing to either RVS or MSRV (both to be incubated at 41,5 C). It was discussed whether such a choice would be allowed. There were no strong feelings not to allow this as both media are very similar. Furthermore, the situation is comparable to the isolation step where one medium is prescribed and the second one is free for choice. It was agreed that WG9 will continue with this suggested choice and wait for further reactions as soon as a draft is distributed. 4. Postpone the discussion about a further 24h incubation of the selective enrichment media until further information about the choice of the selective enrichment media is available: this will be dealt with later. 5. Retain XLD as the mandatory isolation medium. Clearer direction on suitable media for the second plate should be given in the document: a draft proposal for this has been made in the first draft document. France suggested to replace or give the choice for XLT4 instead of XLD in case of analysing dirty samples like primary production samples. This was further discussed in a meeting of WG9 organised in October By then it became clear that XLT4 is especially of advantage when a culture is plated out from MKTTn. However, in ISO 6579 (current Annex D) only MSRV is used for selective enrichment of primary production samples. Experiments in France had shown that for isolation from MSRV no difference was observed between XLD and XLT4. It was therefore again concluded to retain XLD as the mandatory isolation medium. To the draft amended version of ISO 6579 tables were added in an annex to clarify on what selective principles several different isolation media are based. With this information it may be more easy for a laboratory to choose a second isolation medium which will give additional information to XLD. 6. The SC9 secretariat will launch an enquiry to collect data to support the possibility of refrigerating BPW and/or selective enrichment media before subculture: a call was launched in April Some data have become available and it seems to be no problem to store cultured BPW or selective enrichment media for 72 h in the refrigerator. This information will be added in a note to the amended version of ISO Make the plating stage less prescriptive: a text proposal has been made for the first draft. 8. Make the confirmation stage less prescriptive in terms of number of colonies to be confirmed: a text proposal has been made for the first draft. 9. The non-selective medium for purification of colonies should be left to choice: a text proposal has been made for the first draft. 10. Include a note to allow parallel biochemical testing and purity check: a text proposal has been made for the first draft. Furthermore, it was mentioned that it is important that the text is clarified on serological confirmation in ISO and serotyping in ISO Additional, it was stated that it should not be prescribed in a standardised method that isolates have to be further typed at a reference laboratory. This text need to be amended accordingly in ISO Investigate the usefulness of some biochemical tests: In fall 2010 some information was received which indicated that it may not always be necessary to perform all biochemical tests to confirm Salmonella. It was decided still to retain all tests in the document, but to indicate some tests to be optional. A first amended draft text of ISO was sent to WG9 in May The document was further discussed together with additional information at a meeting of WG9 organised in October At this meeting further agreements were made on drafting ISO It was agreed that several members of WG9 would send contributions for the amendment of the document to Kirsten Mooijman by the end of Kirsten will than prepare a new draft proposal of ISO early This new draft proposal may next be sent to the plenary ISO group SC9 for a first vote. At the plenary meeting of SC9 in Buenos Aires and at the last meeting of WG9 it was agreed that Kirsten will draft a report in which all information and research results collected for revision of ISO will be summarised. As soon as this report will be finalised it will be made available to SC9 and also to the NRLs for Salmonella. A first draft 8

12 version of this report was prepared by Kirsten, but as further information is still collected and additional research for improving ISO is still done, the finalisation of the report awaits this additional information and results. At the SC9 meeting in Buenos Aires it was indicated that the work of WG9 will be moved from ISO/TC34/SC9 to CEN/TC275/WG6. This is necessary as the validation of Annex D of ISO 6579 is part of the CEN mandate and in 2006 it has been agreed that work in relation with the CEN mandate will be performed in CEN. Therefore, ISO-WG9 will become dormant and a new group will be raised at CEN level. ISO : NWIP/TS Enumeration of Salmonella by a mini-mpn technique The finalisation of the draft ISO TS document was delayed due to the fact that it was necessary to wait for the information from the ISO working group on statistics (WG2) on the MPN tool. In February 2010 the amended document was sent to the secretariat of SC9. The voting of the document has not been launched yet as it is necessary to have a resolution of CEN to publish the document as a Technical Specification (TS). For this it is necessary to write a justification letter why the document should be published as a TS and not as a full standard. At ISO level it has been discussed whether it would be easier/quicker still to publish the document as a full standard. However, if the document will be published as a full standard it will be necessary to include validation data. Some data are available, but it will take time to collect them and to check whether they fulfil the criteria for inclusion in a standard. If the data do not fulfil the criteria it will further delay the publication of the standard. Therefore, it was decided still to publish the document as a TS and administrative details will be sorted out with CEN. Unfortunately the document was still not published by the end of It is not clear why no further progress is made in ISO and CEN. ISO : Serotyping method for Salmonella The ISO ad hoc group on serotyping met for the first time on 14 December At this meeting it was agreed to prepare a guidance document for serotyping and therefore to publish the document as a Technical Report (TR). At the SC9 meeting it has been asked whether publication of a TR would not cause problems for CEN. As this was not fully clear, it was agreed that it first need to be checked at CEN (by SC9). The draft guidance document which was distributed with the second enquiry was taken as a basis and at the ad hoc group meeting some amendments and additions were agreed. Members of the ad hoc group provided Kirsten with text proposals by March 2010, after which Kirsten prepared an amended document. This latter document was sent to the members of the ad hoc group in the first week of May 2010 and further comments and contributions were asked. In fall 2010 all comments and contributions were received and it is planned that Kirsten will prepare an updated draft document which may by launched for voting as New Work Item Proposal (NWIP) by the secretariat of ISO/TC34/SC9 in CEN mandate on validation of microbiological methods In January 2006 the EC addressed a mandate to CEN/TC275/WG6 for the validation of 15 microbiological methods. CRL-Salmonella has been assigned as project leader for the validation of Annex D of ISO 6579: 2007 (Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage). A draft proposal was sent to CEN in For the validation study, ISO 16140: 2003 need to be followed, or the new draft of this ISO document. For the validation a ring trial shall be organised. The CRL-Salmonella will request some NRLs-Salmonella to participate in such a ring trial, as they are well experienced with the method. The progress with the full proposal of the 15 project leaders in the CEN mandate was very slow, due to bureaucratic problems and inexperience of AFNOR with such a large mandate. At the meeting in Buenos Aires an update was given. After several (small) amendments of the proposal, done by the secretariat of WG6, the final proposal has been sent to CEN central in May However, by the end of May the proposal was still 9

13 not submitted to the EC, due to the fact that the question on the leadership of the revision on ISO 6579 (ISO or CEN?) first needed to be solved. It was clarified that as it was agreed in 2006 that all work under the CEN mandate should be lead by CEN, the revision of part 1 of ISO 6579 should be moved to CEN. The work for part 2 (enumeration) and part 3 (serotyping) could stay in ISO. After solving this question, CEN central promised to submit the proposal as quickly as possible to the EC. Early 2011 the project leaders were surprised by the information that the contract was signed for the project. Therefore it may be possible that some primary work for the CEN mandate may start in Methods For the revision of ISO 6579 part 1 several activities have been performed: - The yield of pure cultures of different Salmonella serovars, as well as after addition to minced pork was tested in different formulations of tetrathionate broths (formulations according to BAM/USP and ISO 6579). The results of the pure cultures as well as of the artificially contaminated pork were comparable to the results found by other laboratories: MKTTn of ISO 6579 gave the best results (highest yield of Salmonella and lowest yield of the background flora). - Literature has been searched for the possible use of MSRV for the detection of Salmonella spp. in food and animal feed samples. In total 7 (large) datasets were screened, of which one concerned a full validation study according to ISO In these datasets also the results of the interlaboratory comparison studies as organised by the CRL-Salmonella have been used. All results showed that MSRV gave equal or better results than ISO The effect of storage time was tested on several media for the detection of Salmonella. In the current ISO 6579 it is indicated that most media need to be prepared fresh and storage at 5 C is only allowed for a few days. Large batches of ready-to-use selective enrichment media MSRV, MKTTn and RVS and plating out media XLD and Smid2 were stored at 5 C and regularly tested (weekly to monthly) with 5 samples of minced meat (mixed pork and beef), artificially contaminated with Salmonella Enteritidis at a level of approximately 9 cfp/25 g. All samples still show positive results after 6 months of storage of the media, except for MSRV, for which a maximum storage time of 4 weeks was shown (which is a prolongation of the current storage time with 2 weeks). - A first screening of the literature was performed to check the possibility of refrigerating cultured BPW and/or selective enrichment media before subculture. Some information was found which supports the possibility of refrigerating cultured BPW and cultured selective enrichment media up to 72 h. When necessary some additional experiments will be performed at the laboratory of the CRL; - Two times a pooling experiment was performed. For this the so-called dry pooling as well as the wet pooling of samples was tested. The set-up and results are described below. Pooling experiment Tested matrices: minced chicken meat and minced pork/beef. Reference materials: a. For the test with minced chicken meat: Salmonella Enteritidis in skim milk, frozen at - 80 C at a level of approximately 9 cfp/sample. b. For the test with minced pork/beef: Salmonella Enteritidis in lenticules, frozen at -20 C at a level of approximately 5 cfp/sample. Set-up experiment: 3x 25 g of matrix were each artificially contaminated with 1 reference material and stored at 5 C for 3 days; 1. Dry pooling: 25 g artificially contaminated matrix g non-contaminated matrix (=4x 25 g) added to 1125 ml BPW. After normal incubation time (37 C for 18 h) the procedure of ISO 6579 (and Annex D) was followed to inoculate the selective 10

14 enrichment media: 0,1 ml onto MSRV agar; 0,1 ml in 10 ml RVS and 1 ml in 10 ml MKTTn. 2. Wet pooling: 1x 25 g artificially contaminated matrix and 4x 25 g non-contaminated matrix were each added to 225 ml BPW and incubated at 37 C for 18 h. From each cultured BPW, 5 ml was taken and the five BPW cultures were mixed in one tube. Of the 25 ml pooled BPW cultures, 0,1 ml was used to inoculate one plate of MSRV agar; 0,5 ml was used to inoculate 50 ml RVS and 5 ml was used to inoculate 50 ml MKTTn. 3. Control: 1x 25 g artificially contaminated matrix was pre-enriched in 225 ml BPW and tested in the normal way on RVS, MKTTn and MSRV. The dry pooling as well as the wet pooling showed positive results for all tested samples of minced chicken meat and minced pork/beef on all tested media. In July 2010 a draft protocol for the (statistical) set-up of pool experiments has been received from the working group on statistics of ISO. This protocol was used to draft a plan to perform some further pooling experiments. In summary this plan looks as follows: - Two strains of each of the following Salmonella serovars will be tested: S. Typhimurium, S. Enteritidis, monophasic S. Typhimurium: 1,4,[5],12:i:- - Four types of poultry matrices will be tested (skin of chicken and turkey, minced meat of chicken and turkey). Of each type of matrix, 5 different samples will be tested. - Each matrix sample will be contaminated with each of the six strains at a level of approximately 5 cfp/25 g. Prior to this contamination step, each strain will be sublethally injured by following a spiking protocol (e.g. storage of a culture at -20 C for 3 days). - Dry pooling and wet pooling will be tested (see above). For each type of pooling one artificially contaminated sample of 25 g will be mixed with 4 non-contaminated samples of 25 g, either at the sample level (dry pooling) or at the level of the BPW cultures (wet pooling). - The method for analyses will be ISO 6579 and Annex D of ISO In the second half of 2010 it was tested what type of stress would give the highest amount of sublethally injuries to the 6 Salmonella strains as mentioned above. According to a draft protocol of MicroVal Technical Committee of September 2010 injury efficiency is usually evaluated by enumerating the pure culture on a selective and on a non selective agar medium. More than 0,5 log cfp/g difference between the result found on the nonselective medium and the results found on the selective medium is expected after performing a stress protocol. Much differences were seen between the stress protocols but also between the strains. For one Salmonella Enteritidis (SE) strain the highest amount of injury was found after storage at -20 C for 3 days. However, for a second SE strain the highest effect was seen after 15 min at 50 C. Similar results were found for the two monophasic S. Typhimurium strains. The two S. Typhimurium (STM) strains behaved more comparable (highest injuries after storage at 50 C for 15 min). The results are summarised in Annex 1. The information on injury efficiencies will be used for the pooling experiments which are planned to start early PCR technique 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 also tested with a Real-Time PCR technique in fall Additional also some fresh artificially contaminated minced meat samples were tested with the bacteriological detection method (ISO 6579) and with the Real-Time PCR technique. The first step in the bacteriological method as well as for the PCR technique was the same. The samples were pre-enriched in BPW at 37 C for 18 hours after which either a selective enrichment 11

15 medium was inoculated (bacteriological detection) or DNA was isolated to be tested with the PCR method. The used PCR technique has been described by Malorny et al, In general comparable results were found between the bacteriological method and the PCR technique. However, some samples containing low numbers of Salmonella in a dirty matrix like chicken faeces gave some unclear results with PCR. It is most likely that the matrix and or the background flora disturb the PCR in these samples. It is planned to perform a few more tests, like dilution of the samples, early Samples for interlaboratory comparison studies Matrix mixed with antibiotic For the current protocol of the interlaboratory comparison studies, the participating laboratories have to mix reference materials (capsules) and matrix in their own laboratories. It has been chosen to perform the studies in this way to better guarantee the contamination level of the reference materials during transport. However, it also has a risk that participants do not add any matrix, which will make the analyses (of only the reference materials) much easier. Therefore, it was decided to include with one of the interlaboratory comparison studies a (blind) check on whether the participants follow the protocol correctly and do add matrix to the reference materials. For this purpose the CRL mixed the matrix with an antibiotic to which the Salmonella strain in the reference materials was sensitive and the background flora in the matrix (preferably) not. If participants would then follow the protocol correctly, the samples would be tested negative for Salmonella. However, if no matrix was added to the reference materials, the results would be positive. Several experiments were needed to be able to prepare such a matrix mixture: - A literature review was done and experts were consulted to find the most optimal choice for the antibiotic and its concentration. - After the choice for the antibiotic was made, several concentrations were tested on the Salmonella reference materials without the addition of matrix. - Experiments were done to find the most optimal way to mix several kilo s of matrix with the antibiotic solution. - Mixtures of matrix with antibiotic were tested with different reference materials at different levels of Salmonella. Also the influence of the antibiotic on the background flora was tested, by checking the number of Enterobacteriaceae and the total amount of aerobic bacteria in the mixed matrix. - The stability of the mixed matrix was also tested, as the antibiotic should still inhibit the growth of Salmonella after mailing the samples and after storage at the participating laboratory. The desired effect was obtained by mixing 1 kg chicken faeces with 1,5 g active Gentamicine. When 25 g of this mixture was added to reference materials containing Salmonella Enteritidis at a level of approximately 20 cfp/capsule, most of the samples were tested negative. Even after storage of the mixed faeces at 5 C for 3 weeks, only 1/25 samples was tested positive. The mixed faeces was used in the interlaboratory comparison study of March 2010 and only a few laboratories found occasionally 1/4 samples positive, which was acceptable, showing that the participants did follow the protocol. Only one laboratory found 4/4 samples positive (see 1. Interlaboratory comparison studies ). Either this was caused by the fact that no mixed matrix was added to the reference materials or by the fact that non-mixed matrix was added. Improve relation with routine samples The current treatment of the samples for the interlaboratory comparison studies deviate from the treatment of routine samples in the participating laboratories. Therefore the following was proposed to improve the relation with real life samples for amount and treatment of the samples: 1 Malorny, B., Paccassoni, E., Fach, P., Bunge, C., Martin, A. and Helmuth, R., Diagnostic Real-Time PCR for detection of Salmonella in food. Appl. Env. Microbiol., Vol. 70, 12,

16 Analyses of 25 g matrix instead of 10 g; Optimisation of the procedure for artificial contamination of samples; Make it possible to treat samples similar as routine samples (e.g. use of stomacher). To test the possibilities, several experiments were carried out in which two types of reference materials (capsules and lenticules) were tested. In experiments performed several years ago it was shown that for capsules the best results (highest number of positive samples) were obtained when the capsules first were dissolved in BPW at 37 C for 45 min, after which the matrix was added. Furthermore, it seems to be better not to mix the matrix with the dissolved capsule. Hence, the current experiments with the capsules were limited to tests with higher amounts of matrix (25 g). The lenticules were tested with the different treatments as well. For the tests with the capsules the following was performed: 4 capsules containing Salmonella Typhimurium at a level of approximately 5 cfp/capsule were each reconstituted in 225 ml BPW at 37 C for 45 min. Next to each jar with BPW, 25 g minced pork was added. Next the procedures as described in ISO 6579 (selective enrichment in RVS and MKTTn) and in Annex D of ISO 6579 (selective enrichment on MSRV) were followed. All samples showed to be positive, where the yield of Salmonella in the MKTTn was approximately 10 7 cfp/ml after 24 h of incubation. The yield of the background flora in the same MKTTn cultures varied from 10 4 to 10 8 cfp/ml. Hence, no major problems were expected by enhancing the amount of matrix from 10 g to 25 g in the interlaboratory comparison studies in which capsule reference materials are used. For the tests with lenticules the following was performed: Tested lenticules: Salmonella Typhimurium at a level of approximately 9 cfp/lenticule (STM9) and Salmonella Enteritidis at a level of approximately 7 cfp/ lenticule (STM7). Tested matrices: mince meat (beef or mixed beef/pork) and chicken faeces. Set-up experiments: a. To each of 5 portions of 25 g matrix, 1 lenticule is added and stored at 3 C for 3-4 days. Next, each sample is added to 225 ml BPW. b. 5 portions of 25 g matrix are stored at 3 C for 3-4 days. At the day of analyses, 25 g of matrix together with 1 lenticule is added to 225 ml BPW. After adding the samples to BPW the food samples are mixed in a pulsifier for 1 min. Of the faeces samples 5 x 25 g (+lenticule) is also mixed in a pulsifier and 5x 25 g (+lenticule) is not mixed at all (which is more usual for faeces samples). The food samples have been tested by following ISO 6579 (selective enrichment in RVS and MKTTn) and Annex D of ISO 6579 (selective enrichment on MSRV). The faeces samples were tested by following Annex D of ISO 6579 only. In the minced meat approximately 10 3 cfp/g Enterobacteriaceae and approximately 10 7 cfp/g total aerobic bacteria were present. In the chicken faeces this was respectively 10 cfp/g and 10 7 cfp/g. The STM9 samples were tested positive for all tested matrices and media. The SE7 minced meat samples were all tested positive on all media, although some samples were tested negative with one plating-out medium, but positive with the other plating-out medium. Of the SE7 faeces samples one was tested negative after mixing with the pulsifier. All other samples were found positive. As mixing is not a usual procedure for faecal samples, this result was not considered problematic. Additional to the tests mentioned above, the lenticules were also tested in the set-up of an interlaboratory comparison study. For this the following was tested: - 25 lenticules were each tested with 25 g of chicken faeces (negative for Salmonella). These 25 lenticules existed of 5 lenticules containing Salmonella Enteritidis at a level of approximately 6 cfp/lenticule (SE6), 5 lenticules with SE60, 5 lenticules with Salmonella Typhimurium at a level of approximately 6 cfp/lenticule (STM6), 5 lenticules with STM60 and 5 blank lenticules. It was not necessary to first reconstitute the lenticules before adding the faeces. At first 25 g faeces was added to 225 ml BPW, immediately followed by the addition of a lenticule. The BPW with sample was left at 13

17 room temperature for min to rehydrate the lenticule, followed by the normal procedure of Annex D of ISO lenticules were tested without faeces (control samples), being 2 blank lenticules, 2 lenticules with SE6, 1 lenticule with SE60 and 2 lenticules with STM6. - Method: Annex D of ISO 6579, with selective enrichment on MSRV and plating out on XLD and Brilliance agar. All samples showed the expected results. The results of this test ring trial encourage the use of the lenticules in the full studies. It is planned to use lenticules for the first time in the interlaboratory comparison study for the detection of Salmonella in chicken faeces, which will be organised in February The above mentioned results show that lenticules may be a good alternative to the capsule reference materials. To be sure that abuse temperatures during transport do not negatively influence the number of Salmonella in the lenticules, a challenge test was done. For this test lenticules containing SE75, SE5 and STM65 were stored at 5 C, 22 C and at 30 C. The numbers of Salmonella in the stored lenticules were tested at day 0, after 3 days and after 7 days of storage. The lenticules containing Salmonella Typhimurium showed to be stable at all tested temperatures. The SE5 lenticules were only stored at 22 C, and no effect was seen on the number of SE after one week of storage at this temperature. The SE75 lenticules were stable at 5 C, but showed some decrease when stored at 22 C or at 30 C. However, after a week of storage at these temperatures the numbers were still sufficient high to be well able to detect Salmonella in the samples. The results are summarised in a figure in Annex Giving assistance to the Commission and ad hoc activities Several questions were received from several parties (European Commission, NRLs- Salmonella, other institutes inside and outside the EU) on subjects like methods (qualitative and quantitative, molecular, serological), validation of methods, typing of Salmonella, storage of strains, reference materials, advice on ring trials, sampling, ISO and CEN activities, certain Salmonella serovars, accreditation. When possible, the questions were answered as quickly as possible, or were forwarded to other experts. To give assistance to the Commission, Arjen van de Giessen and Kirsten Mooijman participated in several meetings of working groups of DG-Sanco (working group on Zoonoses and occasionally working group on microbiological criteria) as well as of EFSA. In 2009 the question was raised on the possibility of pooling of samples. Literature was reviewed as well as some experiments were performed, as indicated in clause 3 (Research). The question is still a current topic for the revision of EC Regulation 2073/2005 on the microbiological criteria of Salmonella in poultry meat. A protocol for experiments on pooling of samples has been written and first experiments were performed (see 3. Research). In 2009 it was also discussed how to deal with monophasic Salmonella Typhimurium (STM-like) strains. For this item a working group was raised in EFSA, in which Kirsten Mooijman has participated. The opinion of the EFSA group was published in September In March 2010, DG-Sanco was contacted by ECDC about a Salmonella outbreak investigation. In 2009 and ongoing in 2010 an outbreak of S. Goldcoast in 6 EU countries was seen with pork containing products as possible source. A high proportion of human cases shared indistinguishable PFGE profile and ECDC wanted to check PFGE profiles from animals (especially pigs) and food in the EU. It was therefore requested if it would be possible to use the CRL/NRL-Salmonella network to: - Collect S. Goldcoast isolates to perform PFGE at the CRL - Collect PFGE profiles of S. Goldcoast from NRLs 14

18 Immediately after this request, the CRL-Salmonella contacted the NRLs to ask for available strains/pfge-profiles. Within 2 weeks, replies were received from 22 NRLs indicating an availability of in total 600 isolates of S. Goldcoast. In the following months, several NRLs sent isolates and PFGE profiles to the CRL. Furthermore, at the CRL- Salmonella also several S. Goldcoast isolates were available in stock from, amongst others, baseline surveys. By the end of May, 118 PFGE profiles were available at the CRL, originating from 14 countries, from humans, pigs, cattle and dogs. The results from the different isolates showed very similar PFGE profiles, with only minute differences. Therefore, the information from PFGE for S. Goldcoast seems to be limited. This was further discussed in a teleconference with ECDC and the laboratory in Italy who analysed the human outbreak strains. In fall 2010 the PFGE profiles collected at the CRL and the PFGE profiles of the outbreak investigations were further compared at the CRL and also these results showed a great similarity between the PFGE profiles, confirming that the information from PFGE for S. Goldcoast is limited. This information was communicated to the ECDC. 5. Communication Every three months a newsletter is published through the CRL-Salmonella website. In April 2010, volume 16 no 1 of the newsletter was published, which included information on the workshop of 2010, the technical report of 2009 and the reports of the meetings of ISO/TC34/SC9-WG9 on revision of ISO 6579 and the ad hoc group on serotyping of Salmonella (both met in December 2009). In July 2010, volume 16 no 2 of the newsletter was published, which included the timetable of the interlaboratory comparison studies on the detection of Salmonella spp. in a Food matrix IV. In October 2010, volume 16 no 3 of the newsletter was published, which included the timetable of the fifteenth interlaboratory comparison study on typing of Salmonella (November 2010). Furthermore, the draft working plan of 2011 and the progress report of the activities performed up to August 2010 were published in this newsletter. In January 2011, volume 16 no 4 of the newsletter was published, which included the time table of the interlaboratory comparison studies on the detection of Salmonella spp. in animal faeces XIV (February 2011) Communication with the NRLs is performed as much as possible electronically (through e- mail and through the CRL-Salmonella website). For this purpose one staff member of the CRL takes care of keeping the website up to date ( In November 2010 the CRL was visited by 11 delegates of Mercosur countries: Argentina, Brasil, Uruguay and Paraguay, to receive information on the system of CRL and NRLs in Europe and especially on the activities performed by the CRL-Salmonella. 6. Training Up to August 2009, 5 trainings have been organised. Three trainings were organised on request of 3 NRLs and were given at the laboratory of the CRL and focused on serotyping and/or molecular typing. These trainings were given in January, February and March In each training, 2 staff members of one NRL participated. Two trainings were organised as a follow-up for poor performance of a NRL in more than one interlaboratory comparison study (also see 1. Interlaboratory comparison studies ). Both trainings concerned a visit of 2 staff members of the CRL at the laboratory of the NRL. One visit was organised in January 2010 and focused on detection of Salmonella in food and animal faeces. The other visit was organised in April 2010 and focused on serotyping of Salmonella. 15

19 7. Missions Participation in Working Groups of DG-Sanco and EFSA Arjen van de Giessen and Kirsten Mooijman participated in meetings of working groups of DG-Sanco and EFSA: 20 January 2010: EFSA working group, Parma 12 February 2010: DG-Sanco working group Zoonoses, Brussels 19 March 2010: EFSA working group, Brussels 13 July 2010: DG-Sanco working group microbiological criteria, Brussels 9 September 2010: EFSA working group Parma 29 October 2010: DG-Sanco working group microbiological criteria, Brussels Budget: DG-Sanco or EFSA The following missions in relation with the CRL-Salmonella activities were performed in 2010: January 2010: Visit to a poor performing NRL (detection of Salmonella) Participants: Angelina Kuijpers and Kirsten Mooijman 9-10 February 2010: CRL forum, organised by IRMM, Geel, Belgium Participant: Kirsten Mooijman (presentation given) April 2010: Visit to a poor performing NRL (serotyping of Salmonella) Participants: Henny Maas and Anjo Verbruggen 31 May 4 June 2010: Meetings of CEN/TC275/WG6, ISO/TC34/SC9 (Food microbiology) in Buenos Aires, Argentina. A joint report of several CRLs was sent to DG-Sanco in July Participant: Kirsten Mooijman 27 June 2010: CRL-Salmonella workshop, St. Malo, France Participants: Angelina Kuijpers, Wilma Jacobs and Kirsten Mooijman June 2010: International Symposium on Salmonella and Salmonellosis (I3S), St. Malo, France Participants: Angelina Kuijpers (poster presentation), Wilma Jacobs and Kirsten Mooijman (oral presentation and co-chair of a session) Budget: For Kirsten Mooijman from I3S. For Angelina Kuijpers and Wilma Jacobs: transport from budget workshop CRL, daily allowances and hotel costs from budget RIVM 5 October 2010 Meeting of WG9 of ISO/TC34/SC9 on revision of ISO in Brussels, Belgium Participant: Kirsten Mooijman Mrs. Drs. Kirsten Mooijman Head CRL-Salmonella Bilthoven, 25 March

20 Annex 1 Results stress protocol on the injury efficiency of 6 different Salmonella strains Before stress 15 min 50 C 3 days -20 C 1,5 week 4 C cfp/ml log cfp cfp/ml log cfp cfp/ml log cfp cfp/ml logcfp SE ATCC on NA 8,40E+08 8,92 4,40E+07 7,64 2,30E+06 6,36 5,40E+07 7,73 SE ATCC on XLD 8,20E+08 8,91 1,00E+07 7,00 4,00E+05 5,60 2,50E+07 7,40 Difference logcfp NA-XLD 0,01 0,64 0,76 0,33 SE 532 on NA 7,80E+08 8,89 5,00E+08 8,70 9,00E+05 5,95 2,40E+08 8,38 SE 532 on XLD 8,20E+08 8,91 3,40E+08 8,53 7,30E+05 5,86 1,80E+08 8,26 Difference logcfp NA-XLD -0,02 0,17 0,09 0,12 STM ATCC on NA 1,20E+09 9,08 9,30E+08 8,97 1,90E+07 7,28 6,50E+08 8,81 STM ATCC on XLD 1,10E+09 9,04 3,50E+08 8,54 8,80E+06 6,94 2,90E+08 8,46 Difference logcfp NA-XLD 0,04 0,42 0,33 0,35 STM 756 on NA 7,80E+08 8,89 4,00E+08 8,60 3,50E+06 6,54 2,70E+08 8,43 STM 756 on XLD 6,90E+08 8,84 1,40E+08 8,15 1,80E+06 6,26 1,00E+08 8,00 Difference logcfp NA-XLD 0,05 0,46 0,29 0,43 STM-like S18 on NA 5,80E+08 8,76 2,10E+08 8,32 3,10E+06 6,49 1,50E+08 8,18 STM-like S18 on XLD 5,30E+08 8,72 1,10E+08 8,04 5,60E+05 5,75 1,00E+08 8,00 Difference logcfp NA-XLD 0,04 0,28 0,74 0,18 STM-like LIS on NA 1,10E+09 9,04 3,00E+08 8,48 7,00E+06 6,85 5,40E+08 8,73 STM-like LIS on XLD 9,70E+08 8,99 1,40E+08 8,15 5,80E+06 6,76 1,70E+08 8,23 Difference logcfp NA-XLD 0,05 0,33 0,08 0,50 Tested strains: SE: Salmonella Enteritidis ATCC and Salmonella Enteritidis No 532 isolated from poultry STM: Salmonella Typhimurium ATCC and Salmonella Typhimurium No 756 from poultry STM-like: 4,5,12:i:- No S18 (also used in the interlaboratory comparison study on typing of 2010) and 4,5,12:i:- from LIS (Laboratory for Infectious Diseases and Perinatal Screening of the RIVM). Media: NA: Nutrient Agar (non-selective) XLD: Xylose Desoxycholate agar (XLD) 17

21 Annex 2 Results challenge test lenticules cfp SE75 5 C SE75 22 C SE75 30 C SE5 22 C STM65 5 C STM65 22 C STM65 30 C days 18

22 LITERATURE INFORMATION Literature related to Salmonella Scopus week 1 to 13, 2011 Golberg, D., Kroupitski, Y., Belausov, E., Pinto, R., Sela, S. Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs (2011) International Journal of Food Microbiology, 145 (1), pp ABSTRACT: Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring 1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and redlettuce (20 ± 15%), showed significantly lower incidence (P < 0.05). Internalization incidence in fresh basil was 46 ± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0-100%) through a 2. year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization. ISSN: Leleu, S., Herman, L., Heyndrickx, M., De Reu, K., Michiels, C.W., De Baerdemaeker, J., Messens, W. Effects on Salmonella shell contamination and trans-shell penetration of coating hens' eggs with chitosan (2011) International Journal of Food Microbiology, 145 (1), pp ABSTRACT: Chitosan is a biopolymer with antimicrobial activity and film-forming properties. In this study, the effects on Salmonella shell contamination and trans-shell penetration of coating hens' eggs with chitosan was evaluated. A chitosan was selected from eight types (four non-commercial and four commercial) based on its antimicrobial activity against Salmonella enterica serovar Enteritidis (S. Enteritidis). For this purpose, a contact plate method was developed and chitosans were applied at a concentration of 0.25% (w/v). A commercial type with a molecular weight of kDa and a deacetylation degree of 75% that reduced S. Enteritidis by 0.71log10 colony forming units compared to the control (without chitosan) was selected for further studies. The chitosan was shown to have antimicrobial activity against other egg borne bacteria, i.e., Acinetobacter baumannii, Alcaligenes sp., Carnobacterium sp., Pseudomonas sp., Serratia marcescens and Staphylococcus warneri, and against S. enterica serovar Typhimurium, Escherichia coli and Listeria monocytogenes. The effects of various concentrations of the selected chitosan (0.25%, 1% and 2%) on Salmonella shell contamination and trans-shell penetration were assessed using the agar molding technique. Effective reduction of eggshell contamination could not be demonstrated, but trans-shell penetration was significantly reduced in the presence of a 2% chitosan eggshell coating, with only 6.1% of the eggs being penetrated compared to 24.5% of the uncoated eggs. It was concluded that the 2% chitosan coating has the potential to reduce contamination of egg contents resulting from trans-shell penetration by S. Enteritidis. ISSN: Lu, Y., Turley, A., Dong, X., Wu, C. Reduction of Salmonella enterica on grape tomatoes using microwave heating (2011) International Journal of Food Microbiology, 145 (1), pp ABSTRACT: Grape tomatoes were surface inoculated with Salmonella enterica serovars Typhimurium, Senftenburg, Kentucky and Enteritidis and heated for 10, 20, 30, 40 and 50 s using a household microwave oven at two different power levels (medium and high). Following heating, viable counts, temperature measurements and quality measurements were performed on the tomatoes. At high power level, more than 2 log reduction of Salmonella enterica was detected on 19

23 grape tomatoes after 50 s but the texture were damaged. Three heating treatments, 40 s heating at high power level, 40 and 50 s heating at medium power level, could achieve more than 1.45 log reduction of Salmonella enterica on grape tomatoes, and all the treatments except for 50 s at high power level did not affect the color, ph value and nutritional quality of grape tomato after heating (p> 0.05). However, 40 s heating at medium power was the only treatment among the three that did not affect the texture quality of grape tomato. Therefore, it might be a potential way for consumers to use microwave heating at medium power level (700. W) for 40 s to reduce Salmonella population on water immersed grape tomatoes. ISSN: Morild, R.K., Olsen, J.E., Aabo, S. Change in attachment of Salmonella Typhimurium, Yersinia enterocolitica, and Listeria monocytogenes to pork skin and muscle after hot water and lactic acid decontamination (2011) International Journal of Food Microbiology, 145 (1), pp ABSTRACT: The attachment of Salmonella enterica subsp. enterica serovar Typhimurium, Yersinia enterocolitica, and Listeria monocytogenes to pig skin and muscle tissue decontaminated with 80 C water or 55 C, 1% lactic acid for 5 and 15. s was investigated. Attachment properties differed between skin and muscle surfaces. A significantly higher number of firmly attached bacteria was found on the decontaminated skin surface compared to the non-treated skin surface, both on hot water (P < ) and on lactic acid treated skin (P < 0.001). At the muscle surfaces, no such difference in attachment were shown between hot water treated surfaces and non-treated surfaces. In contrast, for lactic acid decontamination, significantly fewer bacteria attached to the treated muscle surfaces (P < ). The study did not show significant differences in surface attachment, between Salmonella, Yersinia and Listeria, which indicate that surface and environmental factors may influence attachment more than bacterial properties. A more profound location of attached bacteria at muscle compared to skin was indicated. Confocal laser scanning microscopy studies showed that bacteria located in deep tissue structures of non-decontaminated and decontaminated skin and muscle surfaces. In the latter, bacteria tended to "hide" between the muscle fibres and may be entrapped at those sites. The finding of changed attachment properties at skin after decontamination may play a role in cross- and recontamination, during subsequent meat processing. ISSN: De Busser, E.V., Maes, D., Houf, K., Dewulf, J., Imberechts, H., Bertrand, S., De Zutter, L. Detection and characterization of Salmonella in lairage, on pig carcasses and intestines in five slaughterhouses. (2011) International Journal of Food Microbiology, 145 (1), pp ABSTRACT: In this study, conducted at five slaughterhouses, individual pigs were sampled and followed up from stunning to cooling down of the carcasses. In this way, Salmonella prevalence and possible risk points were described. At the lairage area, pens were sampled using overshoes. At stunning and bleeding, pigs were individually identified and subsequently swabs were taken of the oral cavity and the carcass after polishing, splitting and forced chilling. Additionally, duodenum, ileum, rectum and mesenteric lymph nodes were extracted and samples were taken of the scalding water. All samples were submitted to Salmonella isolation and Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). Of all samples taken (n = 1953), 14.1% were Salmonella positive. The prevalence of S. in the lairage area varied widely (from 0 to 100%) between the slaughterhouses. Of the sampled pigs (n = 226), 48.2% were positive in at least one sample. Statistical analysis revealed that the contamination of the lairage area was related to a higher amount of positive carcasses after polishing. Furthermore, the contamination of the carcasses after splitting and forced chilling was related to the contamination level of the carcass after polishing. A relation between the outer (carcass) contamination and the inner (gut content and lymph nodes) contamination of a pig could not be established. The predominant serotypes were S. Typhimurium (58.7%) and S. Derby (17.4%). Genotyping revealed 46 different PFGE profiles among the 276 Salmonella isolates. The same genotype at the lairage area as in the oral cavity of the pigs was found in 95%. The results indicate that the lairage area is a primary source of Salmonella in slaughter pigs and that carcass contamination originates from the environment rather than from the pig (inner contamination) itself. It further shows that slaughterhouses vary in their capability of dealing with Salmonella positive pigs. A slaughterhouse specific approach is needed, however, general guidelines should be provided to decrease the contamination level of the lairage area and the slaughter environment. ISSN:

24 Brouwer, A., Hill, A., Woodward, M.J. Papers: What makes a Salmonella strain epidemic? An expert opinion workshop. (2011) Veterinary Record, 168 (4), p. 98. ABSTRACT: An expert opinion workshop was held on the subject of the cause, identification and control of new and emerging Salmonella strains. Experts were invited to complete questionnaires, contribute to structured discussions and take part in cluster group tasks. Outputs of the workshop included that, with current surveillance methods, it might take up to 2.5 years from the first introduction of a new strain into the UK livestock population to its identification as a human epidemic strain. In order to reduce the time to detection and provide more effective control options, several recommendations were made, including better back-tracing of human cases to their source, which would require more effective communication between those responsible for human and veterinary surveillance. ISSN: Matsuda, K., Chaudhari, A.A., Lee, J.H. Evaluation of safety and protection efficacy on cpxr and lon deleted mutant of Salmonella Gallinarum as a live vaccine candidate for fowl typhoid. (2011) Vaccine, 29 (4), pp ABSTRACT: We evaluated a recently developed live fowl typhoid (FT) vaccine candidate, JOL916, the cpxr/lon mutant of Salmonella Gallinarum (SG), for safety and protection efficacy in 5-weekold layer chickens. Intramuscular vaccination with JOL916 revealed no or very few lesions in livers and spleens of the animals until the fourth week post-vaccination (wpv). This candidate clearly induced cellular immune responses in 5 of 5 chickens on the first and second wpv based on the peripheral lymphocyte proliferation assay. Systemic IgG responses were observed in 5 of 5 chickens from the first wpv and dramatic elevations were observed on the second and third wpv. Vaccination of chickens offered efficient protection against challenge by a wild-type SG; only slight anorexia and depression were temporarily observed after challenge in the vaccinated group while 100% mortality was observed in the positive control group. Body weight increases per day were slightly reduced between the 3rd and 6th day post challenge (dpc) compared to the negative control group; it was recovered from the 6th dpc. Collectively, these results demonstrate the safety and protective efficacy of JOL916 as a live vaccine for systemic FT. ISSN: X Kang, M.-S., Kwon, Y.-K., Jung, B.-Y., Kim, A., Lee, K.-M., An, B.-K., Song, E.-A., Kwon, J.- H., Chung, G.-S. Differential identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgc and spec genes. (2011) Veterinary Microbiology, 147 (1-2), pp ABSTRACT: Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgc and spec genes showing multiple mutations in the sequenced S. enterica subsp. enterca serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n= 53) and Pullorum (n= 21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n= 57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates. ISSN: Meyer, T., Stratmann-Selke, J., Meens, J., Schirrmann, T., Gerlach, G.F., Frank, R., Dübel, S., Strutzberg-Minder, K., Hust, M. Isolation of scfv fragments specific to OmpD of Salmonella Typhimurium. (2011) Veterinary Microbiology, 147 (1-2), pp ABSTRACT: Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to 21

25 identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-salmonella antibodies in serum. Recombinant antibody fragments (scfvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scfv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-ompd IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs. ISSN: Kober, M.V., Abreu, M.B., Bogo, M.R., Ferreira, C.A.S., Oliveira, S.D. Differentiation of salmonella enteritidis isolates by fluorescent amplified fragment length polymorphism. (2011) Foodborne Pathogens and Disease, 8 (1), pp ABSTRACT: Salmonella Enteritidis is responsible for human gastroenteritis outbreaks worldwide, and the molecular characterization of isolates is an important tool for epidemiological studies. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 31 Salmonella Enteritidis strains from South Brazil isolated from human, foods, swine, broiler carcasses, and other poultry-related samples to subtype isolates in comparison to pulsed-field gel electrophoresis (PFGE) analysis. Five strains of Salmonella Enteritidis from different geographical regions, Salmonella Enteritidis ATCC 13076, and four isolates of different Salmonella serovars were also tested. Among the 41 isolates tested, 96 polymorphic AFs and 40 distinct profiles were obtained, displaying a Simpson's index of diversity of 0.99; whereas the PFGE analysis presented 13 patterns and the resulting Simpson's index was Nine FAFLP and seven PFGE clusters could be inferred based in Dice similarity coefficient. FAFLP clustering readily identified different serotypes of Salmonella but did not distinguish isolates epidemiologically nonrelated or distinct phage types. Therefore, these results indicate that FAFLP is a rapid method for epidemiological investigations of Salmonella outbreaks, presenting a high discriminatory power for subtyping of Salmonella Enteritidis. ISSN: Holt, P.S., Davies, R.H., Dewulf, J., Gast, R.K., Huwe, J.K., Jones, D.R., Waltman, D., Willian, K.R. The impact of different housing systems on egg safety and quality. (2011) Poultry Science, 90 (1), pp ABSTRACT: A move from conventional cages to either an enriched cage or a noncage system may affect the safety or quality, or both, of the eggs laid by hens raised in this new environment. The safety of the eggs may be altered either microbiologically through contamination of internal contents with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. Quality may be affected through changes in the integrity of the shell, yolk, or albumen along with changes in function, composition, or nutrition. Season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account. An understanding of these different effects is prudent before any large-scale move to an alternative housing system is undertaken. ISSN: Borsoi, A., do Santos, L.R., Rodrigues, L.B., de Souza Moraes, H.L., Salle, C.T.P., do Nascimento, V.P. Behavior of salmonella heidelberg and salmonella enteritidis strains following broiler chick inoculation: Evaluation of cecal morphometry, liver and cecum bacterial counts and fecal excretion patterns (2011) Brazilian Journal of Microbiology, 42 (1), pp ABSTRACT: Over the years, Salmonella Heidelberg (SH) has gained prominence in North America poultry production and in the poultry production of other countries. Salmonella Heidelberg has been isolated and reported from poultry and poultry products in Brazil since 1962, whereas Salmonella Enteritidis (SE) has only emerged as a serious problem in poultry and public health since These strains of Salmonella can cause intestinal problems in newly hatched chicks, and infection may persist until adulthood. Upon slaughter of chickens, Salmonella can contaminate carcasses, a condition that poses a threat to human health. The aim of this study was to compare the fecal excretion of Salmonella Enteritidis and Salmonella Heidelberg in newly hatched chicks (orally inoculated with 105ufc/mL each) until 20 days of age. In addition, the ratio of cecal villus height:crypt depth (morphometry) and liver and cecum cell counts was analyzed in chicks ranging 22

26 from 0 to 3 days of age and infected with these two Salmonella strains. One hundred seventeen chicks were separated into one of three experimental groups: A control group, an SE infected group and an SH-infected group. Eight chicks per group were euthanized at 6, 12 and 72 hours post-inoculation (pi) to allow for Salmonella isolation from the liver and cecum and for the collection of the cecum for villi and crypt analysis. Other birds were allowed to mature to 20 days of age and cloacal swabs were taken at 2, 6, 13 and 20 days pi to compare the fecal excretion of inoculated strains. The Salmonella Enteritidis group had a higher number of cells excreted during the trial. Both strains were isolated from the liver and cecum by 6h pi. At 12h pi the Salmonella Heidelberg group had high cell counts in the cecum. No difference was found in liver cell counts. Both strains showed lower villus height:crypt depth ratio than the control group post-infection. ISSN: de Souza, R.B., Magnani, M., Ferrari, R.G., Kottwitz, L.B.M., Sartori, D., Tognim, M.C.B., de Oliveira, T.C.R.M. Detection of quinolone-resistance mutations in Salmonella spp. strains of epidemic and poultry origin. (2011) Brazilian Journal of Microbiology, 42 (1), pp ABSTRACT: Mutations into codons Aspartate-87 (62%) and Serine-83 (38%) in QRDR of gyra were identified in 105 Salmonella strains resistant to nalidixic acid (94 epidemic and 11 of poultry origin). The results show a high incidence of mutations associated to quinolone resistance but suggest association with others mechanisms of resistance. ISSN: Bell, R.L., González-Escalona, N., Stones, R., Brown, E.W. Phylogenetic evaluation of the 'Typhimurium' complex of Salmonella strains using a seven-gene multi-locus sequence analysis. (2011) Infection, Genetics and Evolution, 11 (1), pp ABSTRACT: Salmonella enterica comprises over 2500 serovars, many of which are significant foodborne pathogens in humans. The ability to subtype these microbes is difficult due to the highly clonal nature of many Salmonella strains and a lack of congruence among traditional typing approaches. This work examines the phylogenetic utility of a multi-locus sequence typing (MLST) approach to discriminate between members of a closely related collection of salmonellae, the Salmonella reference collection A (SARA). This 72 strain collection, referred to as the 'Typhimurium' complex, consists of S. Typhimurium and its four closest serological relatives. In this analysis, nucleotide sequences from seven housekeeping genes (aroc, dnan, hemd, hisd, pure, suca, and thra) were PCR amplified, sequenced, and combined into a single concatenated character matrix providing bp for cladistic analysis. The resultant most parsimonious tree yielded seven clades of Salmonella strains that partitioned largely along serovar divisions within the collection except for five 'Paratyphi B' strains, two 'Saintpaul' strains, and two 'Typhimurium' strains. Convergence in the SARA tree was approximately 20% indicating that the vast majority of sequence changes were phylogenetically informative. Despite a high consistency among nucleotide substitutions, analysis of congruence identified several SARA strains with recombinant alleles in the concatenated matrix. These findings point to important differences among phylogenetic contributions made by the individual genes comprising this MLST dataset. ISSN: Lu, Y., Wu, C.-M., Wu, G.-J., Zhao, H.-Y., He, T., Cao, X.-Y., Dai, L., Xia, L.-N., Qin, S.-S., Shen, J.-Z. Prevalence of antimicrobial resistance among salmonella isolates from chicken in China. (2011) Foodborne Pathogens and Disease, 8 (1), pp ABSTRACT: We evaluated the antimicrobial resistance of Salmonella isolated in 2008 from a chicken hatchery, chicken farms, and chicken slaughterhouses in China. A total of 311 Salmonella isolates were collected from the three sources, and two serogroups of Salmonella were detected, of which 133 (42.8%) consisted of Salmonella indiana and 178 (57.2%) of Salmonella enteritidis. The lowest percentage of S. indiana isolates was found in the chicken hatchery (4.2%), followed by the chicken farms (54.9%) and the slaughterhouses (71.4%). More than 80% of the S. indiana isolates were highly resistant to ampicillin (97.7%), amoxicillin/clavulanic acid (87.9%), cephalothin (87.9%), ceftiofur (85.7%), chloramphenicol (84.9%), florfenicol (90.9%), tetracycline (97.7%), doxycycline (98.5%), kanamycin (90.2%), and gentamicin (92.5%). About 60% of the S. indiana isolates were resistant to enrofloxacin (65.4%), norfloxacin (78.9%), and ciprofloxacin (59.4%). Of the S. indiana isolates, 4.5% were susceptible to amikacin and 5.3% to colistin. Of the S. enteritidis isolates, 73% were resistant to ampicillin, 33.1% to amoxicillin/clavulanic acid, 66.3% to tetracycline, and 65.3% to doxycycline, whereas all of these isolates were susceptible to the other drugs used in the study. The S. indiana isolates showed resistance to 16 antimicrobial agents. Strains of Salmonella (n = 108) carrying the resistance genes flor, aac(6')-ib-cr, and 23

27 blatem were most prevalent among the 133 isolates of S. indiana, at a frequency of 81.2%. The use of pulsed-field gel electrophoresis to analyze the S. indiana isolates that showed similar antimicrobial resistance patterns and carried resistance genes revealed six genotypes of these organisms. Most of these isolates had the common pulsed-field gel electrophoresis patterns found in the chicken hatchery, chicken farms, and slaughterhouses, suggesting that many multidrugresistant isolates of S. indiana prevailed in the three sources. Some of these isolates were not derived from a specific clone, but represented a variety of genotypes of S. indiana ISSN: Li, J., Maclehose, R., Smith, K., Kaehler, D., Hedberg, C. Development of a Salmonella screening tool for consumer complaint-based foodborne illness surveillance systems. (2011) Journal of Food Protection, 74 (1), pp ABSTRACT: Foodborne illness surveillance based on consumer complaints detects outbreaks by finding common exposures among callers, but this process is often difficult. Laboratory testing of ill callers could also help identify potential outbreaks. However, collection of stool samples from all callers is not feasible. Methods to help screen calls for etiology are needed to increase the efficiency of complaint surveillance systems and increase the likelihood of detecting foodborne outbreaks caused by Salmonella. Data from the Minnesota Department of Health foodborne illness surveillance database (2000 to 2008) were analyzed. Complaints with identified etiologies were examined to create a predictive model for Salmonella. Bootstrap methods were used to internally validate the model. Seventy-one percent of complaints in the foodborne illness database with known etiologies were due to norovirus. The predictive model had a good discriminatory ability to identify Salmonella calls. Three cutoffs for the predictive model were tested: one that maximized sensitivity, one that maximized specificity, and one that maximized predictive ability, providing sensitivities and specificities of 32 and 96%, 100 and 54%, and 89 and 72%, respectively. Development of a predictive model for Salmonella could help screen calls for etiology. The cutoff that provided the best predictive ability for Salmonella corresponded to a caller reporting diarrhea and fever with no vomiting, and five or fewer people ill. Screening calls for etiology would help identify complaints for further follow-up and result in identifying Salmonella cases that would otherwise go unconfirmed; in turn, this could lead to the identification of more outbreaks. ISSN: X Tien, Y.-Y., Wang, Y.-W., Tung, S.K., Liang, S.-Y., Chiou, C.-S. Comparison of multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in molecular subtyping of Salmonella enterica serovars Paratyphi A. (2011) Diagnostic Microbiology and Infectious Disease, 69 (1), pp ABSTRACT: Salmonella enterica serotype Paratyphi A is a highly clonal organism; pulsed-field gel electrophoresis (PFGE) is insufficient in discriminating isolates. A multilocus variable-number tandem repeat analysis (MLVA) was developed, and its usefulness in discriminating isolates was compared. PFGE analysis with XbaI and BlnI discriminated 55 isolates into 14 types, with a discriminatory index (DI) of (confidence interval [CI], ). MLVA divided the isolates into 23 types, with a DI of (CI, ), which was significantly higher than that for PFGE. Clustering analysis of PFGE and MLVA patterns indicated that S. Paratyphi A isolates recovered from 2000 to 2010 in South and Southeast Asia were highly clonal. Although MLVA is not sufficiently powerful in discriminating epidemiologically unrelated isolates, it can complement PFGE for epidemiologic investigation of S. Paratyphi A infections. ISSN: McCabe, E.M., Burgess, C.M., Walsh, D., O'Regan, E., McGuinness, S., Barry, T., Fanning, S., Duffy, G. Validation of DNA and RNA real-time assays for food analysis using the hila gene of Salmonella enterica serovars. (2011) Journal of Microbiological Methods, 84 (1), pp ABSTRACT: In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR technology in tandem with an adapted two-step enrichment protocol were assessed and validated against a reference culture method, ISO 6579, The DNA and RNA real-time PCR assays amplified a 270. bp region of the hila gene of Salmonella enterica serovars, and incorporated an internal amplification control (IAC) which was co-amplified with the hila gene to monitor potential PCR inhibitors and ensure successful amplification. The inclusivity and exclusivity of the hila primer set was examined for both the DNA and RNA methods and detected the 30 S. enterica serovars but not the 30 non-salmonellae strains. The inoculation of meat carcass swabs with five different S. enterica serovars at five different inocula, indicated both 24

28 PCR methods were able to detect between 1 and 10. CFU per carcass swab. The real-time DNA PCR assay performed as well as the traditional cultural method in detecting Salmonella sp. in artificially contaminated salad, chocolate, fish and cheese samples. The relative accuracy, relative sensitivity and relative specificity of the DNA PCR real-time method were determined to be 98.5, 98.1 and 100%, respectively. The DNA method was further validated in a collaborative inter-laboratory trial according to ISO 16140, The validated methods provide an accurate means for the rapid detection and tracking of S. enterica serovars giving equivalent results to the standard method within three days, thus providing an alternative testing method to the reference microbiological method. The real-time PCR methodology not only offers significant time-saving advantages compared to traditional methods, it can also be applied to a wide range of samples types ISSN: Greiner, M., Alter, T., Johne, A., Schlichting, D., Fetsch, A., Bräunig, J., Käsbohrer, A., Knobloch, S., Braun, P., Appel, B. Studies on the growth of Salmonella in table eggs under different storage temperatures. (2011) Archiv fur Lebensmittelhygiene, 62 (1), pp ABSTRACT: In Germany salmonellosis is one of the most common foodborne infections in humans. Contaminated table eggs are one of the main sources of the infection. The aim of the study was to obtain experimental data on the impact of temperature conditions on the behaviour of Salmonella Enteritidis (SE) in table eggs which may occur under practical conditions. For this purpose, 170 fresh eggs were contaminated by inoculating SE into the albumen of intact eggs and eggs were stored at different temperatures (group A: 5 C; group B: 25 C; group C: 25 C/5 C in alternating sequences of three-day intervals). Every six days, qualitative and quantitative examinations to detect SE were carried out during the following 30 days. Salmonella, which were inoculated into the albumen could be detected over the whole study period, i. e. it was not inactivated; however, only during storage at 5 C, there was no growth of SE. By qualitative examination, SE could be detected in the egg yolk in all groups within six days after inoculation. By quantitative testing, a considerable bacterial growth was confirmed during storage at 25 C or changing temperatures (25 C/5 C). By alternating storage temperatures, the highest migration rate into the yolk could be detected (64%), but changing temperatures had no additional effect on the detection or concentration of Salmonella in fresh eggs. This study shows that eggs should be chilled immediately after laying; a storage temperature of 5 C is recommended. M. & H. Schaper GmbH & Co. ISSN: X Alabdeh, M., Lechevalier, V., Nau, F., Gautier, M., Cochet, M.-F., Gonnet, F., Jan, S., Baron, F. Role of incubation conditions and protein fraction on the antimicrobial activity of egg white against Salmonella Enteritidis and Escherichia coli. (2011) Journal of Food Protection, 74 (1), pp ABSTRACT: The mechanism of egg white antimicrobial activity involves specific molecules and environmental factors. However, it is difficult to compare the data from the literature because of the use of various bacterial strains and incubation conditions. The aim of our study was to determine the effect of temperature, ph, inoculum size, and egg white protein concentration on egg white antimicrobial activity and to investigate the putative interactions among these factors by conducting a complete factorial design analysis. The behavior of Salmonella Enteritidis and Escherichia coli was studied after precultivation in tryptic soy broth and Luria-Bertani broth, respectively, using three different egg white protein concentrations (0, 10, and 100%), five temperatures (37, 40, 42, 45, and 48 C), two phs (7.8 and 9.3), and six inoculum levels (3 to 8 log CFU/ml). The essential role of temperature was identified. An inverse relationship was observed between bacterial growth and an increase in temperature. The role of egg white proteins was clearly demonstrated. In the absence of egg white proteins, bacterial growth occurred under most incubation conditions, whereas the presence of 10 and 100% protein produced bacteriostatic or bactericidal effects. The interaction between temperature and protein concentration was significant. At the highest tested temperatures, proteins were less involved in the bactericidal effect. Bacterial destruction was higher at ph 9.3 than at ph 7.8. Under our experimental conditions, Salmonella Enteritidis was more resistant to inactivation by egg white than was E. coli. Copyright International Association for Food Protection. ISSN: X 25

29 Hyeon, J.-Y., Chon, J.-W., Hwang, I.-G., Kwak, H.-S., Kim, M.-S., Kim, S.-K., Choi, I.-S., Song, C.-S., Park, C., Seo, K.-H. Prevalence, antibiotic resistance, and molecular characterization of Salmonella serovars in retail meat products. (2011) Journal of Food Protection, 74 (1), pp ABSTRACT: The prevalence of Salmonella was determined in chicken meat (n = 26), beef (n = 49), and pork (n = 56) collected from wholesale markets, retail stores, and traditional markets in Seoul, South Korea, in Antibiotic resistance was assessed, and the molecular subtypes of Salmonella isolates were ascertained using an automated repetitive sequence-based PCR (rep-pcr) system (DiversiLab). A total of 18 Salmonella strains were isolated from 17 of 131 samples: 16 strains from each of 16 samples and 2 strains from the same pork sample. The prevalence of Salmonella from the retail meats was 2.0% in beef, 8.9% in pork, and 42.3% in chicken meat. Among 10 different serotypes, Salmonella enterica Panama was recovered from a beef sample, and Salmonella London and Salmonella Montevideo were the predominant serotypes from pork and chicken meat, respectively. The highest antibiotic resistance observed was to erythromycin (100%) followed by streptomycin (22.2%) and tetracycline and chloramphenicol (16.7%). Of the 18 isolates, 5 (27.8%) were resistant to two or more antibiotics, and 1 isolate from chicken meat was resistant to eight antibiotics, including cephalosporins. Differentiation between all of the Salmonella isolates except between Salmonella Montevideo and Salmonella London was successfully performed with the automated rep-pcr system, indicating that it can be added to the toolbox for source tracking of foodborne pathogens associated with outbreaks. Copyright International Association for Food Protection. ISSN: X Wilkinson, K.G., Tee, E., Tomkins, R.B., Hepworth, G., Premier, R. Effect of heating and aging of poultry litter on the persistence of enteric bacteria. (2011) Poultry Science, 90 (1), pp ABSTRACT: Food-borne illnesses have rarely been associated with the reuse of poultry litter as an organic fertilizer and soil amendment in agriculture. Yet farming practices in many countries have come under increased scrutiny because of heightened consumer awareness of food safety and environmental issues. This study was conducted to determine whether simple on-farm management practices could improve the microbiological safety of poultry litter. First, the effects of heat and moisture on the survival of Escherichia coli and Salmonella enterica serovar Typhimurium in poultry litter were investigated under laboratory conditions. Second, the persistence and regrowth of enteric bacteria were examined in poultry litter that had been aged for up to 12 wk in either a turned or static (unturned) windrow. Escherichia coli and Salmonella counts in poultry litter were reduced by >99% in 1 h at 55 or 65 C under laboratory conditions. At 35 C, both persisted longer under moist (65% wt/wt, wet basis) than dry (30% wt/wt) conditions. Poultry litter aged for 3 wk in a turned windrow, and up to 6 wk in a static windrow, supported increased E. coli densities when incubated in the laboratory at 37 C for 21 d. Peak temperatures >65 C were observed in both windrows within the first 3 wk of aging; after this point, the turned windrow was more consistently exposed to temperatures >45 C than the static windrow. By 12 wk, however, E. coli counts were very similar (3 to 3.6 log10) in the outside edge of both windrows. This study highlights the need for a better understanding of the interrelationship between spontaneous heating in organic waste streams, organic matter stabilization, and pathogen reduction Poultry Science Association Inc. ISSN: Trujillo, S., Keys, C.E., Brown, E.W. Evaluation of the taxonomic utility of six-enzyme pulsed-field gel electrophoresis in reconstructing Salmonella subspecies phylogeny. (2011) Infection, Genetics and Evolution, 11 (1), pp ABSTRACT: Pulsed-field gel electrophoresis (PFGE) remains an important tool in the molecular epidemiological evaluation of strains emerging from disease outbreak clusters. Recent studies of Escherichia coli O157:H7 and Salmonella Enteritidis have noted marked improvements in the discriminatory power of PFGE when combining band profiles from up to six restriction enzyme datasets into a single concatenated analysis. This approach has provided more accurate assignments of genetic relationships among closely related strains and allowed effective phylogenetic inference of host and geographical reservoirs. Although this approach enhances epidemiological congruence among closely related strains, it remains unclear to what extent sixenzyme PFGE pattern similarity reiterates evolutionary relatedness among more distantly related Salmonella strains (i.e., serovar or subspecies levels). Here, taxonomic accuracy of six-enzyme PFGE is tested phylogenetically across two distinct Salmonella enterica populations-salmonella reference collection B (SARB), representing the breadth of taxonomic diversity of S. enterica subspecies I only, and Salmonella reference collection C (SARC), comprising the seven disparate 26

30 subspecies of S. enterica plus S. bongori. Cladistic analysis of SAR strains revealed substantial polyphyly between the two strain collections such that numerous SARB strains clustered more closely with diverged SARC subspecies rather than with other members of subspecies I. Also, in several cases, SARC sibling strains from the same subspecies were evolutionary obscured-broken into distant locales on the most parsimonious six-enzyme trees. Genetic diversity among SARB and SARC strains was comparable at 45% and 47%, respectively, while congruence testing revealed discordance among individual enzyme datasets. While six-enzyme PFGE is effective in ascertaining accurate genetic relationships for more closely related strains (e.g., strains within the same serovar), reconstitution of evolutionarily meaningful strain groupings may be elusive for Salmonella at the serovar level and above. Thus, caution is warranted when applying PFGE with a limited number of enzymes as the primary phylogenetic marker in these instances ISSN: Adley, C., Dillon, C., Morris, C.P., Delappe, N., Cormican, M. Prevalence of Salmonella in pig ear pet treats. (2011) Food Research International, 44 (1), pp ABSTRACT: Salmonella is one of the most important causes of foodborne disease throughout the world. In recent years there have been documented outbreaks of Salmonella infection in humans associated with pet treats of animal origin; in particular pig ear pet treats in Canada and the United States. The main objectives of this study were to determine the degree of Salmonella contamination in dried pig ear dog treats on sale in Limerick City, Ireland and to examine pet dogs for Salmonella carriage. Over a 12. month period, from October 2008 to September 2009, 102 pig ear pet treats were sampled using both a conventional culture detection method and a PCR method. Salmonella was detected in 24.5% of samples by the culture method, while 28.4% tested positive using a PCR method. Resistance to at least one antimicrobial agent was observed in 50% of Salmonella isolates. Salmonella was not detected in any of 86 rectal swabs from dogs attending 2 different veterinary clinics between February and April and August and October of that period. The contaminated ears were traced back to a single distributor. This study emphasises that there is a long term continuing risk of human exposure to Salmonella associated with pig ear treats. ISSN: Marin, C., Balasch, S., Vega, S., Lainez, M. Sources of Salmonella contamination during broiler production in Eastern Spain. (2011) Preventive Veterinary Medicine, 98 (1), pp ABSTRACT: Prevention of Salmonella contamination of poultry products requires detailed knowledge of the main sources associated with its presence in the production system. The aims of this study were to determine the main sources of Salmonella contamination in broiler production during growing, to assess the risk factors for Salmonella contamination at the end of the rearing period and to determine the main serovars involved in broiler production systems in Eastern Spain. A total of 65 different broiler houses from different farms were sampled. Each house was sampled at different times during the rearing period. First, when the previous flock was taken to the slaughterhouse, samples of dust, surfaces and previous flock faeces were collected. After cleaning and disinfection (C&D), samples of dust and surfaces were also taken. On the first day of rearing, samples of water, bedding, farming boots, meconiums, delivery-box liners and feed were collected. During rearing, feed samples were taken directly from the truck and from feeders. On slaughter day, samples of dust, surfaces, water, feed and faeces were also collected. Finally, two days after slaughter, carriers (rodents, flies and beetles) were trapped. All samples collected were analysed according to ISO 6579:2002 (Annex D) and positive samples were serotyped in accordance with Kauffman-White-Le-Minor technique. Our results showed that all different types of samples collected were contaminated with Salmonella (prevalence ranged between 1.5% and 38.6%). The most contaminated samples related with poultry production were: delivery-box liners (32.0%), faeces samples (31.2%), dust samples (25.0%), farming boots (19.7%) and feed from feeders (16.0%). However, the most important risk factors for Salmonella contamination of the flocks at the end of the rearing period were Salmonella status of the house after cleaning and disinfection, Salmonella status of day-old chick flocks and feed from feeders. Twenty-one different serovars were isolated from the samples analysed. The most prevalent were in decreasing order: Salmonella Enteritidis (52.9%), S. Hadar (17.8%), S. Virchow (8.9%) and S. Ohio (5.4%). The study suggested that there are many sources for Salmonella contamination and persistence in broiler production. Hence, the whole production chain needs to be controlled to eradicate the bacteria from primary production. ISSN:

31 Jasson, V., Baert, L., Uyttendaele, M. Detection of low numbers of healthy and sub-lethally injured Salmonella enterica in chocolate (2011). International Journal of Food Microbiology, 145 (2-3), pp ABSTRACT: The capacity to detect low levels of healthy and sub-lethally injured Salmonella enterica cells in chocolate by two alternative rapid detection methods iq-checktm Salmonella II real-time PCR (Bio-Rad) and VIDAS Easy SLM (BioMérieux) was assessed and compared with ISO 6579:2005. Chocolate, a low moisture food known to support the survival of Salmonella, was challenged as food matrix. Buffered peptone water (BPW) did not support the recovery of low levels of sub-lethally injured S. enterica independent of the detection method, while BPW supplemented with milk powder enabled detection by the three examined methods. However, inhibition of real-time PCR was observed since for one out of three repetitions of chocolate inoculated with a low number of sub-lethally injured S. enterica cells, no PCR signal was obtained. Therefore, attention should be paid to the enrichment step to avoid false negative results due to the presence of especially sub-lethally injured Salmonella cells in chocolate. An appropriate sample preparation (such as enrichment media and conditions for incubation) remains the key factor for reliable detection including sub-lethally injured cells and should be evaluated, if necessary optimized, for each detection assay. ISSN: Monfort, S., Gayán, E., Condón, S., Raso, J., Álvarez, I. Design of a combined process for the inactivation of Salmonella Enteritidis in liquid whole egg at 55 C (2011). International Journal of Food Microbiology, 145 (2-3), pp ABSTRACT: This paper is an evaluation of the lethal effectiveness of a successive application of pulsed electric fields (PEFs) and heat treatment in liquid whole egg (LWE) in the presence of different additives on the population of Salmonella serovar Enteritidis. Synergistic reductions of the Salmonella Enteritidis population were observed when LWE samples containing additives were treated with PEF (25kV/cm; 100 and 200kJ/kg), heat (55 C), or PEF followed by heat. The presence of additives, such as 10mM EDTA or 2% triethyl citrate, increased the PEF lethality 1 log10 cycle and generated around 1.5log10 cycles of cell damage, resulting in the reduction of undamaged cells of 4.4 and 3.1log10 cycles, respectively. The application of PEF followed by heat treatment significantly (p<0.05) reduced D55oC from 3.9±0.2min in LWE to 1.40±0.06min or 0.24±0.02min in the presence of 10mM EDTA or 2% triethyl citrate, respectively. A PEF treatment of 25kV/cm and 200kJ/kg followed by a heat treatment of 55 C and 2min reduced more than 8log10 cycles of the population of Salmonella Enteritidis in LWE combined with 2% triethyl citrate, with a minimal impact on its protein soluble content. The heat sensitizing effect of PEF treatments in the presence of 2% triethyl citrate on the Salmonella population could enable LWE producers to reduce the temperature or processing time of thermal treatments (current standards are 60 C for 3.5min in the United States), increasing the level of Salmonella inactivation and retaining the quality of non-treated LWE. ISSN: Hur, J., Song, S.O., Lim, J.S., Chung, I.K., Lee, J.H. Efficacy of a novel virulence gene-deleted Salmonella Typhimurium vaccine for protection against Salmonella infections in growing piglets (2011). Veterinary Immunology and Immunopathology, 139 (2-4), pp ABSTRACT: We have previously developed a novel attenuated Salmonella Typhimurium (S. Typhimurium) cpxr lon vaccine. This study was carried out to examine whether this vaccine could effectively protect growing piglets against Salmonella infection. Attenuated S. Typhimurium secreting the B subunit of Escherichia coli heat-labile enterotoxin was also used as a mucosal adjuvant. Pregnant sows in groups A and B were primed and boosted with the vaccine and mucosal adjuvant, whereas sows in groups C, D and E received PBS. Piglets in groups A and C were intramuscularly primed with formalin-inactivated vaccine and orally boosted with live vaccine, while piglets in groups B, D and E received PBS. Piglets in groups A, B, C, and D were challenged with a wild type virulent S. Typhimurium at the 11th weeks of age. Colostrum siga and IgG titers in vaccinated groups A and B sows were approximately 50 and 40 times higher than those of nonvaccinated groups C, D and E sows (P< 0.001). Serum IgG titers of group A piglets were also significantly higher than those of groups D and E piglets during the study (P< 0.001). Furthermore, no clinical signs were observed in group A piglets during the entire experimental period after the challenge, while diarrhea was observed in many of the piglets in groups B, C, and D. No Salmonella was isolated from fecal samples of the groups A and C piglets on day 14 after challenge, whereas the challenge strain was isolated from several piglets in groups B and D. 28

32 These results indicate that vaccination of the piglets with the vaccine and mucosal adjuvant in addition to vaccination of their sows induced effective protection against Salmonella infections in the growing piglets. ISSN: Elizaquível, P., Gabaldón, J.A., Aznar, R. Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in nonspiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis (2011). Food Control, 22 (2), pp ABSTRACT: Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. are foodborne pathogens frequently associated with foods such as poultry, ready-to-eat products, fruits and vegetables. PCR-based procedures are rapid, sensitive and accurate; in particular, real-time PCR (qpcr), which besides being an automated high-throughput technique, allows quantification of foodborne pathogens. In the present work, qpcr-based methods were applied for the quantitative detection of E. coli O157:H7, Salmonella spp. and L. monocytogenes in a total of 306 non-spiked food samples in a study carried out in two laboratories simultaneously. qpcr allowed the detection of the three pathogens in around 20% of the analyzed samples for each pathogen. Quantification results revealed the presence of the three pathogens mostly at levels between 102 and 104 cells/g. Besides quantification, the qpcr results (presence/absence) were compared with those of the standard mini-vidas system. In order to determine which were the "true" positive samples, conventional PCR was carried out after the corresponding enrichment for each pathogen. These results were considered as the gold standard for further analysis. The statistical analysis of global data recording the presence of E. coli O157:H7 and L. monocytogenes, together with data previously obtained for Salmonella spp., revealed that qpcr outperformed the mini-vidas procedures, in terms of both time and accuracy. Thus, these results proved qpcr to be useful as a rapid diagnostic test for the direct detection of pathogens in food, without the need for enrichment steps. ISSN: Bhowmick, P.P., Devegowda, D., Ruwandeepika, H.A.D., Fuchs, T.M., Srikumar, S., Karunasagar, I., Karunasagar, I. GcpA (stm1987) is critical for cellulose production and biofilm formation on polystyrene surface by Salmonella enterica serovar Weltevreden in both high and low nutrient medium (2011). Microbial Pathogenesis, 50 (2), pp ABSTRACT: Biofilm formation by Salmonella is a serious concern in the food-processing industry and the persistence of the organism in biofilms becomes a constant source of contamination. Since there is zero tolerance for Salmonella in foods, it is important to understand the mechanism of biofilm formation and to prevent the formation. Therefore, this study aimed at investigating the biofilm-forming ability of seafood isolates of Salmonella enterica serovar Weltevreden (S. Weltevreden) under two different nutrient conditions (normal strength trypticase soy broth (TSB) and 1:100 diluted TSB). The role of cellulose production in biofilm formation and in the expression of multicellular behavior (rough, dark, red morphotype: rdar) was investigated. Fourteen isolates of seafood associated S. Weltevreden were studied for biofilm production in polystyrene microtitre plates. Only one (SW49) of 14 was a strong biofilm former on polystyrene template and was able to produce biofilm in both undiluted TSB and 1:100 diluted TSB at 24. h. All others produced moderate or weak biofilms which was higher in 1:100 diluted TSB compared to undiluted medium. All the isolates except one were positive by PCR for the three genes, gcpa (stm1987), adra (yaic) and csgd. Gene expression of gcpa, adra and csgd was studied by real-time PCR with the one strong (SW49) and one representative weak (SW30) biofilm former. In SW49 at 24. h of incubation, the expression of gcpa from biofilm cells was 33 and 36 times higher than from planktonic cells grown in TSB and diluted TSB respectively and at 72. h the expression from biofilm cells was 57 and 61 times higher than that from planktonic cells. Quantification of gene expression did not reveal any significant difference in the expression of csgd and adra gene. Deletion of gcpa in SW49 resulted in its inability to produce cellulose and consequent inability to bind calcoflour, inability to form rdar colony on Congo Red-agar plates and failure to produce biofilm on polystyrene substrate. The data indicated that, in case of S. Weltevreden, gcpa is critical for activating cellulose synthesis and biofilm formation both in undiluted and diluted TSB. The results of this study suggest the existence of an alternative biofilm regulatory pathway in S. Weltevreden. Role of adra in cellulose production in nutrient rich medium is known but role of gepa in the above phenomenon is proved in this study. An understanding of the genes involved would help in looking at strategies of repression of the gene to control formation of biofilm. ISSN:

33 McQuiston, J.R., Waters, R.J., Dinsmore, B.A., Mikoleit, M.L., Fields, P.I. Molecular determination of H antigens of Salmonella by use of a microsphere-based liquid array (2011). Journal of Clinical Microbiology, 49 (2), pp ABSTRACT: Serotyping of Salmonella has been an invaluable subtyping method for epidemiologic studies for more than 70 years. The technical difficulties of serotyping, primarily in antiserum production and quality control, can be overcome with modern molecular methods. We developed a DNA-based assay targeting the genes encoding the flagellar antigens (flic and fljb) of the Kauffmann-White serotyping scheme. Fifteen H antigens (H:a, -b, -c, -d, -d/j, -e,h, -i, -k, -r, -y, -z, -z10, -z29, -z35, and -z6), 5 complex major antigens (H:G, -EN, -Z4, -1, and -L) and 16 complex secondary antigens (H:2, -5, -6, -7, -f, -m/g,m, -m/m,t, -p, -s, -t/m,t, -v, -x, -z15, -z24, -z28, and -z51) were targeted in the assay. DNA probes targeting these antigens were designed and evaluated on 500 isolates tested in parallel with traditional serotyping methods. The assay correctly identified 461 (92.2%) isolates based on the 36 antigens detected in the assay. Among the isolates considered correctly identified, 47 (9.4%) were partially serotyped because probes corresponding to some antigens in the strains were not in the assay, and 13 (2.6%) were monophasic or nonmotile strains that possessed flagellar antigen genes that were not expressed but were detected in the assay. The 39 (7.8%) strains that were not correctly identified possessed an antigen that should have been detected by the assay but was not. Apparent false-negative results may be attributed to allelic divergence. The molecular assay provided results that paralleled traditional methods with a much greater throughput, while maintaining the integrity of the Kauffmann-White serotyping scheme, thus providing backwards-compatible epidemiologic data. This assay should greatly enhance the ability of clinical and public health laboratories to serotype Salmonella. ISSN: Techathuvanan, C., Draughon, F.A., D'Souza, O.H. Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for salmonella detection from pork processing environments (2011). Journal of Food Protection, 74 (2), pp ABSTRACT: Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Realtime reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT- LAMP, RT-PCR, and culturebased assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described inva primers was conducted at 62 C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT- PCR was carried out with inva primers followed by melt temperature analysis. The results of RT- LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry. ISSN: X Ido, N., Kudo, T., Sasaki, K., Motokawa, M., Iwabuchi, K., Matsudate, H., Seimiya, Y.M., Akiba, M. Molecular and phenotypic characteristics of salmonella enterica serovar 4,5,12:i:- isolated from cattle and humans in Iwate Prefecture, Japan (2011). Journal of Veterinary Medical Science, 73 (2), pp ABSTRACT: A total of 15 isolates of Salmonella enterica serovar 4,5,12:i:- obtained from diseased cows and patients in Iwate Prefecture, Japan, were characterized to clarify the genetic basis of this serovar. S. Typhimurium- specific IS200 was detected from all the isolates. A 94-kb plasmid and 30

34 the spvb gene were detected from all but one of the 15 isolates. The results of PCR mapping of the fljab operon and its flanking regions indicate that there are deletions or mutations in this chromosomal region. These data suggest that the 15 isolates are monophasic variants of S. Typhimurium. Epidemiological relationships between the isolates obtained from cattle and humans were not suspected based on the comparison of data employing plasmid profiling and pulsed-field gel electrophoresis. ISSN: Hannah, J.F., Cason, J.A., Richardson, J.R., Cox, N.A., Hinton, A., Buhr, R.J., Smith, D.P. Effect of stomaching on numbers of bacteria recovered from chicken skin (2011). Poultry Science, 90 (2), pp ABSTRACT: Stomaching of skin samples releases only slightly more bacteria than a single rinse. Successive rinses, however, continue to remove almost as many bacteria as the first rinse. One hypothesis to explain this observation is that relatively violent treatment of skin generates smaller pieces of skin, thus increasing the net surface area and effectively sequestering bacteria in a water film on the skin pieces so that numbers of bacteria suspended in the rinsate do not increase. An experiment was conducted to determine whether inoculated marker bacteria are removed from the rinse liquid as skin pieces are stomached and naturally occurring bacteria are released. In each of 4 replications, 5 prechill broiler carcasses were collected from a commercial processing plant. Two 5- g pieces (n = 40) of breast skin were removed from each carcass and placed in a stomacher bag. An inoculum of 30 ml of 0.85% saline solution containing approximately 104 of nalidixic acidresistant Salmonella enterica serovar Typhimurium per milliliter was added to each sample. Skin samples were hand-massaged for 30 s to mix the inoculum, after which a 1-mL aliquot was removed for enumeration of bacteria. A similar sample was taken after 4 min of vigorous stomaching of the skin sample. Bacterial counts recovered from the 30-s hand-massage were 4.3, 2.7, 2.6, and 3.7 log10 cfu/ml of rinsate for aerobic bacteria, coliforms, Escherichia coli, and Salmonella, respectively. After stomaching, counts were 4.3, 2.9, 2.8, and 3.8, respectively. There was no difference in aerobic plate counts, but mean coliform and E. coli counts were significantly higher (P < 0.05) after stomaching. Numbers of inoculated Salmonella did not decrease. Breaking up the skin into smaller pieces by stomaching did not reduce the number of inoculated bacteria suspended in the rinsate. ISSN: Iwabuchi, E., Yamamoto, S., Endo, Y., Ochiai, T., Hirai, K. Prevalence of salmonella isolates and antimicrobial resistance patterns in chicken meat throughout Japan (2011). Journal of Food Protection, 74 (2), pp ABSTRACT: We investigated the prevalence of Salmonella in chicken meat from northern, central, and southern Japan. Between 2006 and 2008, 821 samples from these three regions were collected and examined. Salmonella isolates were detected in 164 (20.0%) of these samples, with 15 (10.0%) of 150, 113 (27.5%) of 411, and 36 (13.8%) of 260 recovered from the northern, central, and southern regions, respectively. We recovered 452 Salmonella isolates. From the isolates, 27 serovars were identified; the predominant serovars isolated were Salmonella Infantis (n = 81), Salmonella Kalamu (n = 56), and Salmonella Schwarzengrund (n = 43). Of the 452 isolates, 443 (98.0%) were resistant to one or more antibiotics, and 221 (48.9%) showed multipleantibiotic resistance, thereby implying that multiple-antibiotic resistant Salmonella organisms are widespread in chicken meat in Japan. Resistance to oxytetracycline was most common (72.6%), followed by dihydrostreptomycin (69.2%) and bicozamycin (49.1%). This study, the first to report Salmonella prevalence in chicken meat throughout Japan, could provide valuable data for monitoring and controlling Salmonella infection in the future. ISSN: X Izumiya, H., Sekizuka, T., Nakaya, H., Taguchi, M., Oguchi, A., Ichikawa, N., Nishiko, R., Yamazaki, S., Fujita, N., Watanabe, H., Ohnishi, M., Kuroda, M. Whole-genome analysis of Salmonella enterica serovar typhimurium T reveals the acquisition of a genomic island involved in multidrug resistance via IS1 derivatives on the chromosome (2011). Antimicrobial Agents and Chemotherapy, 55 (2), pp ABSTRACT: Salmonella enterica serovar Typhimurium is frequently associated with life-threatening systemic infections, and the recent global emergence of multidrug resistance in S. enterica isolates from agricultural and clinical settings has raised concerns. In this study, we determined the wholegenome sequence of fluoroquinolone-resistant S. enterica serovar Typhimurium T strain (DT12) isolated from human gastroenteritis in Comparative genome analysis revealed that T displays high sequence similarity to strain LT2, which was originally isolated in 1940, indicating that progeny of LT2 might be reemerging. T possesses a unique 82-kb genomic 31

35 island, designated as GI-DT12, which is composed of multidrug resistance determinants, including a Tn2670-like composite transposon (class 1 integron [inti1, blaoxa-30, aada1, qace 1, and sul1], mercury resistance proteins, and chloramphenicol acetyltransferase), a Tn10-like tetracycline resistance protein (teta), the aerobactin iron-acquisition siderophore system (luta and lucabc), and an iron transporter (sitabcd). Since GI-DT12 is flanked by IS1 derivatives, IS1-mediated recombination likely played a role in the acquisition of this genomic island through horizontal gene transfer. The aminoglycoside-(3)-n- acetyltransferase (aac(3)) gene and a class 1 integron harboring the dfra1 gene cassette responsible for gentamicin and trimethoprim resistance, respectively, were identified on plasmid pstmdt12-l and appeared to have been acquired through homologous recombination with IS26. This study represents the first characterization of the unique genomic island GI-DT12 that appears to be associated with possible IS1-mediated recombination in S. enterica serovar Typhimurium. It is expected that future whole-genome studies will aid in the characterization of the horizontal gene transfer events for the emerging S. enterica serovar Typhimurium strains. ISSN: McLaren, I., Wales, A., Breslin, M., Davies, R. Evaluation of commonly-used farm disinfectants in wet and dry models of Salmonella farm contamination (2011). Avian Pathology, 40 (1), pp ABSTRACT: Two experimental models of Salmonella contamination were used in an attempt to mimic the conditions of disinfectant use on farms. A wet model, for conditions such as boot dips, used disinfectant application to a slurry of poultry faeces inoculated with Salmonella Enteritidis or Salmonella Typhimurium. A dry model, for disinfectant application to surfaces and equipment with adherent or residual organic material, used Salmonella-inoculated poultry faeces that were airdried onto wooden dowels, immersed in disinfectant solution then left in air at room temperature overnight. All samples were subjected to a disinfectant neutralization step and resuscitation in broth, followed by Salmonella culture on semi-solid then indicator media. Disinfectants were tested at 0.5x, 1x and 2x the concentrations specified for the general control of bacterial pathogens on livestock premises in the UK (Defra General Orders rates). Chlorocresol-based disinfectants provided consistently high rates of Salmonella killing in both wet and dry tests. Formaldehydecontaining disinfectants showed very high efficacy in the dry test but were less effective in the shorter wet test, whereas the efficacy of glutaraldehyde without formaldehyde was variable between products. Other chemical classes tested (quaternary ammonium compounds, amphoteric surfactants, iodine preparations, peroxygens and a substituted phenol blend) were only moderately effective. They often required concentrations above General Orders rates to eliminate the test salmonellas, and frequently elimination was not achieved even under maximal conditions of concentration and exposure. ISSN: Zou, W., Al-Khaldi, S.F., Branham, W.S., Han, T., Fuscoe, J.C., Han, J., Foley, S.L., Xu, J., Fang, H., Cerniglia, C.E., Nayak, R. Microarray analysis of virulence gene profiles in Salmonella serovars from food/food animal environment (2011). Journal of Infection in Developing Countries, 5 (2), pp ABSTRACT: Introduction: Rapid, accurate and inexpensive analysis of the disease-causing potential of foodborne pathogens is an important consideration in food safety and biodefense, particularly in developing countries. The objective of this study is to demonstrate the use of a robust and inexpensive microarray platform to assay the virulence gene profiles in Salmonella from food and/or the food animal environment, and then use ArrayTrackTM for data analysis. Methodology: The spotted array consisted of 69 selected Salmonella-specific virulence gene probes (65bp each). These probes were printed on poly-l-lysine-coated slides. Genomic DNA was digested with Sau3AI, labeled with Cy3 dye, hybridized to the gene probes, and the images were captured and analyzed by GenePix 4000B and ArrayTrackTM, a free software developed by Food and Drug Administration (FDA) researchers. Results: Nearly 58% of the virulence-associated genes tested were present in all Salmonella strains tested. In general, genes belonging to inv, pip, prg, sic, sip, spa or ttr families were detected in more than 90% of the isolates, while the iacp, avra, invh, rhum, sira, sopb, sope or sugr genes were detected in 40 to 80% of the isolates. The gene variability was independent of the Salmonella serotype. Conclusions: This hybridization array presents an accurate and cost-effective method for evaluating the disease-causing potential of Salmonella in outbreak investigations by targeting a selective set of Salmonella-associated virulence genes. ISSN:

36 Torres, G.J., Piquer, F.J., Algarra, L., de Frutos, C., Sobrino, O.J. The prevalence of Salmonella enterica in Spanish feed mills and potential feed-related risk factors for contamination (2011). Preventive Veterinary Medicine, 98 (2-3), pp ABSTRACT: A cross-sectional study was conducted in Spain to estimate the prevalence of Salmonella enterica in feed mills and to identify and evaluate potential risk factors associated with feed contamination. A total of 3844 samples were collected from 523 different feed mills using a stratified sampling method. Samples were tested for the presence of Salmonella using conventional culture methods. When the presence of Salmonella was detected, samples were further characterised using serotyping at the National Reference Laboratory (NRL) for animal feed. Additional data about the biosecurity and hygiene measures, feed material used and compound feed produced, were collected by official veterinarians using a questionnaire in situ. In 144 of the feed mills visited (28%), Salmonella were present. However, it was only isolated from 4.8% of samples taken from all of the feed mills (3.5% from feed materials, 3.2% from compound feed and 12.5% from dust of the feed mill facilities). Salmonella serovars of public health importance (Enteritidis, Typhimurium, Infantis, Virchow and Hadar), were detected in only 2.7% of feed mills and in 0.3% of the samples studied. Logistic regression was used to investigate potential feed-mill risk factors for the isolation of Salmonella. Feed mill intake pits were demonstrated to have an increased risk of culture-positive dust samples (OR = 6.4; 95% CI: ). The feed material used in the production of compound feed was associated with recovery of Salmonella. Of the feed material used, cotton seeds were identified as having the highest odds of contamination (OR = 3.8; 95% CI: ). Pelleting appears to reduce the chance of contamination because non-pelleted compound feed is 8 times more likely to be contaminated than pelleted compound feed (OR = 8.2; 95% CI: ). The role of the feed itself in the epidemiology of Salmonella seems to be of limited importance as compound feed is not frequently contaminated at the feed mill level. This should not preclude Salmonella control measures from including all stages of feed production and they should have a risk-based approach according to the findings of this study. ISSN: Callaway, T.R., Edrington, T.S., Brabban, A., Kutter, B., Karriker, L., Stahl, C., Wagstrom, E., Anderson, R., Poole, T.L., Genovese, K., Krueger, N., Harvey, R., Nisbet, D.J. Evaluation of phage treatment as a strategy to reduce Salmonella populations in growing swine (2011). Foodborne Pathogens and Disease, 8 (2), pp ABSTRACT: Salmonella is a foodborne pathogenic bacterium that causes human illnesses and morbidity and mortality in swine. Bacteriophages are viruses that prey on bacteria and are naturally found in many microbial environments, including the gut of food animals, and have been suggested as a potential intervention strategy to reduce Salmonella levels in the live animal. The present study was designed to determine if anti-salmonella phages isolated from the feces of commercial finishing swine could reduce gastrointestinal populations of the foodborne pathogen Salmonella Typhimurium in artificially inoculated swine. Weaned pigs (n=48) were randomly assigned to two treatment groups (control or phage-treated). Each pig was inoculated with Salmonella Typhimurium ( colony forming units/pig) via oral gavage at 0h and fecal samples were collected every 24h. Swine were inoculated with a phage cocktail via oral gavage (3 109 plaque forming units) at 24 and 48h. Pigs were humanely killed at 96h, and cecal and rectal intestinal contents were collected for quantitative and qualitative analysis. Fecal Salmonella populations in phage-treated pigs were lower (p<0.09) than controls after 48h. Phage treatment reduced intestinal populations of inoculated Salmonella Typhimurium in pigs compared to controls at necropsy. Cecal populations were reduced (p=0.07) by phage treatment >1.4log10 colony forming units/g digesta, and rectal populations were numerically reduced. The number of pigs that contained inoculated Salmonella Typhimurium was reduced by phage treatment, but a significant (p<0.05) reduction was only observed in the rectum. We conclude that phages can be a viable tool to reduce Salmonella in swine. Further research needs to be performed to determine the most efficacious dosing regimens and the most effective combinations of phages targeting the diverse Salmonella population found in swine before they can enter the food supply. ISSN: Oscar, T.P. Development and validation of a predictive microbiology model for survival and growth of salmonella on chicken stored at 4 to 12 (2011). Journal of Food Protection, 74 (2), pp ABSTRACT: Salmonella spp. are a leading cause of foodborne illness. Mathematical models that predict Salmonella survival and growth on food from a low initial dose, in response to storage and handling conditions, are valuable tools for helping assess and manage this public health risk. The objective of this study was to develop and to validate the first predictive microbiology model for survival and growth of a low initial dose of Salmonella on chicken during refrigerated storage. 33

37 Chicken skin was inoculated with a low initial dose (0.9 log) of a multiple antibiotic-resistant strain of Salmonella Typhimurium DT104 (ATCC ) and then stored at 4 to 12uC for 0 to 10 days. A general regression neural network (GRNN) model that predicted log change of Salmonella Typhimurium DT104 as a function of time and temperature was developed. Percentage of residuals in an acceptable prediction zone, from 21 (fail-safe) to 0.5 (fail-dangerous) log, was used to validate the GRNN model by using a criterion of 70% acceptable predictions. Survival but not growth of Salmonella Typhimurium DT104 was observed at 4 to 8uC. Maximum growth of Salmonella Typhimurium DT104 during 10 days of storage was 0.7 log at 9uC, 1.1 log at 10uC, 1.8 log at 11uC, and 2.9 log at 12uC. Performance of the GRNN model for predicting dependent data (n = 163) was 85% acceptable predictions, for predicting independent data for interpolation (n = 77) was 84% acceptable predictions, and for predicting independent data for extrapolation (n = 70) to Salmonella Kentucky was 87% acceptable predictions. Thus, the GRNN model provided valid predictions for survival and growth of Salmonella on chicken during refrigerated storage, and therefore the model can be used with confidence to help assess and manage this public health risk. ISSN: X Barrow, P.A., Freitas Neto, O.C. Pullorum disease and fowl typhoid-new thoughts on old diseases: A review (2011). Avian Pathology, 40 (1), pp ABSTRACT: Fowl typhoid and pullorum disease are two distinct septicaemic diseases largely specific to avian species and caused by Salmonella Gallinarum and Salmonella Pullorum, respectively. They were first described more than one century ago. Since their discovery, many efforts have been made to control and prevent their occurrence in commercial farming of birds. However, they remain a serious economic problem to livestock in countries where measures of control are not efficient or in those where the climatic conditions favour the environmental spread of these microorganisms. During the past 15 to 20 years there has been an explosion of genetic and immunological information on the biology of these two organisms, which is beginning to contribute to a better understanding of the organisms and their interaction with the host. However, it is not enough simply to understand the pathology in greater and greater detail. What is needed, in addition to this increase in basic knowledge, is creative thinking to challenge existing paradigms and to develop really novel approaches to infection control. ISSN: Blagojevic, B., Antic, D., Ducic, M., Buncic, S. Ratio between carcass-and skin-microflora as an abattoir process hygiene indicator (2011). Food Control, 22 (2), pp ABSTRACT: In two abattoirs, each slaughtering both cattle and pigs, 100 cattle and 100 pigs were randomly selected and sampled. From each animal, two samples were taken: a) immediately after sticking of bovines or stunning of pigs, approximately 2000cm2 hide (cattle) or 1500cm2 skin (pigs) areas were sponge-swabbed; and b) at the end of slaughter line but before chilling, the same areas on corresponding dressed carcasses were sponge-swabbed. In each swab-sample (400 in total), total viable count (TVC) and Enterobacteriaceae count (EC), as well as Escherichia coli O157 (in cattle) or Salmonella (in pigs) occurrence, were determined and used to assess process hygiene in the abattoirs. The results indicated that simply fitting mean TVC and/or EC on final carcasses into an acceptable, marginal or unacceptable process hygiene category (according to current microbiological EU process hygiene criteria) did not enable characterisation of each process with respect to its ability to reduce the transfer of incoming microbial loads (i.e. on skins) onto dressed carcasses. On the other hand, determining the ratio between mean TVC and/or EC on final carcasses and those on corresponding skins enabled more precise assessment of the hygiene of each abattoir process, as well as more reliable differentiation between abattoirs. On the other hand, occurrence of E. coli O157 in cattle or Salmonella in pigs (skins and/or carcasses), being dependant on varying factors including those on-farm/pre-abattoir, did not appear to be very useful for characterisation of the process hygiene but is valuable for the purposes of consumer exposure assessment and pathogen reduction. ISSN: Wang, Z., Xu, H., Wu, J., Ye, J., Yang, Z. Sensitive detection of Salmonella with fluorescent bioconjugated nanoparticles probe (2011). Food Chemistry, 125 (2), pp pathogen, with Salmonella as model analyte, has been developed basing upon DNA hybridization and fluorescent bioconjugated nanoparticles probe. The indicator probe was designed through bioconjugating the Salmonella sequence-specific oligonucleotide with the fluorescent Ru(bpy)32+-doped silica nanoparticles that 34

38 were prepared by a microemulsion method. Through a sandwich-type DNA hybridization procedure, the target Salmonella DNA was captured and the indicator probe was assembled onto solid matrix so that the Salmonella DNA can be measured by the fluorescent signals of assembled indicator probes. Under optimal conditions, the calibration graph for detection of target Salmonella DNA is linear over the range of 10-10,000. fmol/l with a detection limit of 3. fmol/l. The proposed method was also applied to detect Salmonella in mixed bacteria sample. It gives the detection limit of 30. cfu/ml. The method offers a potential resolution for simple, rapid and sensitive detection of foodborne pathogens in food, clinical and environmental samples. ISSN: Wales, A.D., Cook, A.J.C., Davies, R.H. Producing salmonella-free pigs: A review focusing on interventions at weaning (2011). Veterinary Record, 168 (10), pp ABSTRACT: Salmonella infection in pig production is typically endemic and largely asymptomatic. It is a cause of substantial concern among food safety bodies, prompting voluntary and legislative responses aimed at monitoring and reducing the number of Salmonella-infected animals entering the human food chain. Elimination of the problem at an early stage of production is highly desirable, and to this end the present review examines published evidence on the carriage of Salmonella by piglets before and after weaning, as well as evidence on the dynamics of Salmonella infection in the weaner and grower stages of pig production, the effects of maternal immunity, and risk factors for Salmonella excretion after weaning. Various interventions to reduce or eliminate Salmonella infection in young pigs have been tried, such as vaccination, competitive exclusion, treatments in feed and water, antibiotic administration, disinfection of animals, and segregated weaning to clean accommodation. The evidence on the effectiveness of these is considered, and the last is examined in some detail, as it appears currently to offer the best chance of eliminating Salmonella from growing stock. ISSN: Behravesh, C.B., Mody, R.K., Jungk, J., Gaul, L., Redd, J.T., Chen, S., Cosgrove, S., Hedican, E., Sweat, D., Chávez-Hauser, L., Snow, S.L., Hanson, H., Nguyen, T.-A., Sodha, S.V., Boore, A.L., Russo, E., Mikoleit, M., Theobald, L., Gerner-Smidt, P., Hoekstra, R.M., Angulo, F.J., Swerdlow, D.L., Tauxe, R.V., Griffin, P.M., Williams, I.T Outbreak of Salmonella Saintpaul infections associated with raw produce (2011). New England Journal of Medicine, 364 (10), pp ABSTRACT: BACKGROUND: Raw produce is an increasingly recognized vehicle for salmonellosis. We investigated a nationwide outbreak that occurred in the United States in METHODS: We defined a case as diarrhea in a person with laboratory-confirmed infection with the outbreak strain of Salmonella enterica serotype Saintpaul. Epidemiologic, traceback, and environmental studies were conducted. RESULTS: Among the 1500 case subjects, 21% were hospitalized, and 2 died. In three case-control studies of cases not linked to restaurant clusters, illness was significantly associated with eating raw tomatoes (matched odds ratio, 5.6; 95% confidence interval [CI], 1.6 to 30.3); eating at a Mexican-style restaurant (matched odds ratio, 4.6; 95% CI, 2.1 to ) and eating pico de gallo salsa (matched odds ratio, 4.0; 95% CI, 1.5 to 17.8), corn tortillas (matched odds ratio, 2.3; 95% CI, 1.2 to 5.0), or salsa (matched odds ratio, 2.1; 95% CI, 1.1 to 3.9); and having a raw jalapeño pepper in the household (matched odds ratio, 2.9; 95% CI, 1.2 to 7.6). In nine analyses of clusters associated with restaurants or events, jalapeño peppers were implicated in all three clusters with implicated ingredients, and jalapeño or serrano peppers were an ingredient in an implicated item in the other three clusters. Raw tomatoes were an ingredient in an implicated item in three clusters. The outbreak strain was identified in jalapeño peppers collected in Texas and in agricultural water and serrano peppers on a Mexican farm. Tomato tracebacks did not converge on a source. CONCLUSIONS: Although an epidemiologic association with raw tomatoes was identified early in this investigation, subsequent epidemiologic and microbiologic evidence implicated jalapeño and serrano peppers. This outbreak highlights the importance of preventing raw-produce contamination. Copyright 2011 Massachusetts Medical Society. ISSN: Menconi, A., Wolfenden, A.D., Shivaramaiah, S., Terraes, J.C., Urbano, T., Kuttel, J., Kremer, C., Hargis, B.M., Tellez, G. Effect of lactic acid bacteria probiotic culture for the treatment of Salmonella enterica serovar Heidelberg in neonatal broiler chickens and Turkey poults (2011). Poultry Science, 90 (3), pp ABSTRACT: In the present study, a series of experiments was conducted to evaluate the ability of a commercial probiotic culture (FloraMax, IVS-Wynco LLC, Springdale, AR) to reduce Salmonella 35

39 enterica serovar Heidelberg (SH) in chicks and turkey poults. In experiments 1 and 2, chicks were randomly assigned to treatment groups and then challenged via oral gavage with SH. Chicks were treated 1 h following SH challenge with the probiotic culture via oral gavage. At 24 and 72 h posttreatment, cecal tonsils and ceca were collected for recovery and enumeration of enteric Salmonella Heidelberg, respectively. In experiment 3, day-of-hatch turkeys were randomly assigned to treatment groups and then challenged via oral gavage with SH. Poults were treated 1 h following challenge with the probiotic via oral gavage. At 24 and 72 h post probiotic treatment, cecal tonsils and ceca were collected for recovery and enumeration of enteric SH, respectively. The probiotic culture significantly reduced the incidence of SH in cecal tonsils at both time points in chicks in both experiments (P < 0.05). These data demonstrate that administration of probiotic 1 h post SH challenge significantly reduced the incidence of SH recovery from cecal tonsils of chicks compared with controls 24 and 72 h following treatment. Similarly, probiotic treatment resulted in significant reductions in the concentrations of SH within the ceca in both experiments. Although similar significant results were observed at both 24 and 72 h in experiment 3, it was clear that poults were more susceptible to SH colonization than chicks. Overall, a Lactobacillus-based probiotic significantly reduced Salmonella enterica serovar Heidelberg in chicks and turkey poults. ISSN: Payne, J.B., Li, X., Santos, F.B.O., Williams, M., Sheldon, B.W. Survey of Salmonella populations from swine waste-treatment technologies (2011). Journal of Swine Health and Production, 19 (2), pp ABSTRACT: Salmonella presence, populations, serotypes, and antibiotic susceptibilities in untreated and treated swine manure were determined for farms implementing environmentally superior waste-treatment technologies. The waste-treatment systems surveyed showed potential in reducing Salmonella populations. ISSN: X Sirsat, S.A., Burkholder, K.M., Muthaiyan, A., Dowd, S.E., Bhunia, A.K., Ricke, S.C. Effect of sublethal heat stress on Salmonella Typhimurium virulence (2011). Journal of Applied Microbiology, 110 (3), pp ABSTRACT: Aims: To determine the virulence gene expression of Salmonella Typhimurium in response to sublethal heat stress and determine the adhesion and invasion pattern of heatstressed Salmonella in Caco-2 intestinal epithelial cells. Methods and Results: Transcriptional profiling was employed to capture the virulence gene response of Salm. Typhimurium at 42 C sublethal heat stress. Data indicated an induction of SPI-2 and SPI-5 genes and a repression of SPI-1-encoded genes due to heat stress. Gene expression pattern also showed induced transcription of fimbriae genes and genes present within the stress-associated Rpo regulon. Changes in adhesion and invasion pattern of heat-stressed Salm. Typhimurium were tested in Caco-2 cells. Heat-stressed Salm. Typhimurium showed greater adhesion to Caco-2 cells compared with nonstressed control cells. Conclusions: Salmonella Typhimurium exposed to sublethal heat stress responds by altered virulence gene expression, which further enhances the adhesion of bacterial cells to intestinal Caco-2 cells. Results indicate a role of physiological stress in Salm. Typhimurium in promoting microbial virulence and host cell vulnerability to infection. Significance and Impact of the Study: Studying the Salmonella virulence genes expression in response to sublethal heat stress is crucial for the understanding of the virulence status of Salmonella in temperature-abused foods. Results of this study provide information about the gene response and virulence status of Salmonella pathogenicity factors in response to sublethal heat stress towards host cells. ISSN: Edrington, T.S., Carter, B.H., Farrow, R.L., Islas, A., Hagevoort, G.R., Friend, T.H., Callaway, T.R., Anderson, R.C., Nisbet, D.J. Influence of weaning on fecal shedding of pathogenic bacteria in dairy calves (2011). Foodborne Pathogens and Disease, 8 (3), pp ABSTRACT: The objectives of the current research were to determine the effect of weaning on fecal shedding of Salmonella and Escherichia coli O157:H7 in dairy calves and to examine cultured isolates (to include Enterococcus) for antimicrobial susceptibility. This research was conducted on one large commercial dairy (>3000 head) in the southwestern United States. Two collections were made, during the winter (January 2009) and summer (July 2009) seasons. For the winter collection, two groups of calves were sampled (group 1: n = 18 pens, 69 head, 12 weeks of age; group 2: n = 19 pens, 75 head, 10 weeks of age). Fecal samples were collected from all calves via rectal palpation 2 days pre- and again 2 days postweaning. For the summer collection, one group of calves housed in 40 pens were utilized and 79 and 76 calves sampled 7 days pre- and 5 36

40 days postweaning, respectively. Fecal samples were collected into sterile palpation sleeves, placed on ice, and shipped to our laboratory for bacterial culture of E. coli O157:H7, Salmonella, and Enterococcus. Antimicrobial susceptibility was determined on select isolates. No differences (p > 0.10) in prevalence of Salmonella or E. coli O157:H7 were observed due to weaning in the winter collection. In the summer collection, more (p < 0.01) fecal samples were Salmonella positive preweaning (15.2%) as compared to postweaning (2.6%). No differences were observed for antimicrobial susceptibility in isolates collected pre- as compared to postweaning in either winter or summer collections, with the exception that multidrug-resistant Enterococcus faecium isolates preweaning were resistant to six antibiotics compared to seven or eight antibiotics postweaning (summer collection). Results of the current research indicate that the weaning of dairy calves does not increase the prevalence of pathogenic bacteria or substantially modify antimicrobial susceptibility profiles of these bacteria. ISSN: Park, S.H., Hanning, I., Jarquin, R., Moore, P., Donoghue, D.J., Donoghue, A.M., Ricke, S.C. Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia colio157:h7, and Salmonella serotypes in water samples (2011). FEMS Microbiology Letters, 316 (1), pp ABSTRACT: Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-pcr) using each specific primer pair simultaneously. Under optimized m-pcr conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 C), E. coli O157:H7 (83.3 C), and S. Typhimurium (85.9 C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction. ISSN: Jones, F.T. A review of practical Salmonella control measures in animal feed (2011). Journal of Applied Poultry Research, 20 (1), pp ABSTRACT: Salmonella is a major microbial hazard in animal feed. Salmonella can persist for long periods in a wide range of materials. The lack of uniformity involved in Salmonella contamination and the large volumes of feed produced make accurate assessments of feed contamination rates difficult. Salmonella control principles may be divided into 3 broad categories: efforts to prevent contamination from entering the facility, work to reduce microbial multiplication within the plant, and procedures designed to kill the pathogen. Preventing contamination also involves controlling dust, managing the flow of equipment and humans, reducing rodent infestations, preventing contamination from wild birds, and ensuring the sanitation of transport vehicles. Reducing Salmonella multiplication in feed manufacturing facilities involves discovering microbial growth niches and reducing conditions that lead to growth. Killing Salmonella may involve thermal processing (pelleting) or chemical addition. Pelleting reduces, but may not completely eliminate, Salmonella contamination because of limitations of the process or recontamination after thermal processing. Chemical additions to control Salmonella in feed primarily involve the use of products containing organic acid, formaldehyde, or a combination of such compounds. ISSN: Mørkbak, M.R., Christensen, T., Gyrd-Hansen, D. Consumers' willingness to pay for safer meat depends on the risk reduction methods - A Danish case study on Salmonella risk in minced pork (2011). Food Control, 22 (3-4), pp ABSTRACT: A choice experiment was conducted to elicit the extent to which consumers' willingness to pay (WTP) for reducing Salmonella risks in minced pork depends on how risk reductions are obtained. Three different risk reduction methods were analysed: Risk reduction on the farm and decontamination of carcasses using either hot water/steam or lactic acid. The sample consisted of 844 Danish consumers who participated in an Internet based survey. We found that consumers were willing to pay for risk reductions, but they disliked all the risk reduction methods they were 37

41 confronted with. Indeed, consumers were willing to pay a price premium for a complete elimination of Salmonella in minced pork but only if it was obtained at the farm level. Hence, our results indicate that consumers demand safer meat although it depends on the risk reduction method. We also found that consumers preferred the status quo to all other combinations of risk reduction and risk reduction methods. ISSN: Mainali, C., McFall, M.E., King, R.K. Validation of a real-time polymerase chain reaction assay for the detection of Salmonella in crops of broiler chickens (2011). Poultry Science, 90 (3), pp ABSTRACT: Salmonella is one of the frequent causes of bacterial foodborne diseases with major public health impact in industrialized countries. Food-producing animals, in particular poultry, are major sources of human salmonellosis. Salmonella is normally found in the gastrointestinal tract of animals and can contaminate the carcass during the slaughtering process. In poultry, crops are also colonized by this pathogen. Crops are more likely to get ruptured during evisceration and contaminate the carcass and therefore present a health risk to consumers. Reducing Salmonella colonization in crops could decrease carcass contamination and is considered a potential preharvest critical control point in poultry production. Furthermore, rapid and reliable diagnostic methods to detect Salmonella are needed to monitor crop colonization to help ensure food safety. However, detection of Salmonella by bacteriological methods is time consuming and labor intensive and is not suitable for routine screening of a large number of samples. Therefore, this study was undertaken to validate a real-time PCR (RPCR) assay for the detection of Salmonella spp. in crop samples of broiler chickens. In total, 997 crop samples (35 spiked, 962 field) were processed by both RPCR and culture. The RPCR correctly identified all spiked crop samples. Out of 962 field crop samples, 100 tested positive by RPCR and 88 tested positive by culture for Salmonella, giving a sample level prevalence of 10.4% (95% CI: 8.54 to 12.50%) and 9.1% (95% CI: 7.40 to 11.15%), respectively. The agreement beyond chance between RPCR and culture was 92% (P < 0.001) and 100% (P < 0.001) for field and spiked samples, respectively. Compared with culture, the sensitivity and specificity of RPCR were and 98.51% for field samples and 100 and 100% for spiked samples, respectively. Where bacterial speciation is required, only the positive samples would be cultured. Therefore, RPCR can be used as a good screening tool for Salmonella spp. in crops by eliminating the time-consuming and labor-intensive culture of negative samples. ISSN: