p. 4 Determining coccidiostats using LC-MS

p. 8 Screening method for the simultaneous determination of anabolics and corticosteroids

p. 10 Report and ambiance of the IAG meeting 2010

p. 14 NRL-GMO GMODetec research project (2007-2010)

p. 20 Benzene in foodstuff s

p. 22 The importance of Norovirus detection in foodborne outbreaks

p. 25 Milk and Milk Products

p. 30 Workshops & Symposia

Labinfo

N A T I O N A LR E F E R E N C ELABORATORIESNRL

Newsletter for the approved food safety laboratories

SEMI-ANNUEL NEWSLETTER - N°5 DECEMBER 2010

FASFCAC-Kruidtuin - Food Safety Center, Kruidtuinlaan 55, 1000 Brussels

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LabInfoNewsletter for the approved food safety laboratories

Editors’ groupDirk Courtheyn, Mieke De Mits, Conny De Schepper, Alain Dubois, Marc Evrard, Alain Laure, Bert Vandenborre, Mieke Van de Wiele, Eva Wevers and Marie-Christine Wilem.

Authors of this issueGeert De Poorter, Eva Wevers, Mieke Van de Wiele, Jeroen Vancutsem, Elodie Barbau-Piednoir, Antoon Lievens, Amaya Leunda Casi, Nancy Roosens, Marc Van den Bulcke, Myriam Sneyers, Frédéric Debode, Eric Jansens, Gilbert Berben, Tom Ruttink, Isabel Taverniers, Marc De Loose, Ilse Van Overmeire, Raquel Vinci, Joris Van Loco, Sarah Denay-er, Nadine Botteldoorn, Hadewig Werbrouck, Véronique Ninane, Koen De Reu and Jessy Claeys.

TranslationTranslation Service of the AgencyEditors’ group

Photographs and illustrationsSupplied by the laboratories

LayoutGert Van Kerckhove

Editor’s addressLabInfop.a. D. CourtheynFASFCAC-Kruidtuin – Food Safety Center4de verdieping, bureel K04/120218Kruidtuinlaan 551000 BrusselTel.: 02.211.87.33dirk.courtheyn@favv.be

N A T I O N A LR E F E R E N C ELABORATORIESNRL

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Dear reader,

The end of the year is near, and generally it is a period of yearly review, talks, refl ections about past and future events. For the FASFC 2010 was a festive year : we celebrated our tenth birthday. An anniversary that one may see and experience fully. The FASFC managed to give an image of itself as a steady and effi cient public institution, able to realize its mission on a more than eff ective way. Reacting watchfully to incidents is part of that mission among others, and ‘incidents’ means ‘analyses’. Our own labs, supported by a very large network of approved external laboratories, see to get hundreds of thousands analyses being delivered in a period as short as possible. In that framework an ‘External Dashboard’ has been launched recently, through which all external partners of DG Laboratories can have a view on-line on their performances in their cooperation with the FASFC. A second major web application that received recently its baptism of fi re is the Foodlims module “Profi ciency Testing Schemes”. That module will allow the labmanagement as well as the competent authority and the accreditation bodies to follow up continuously the labperformance.Last but not least, the readability and accessibility of the articles of our e-newsletter have been improved as well. So I hope you will enjoy reading this newsletter, and I wish you a Merry Christmas and a Happy New Year.

Geert De PoorterDirector general Laboratories

EditorialNewsletter for the approved food safety laboratories

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Determining coccidiostats using LC-MS

Coccidiosis and coccidiostats

Coccidiosis is a very contagious infection disease in animals caused by eukaryotic single-celled parasitic orga-nisms (protozoa) referred to as coccidia, genus Eimeria. More than 600 species of coccidia are known today but only a few of them do infect poultry. The most pathogenic among them are E. tenella, E. necatrix, E. maxima, E. acervulina, E. mitis, E. praecox and E. brunetti. In addition to poultry, turkeys and rabbits coccidia may also infect other animal species, such as pigs and sheep, but these are less sensitive. In its acute form, coccidiosis is lethal and the subacute form of the disease leads to a loss of weight due to lower food conversion and to drops in egg production.

The chemical compounds used to fi ght coccidiosis do not have a common denominator. They may aff ect the coccidia at any stage of the parasite’s life cycle. They are mostly administered as medicated feed but sometimes they are added to the drinking water or administered by spraying. Some compounds also have an antibiotic or antibacterial action, in addition to their coccidiostatic eff ect. Whether an antibiotic is used for therapeutic purpo-ses or as a coccidiostat depends on the concentration.

At present, the use of 11 coccidiostats is authorised in chickens, turkeys and rabbits. In a general way, cocci-diostats may be classifi ed into two groups: ionophore coccidiostats (semduramycin[-sodium], lasalocid[-sodium], monensin[-sodium], salinomycin[-sodium], narasin and maduramicin[-ammonium]) and non-ionophore cocci-diostats (halofuginon, nicarbazin, robenidin[-hydrochloride], diclazuril and decoquinate).

Legal background

Regulation (EC) No 1831/2003 provides that manufacturers who want to be authorised to market a feed additive (e.g. a coccidiostats) intended for use and processing should submit an application thereto to the European Com-mission. Detailed rules for the assessment of the application and for issuing authorisations as well as the requi-rements with respect to the application are laid down in Regulation (EC) No 429/2008. Residue studies must in any case be submitted together with the application in order to allow the European Commission to decide, upon recommendation of the European Food Safety Authority, whether maximum residue limits (MRLs) must be set for the foodstuff s obtained from animals to which the additive would have been fed.When the assessment of the application is positive, the authorisation is granted by publishing a Regulation (EC) that lays down specifi c rules for the use of the additive, such as the authorised dose and the target animal species or categories for which it is meant. For fi ve (monensin, salinomycine, narasin, robenidine, diclazuril) of the above-mentioned eleven substances MRLs have already been set for tissues of (some) target animals.References of the relevant Regulations (EC) are to be found in the List of authorised additives.(http://ec.europa.eu/food/food/animalnutrition/feedadditives/registeradditives_en.htm).

Some operators of feed businesses manufacture many diff erent feedstuff s in one establishment whereby one production line is used for many products manufactured one after the other. In this process, it is sometimes impossible to avoid that traces of one product stay behind in the production line and end up in the beginning of the production of the next feed. This phenomenon is known as “carry-over” or “cross-contamination”. It may have as an eff ect that traces of coccidiostats are transferred to feedstuff s for “non target animals” that are manufactured

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next. Carry-over may occur at all stages of the manufacturing and processing of feedstuff s as well as during the storage and the transport of feedstuff s. It was therefore recommendable to set maximum limits for the carry-over of coccidiostats to non-target feed, in accordance with the ALARA principle (As Low As Reasonably Achievable).The unavoidable carry-over of active substances of authorised coccidiostats to non-target feed is considered as a case of undesirable substances in animal feed, as referred to in Directive 2002/32/EC and must therefore not involve any hazard for animal health, human health or the environment. Hence, Commission Directive 2009/08/EC amending Annex I to Directive 2002/32/EC lays down maximum levels of these substances in animal feed. In order to allow feed manufacturers to control this unavoidable carry-over, Directive 2009/8/EC takes into ac-count the distinct authorised carry-over ratios that depend on the fi nal destination of the feed (fed to sensitive animal species or not) when establishing the maximum levels. So, in feed for less sensitive non-target animals a carry-over of some 3 % is authorised. In other cases, a carry-over level of 1 % is accepted, e.g. in feed for sen-sitive non-target species, in compound feed used in the period before slaughter and in feed for target animals into which no coccidiostats have been incorporated. This level must also be observed for non-target animals intended for use as permanently food producing animals such as dairy cows and laying hens when evidence has been given that a carry-over from feed to food of animal origin is possible. When feedstuff s are given directly to the animals or when additional feed is given, the use of those feedstuff s in the daily ration must not increase the exposure of the animal to a coccidiostat beyond the maximum level in force for daily rations containing only com-plete feedstuff s.

Another aspect of this unavoidable carry-over of coccidiostats to non-target feed is the fact that residues of such substances may end up in foodstuff s of animal origin, even if the actual carry-over is lower than the maximum levels set in accordance with Directive 2002/32/EC.Within the framework of Regulation (EC) No 315/93 laying down rules for not intentionally added substances it was therefore necessary to establish maximum tolerances for the presence of active substances of coccidiotstats in food of animal origin in order to protect public health. For a limited number of coccidiostats that may be authorised by the national authorities for therapeutic use in some animal species and animal categories in accordance with Directive 2001/82/EC some MRLs have already been established indirectly for the relevant foodstuff s by means of Regulation (EU) No 37/2010. However, yet another legal initiative had to be taken since Regulation (EU) No 37/2010 does not include a decision on the MRLs for all coccidiostats that may lead to residues in animal tissues due to carry-over from non target feed. At the request of the Commission the EFSA therefore issued some opinions on animal and public health risks re-lated to the possible carry-over of coccidiostats to non target feed. On the whole, the EFSA considers that the pos-sible presence of coccidiostats that are authorised as additives in animal nutrition in non-target feed do not have any negative eff ect on animal health if certain precautionary measures are taken. In this matter, the EFSA refers to the conclusions of several scientifi c opinions. Moreover, the EFSA considers that the health risk for consumers related to the intake of residues of such substances in products of animals that were fed feedstuff s contaminated as a result of carry-over may be neglected. On the basis of the opinions of the EFSA, Regulation (EC) No 124/2009, which took eff ect on 1 July 2009, esta-blished maximum levels in order to protect public health. These maximum levels should always be adjusted to the changes made to the MRLs for the food in question within the context of Regulation (EC) No 37/2010 on pharmacologically active substances and their classifi cation regarding maximum residue limits in foodstuff s of animal origin.

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Determination of coccidiostats by the FLVVT (Federal Food Safety Laboratory in Tervuren)

Some specifi c methods are available at the FLVVT for checking the maximum levels of coccidiostats in feedstuff s. These methods are extremely appropriate to check the levels in terms of mg/kg but their analytical capacity is not suffi cient for detecting concentrations in terms of μg/kg. That is why LC-MS methods have already been develo-ped before in order to detect coccidiostats residues in feedstuff s in terms of μg/kg, in spite of the fact that at the time there was no legal framework for that kind of tests. The LC-MS method for ionophore coccidiostats in feeds-tuff s could be accredited but the LC-MS equipment of the time (ion trap type) was not appropriate to develop a satisfactory test method for non-ionophore coccidiostats. However, since late 2008 the FLVVT has a triple quadru-pole type of LC-MS device which made it possible to develop one single method for the simultaneous detection of both ionophore and non-ionophore coccidiostats in feedstuff s. Besides, Directive 2009/08/EC laying down maximum limits for non-target feed, in terms of μg/kg, came into force on 1 March 2009 requiring that the LC-MS method under construction be adjusted to the new maximum limits.

On the other hand, Regulation (EC) No124/2009 laying down maximum levels of coccidiostats in food, came into force on 1 July 2009. The FLVVT already had an accredited LC-MS method for the detection of residues of iono-phore coccidiostats in eggs and meat, in addition to the method for feedstuff s. But, the reporting level of that method being close to the maximum levels suggested, a decision was made to optimize this method and extend it to non-ionophore coccidiostats.

In other words, a new testing method for determining the levels of coccidiostats was required that was appropria-te for the matrices eggs and feedstuff s. That method also had to be able to determine simultaneously the levels of both ionophore coccidiostats (semduramicin, lasalocid, monensin, salinomycin, narasin and maduramicin) and non-ionophore coccidiostats (halofuginon, robenidin, nicarbazin, diclazuril, decoquinate and amprolium).

For both the matrices ‘feedstuff s’ and ‘eggs’ was developed a testing method that allowed the testing of samples as referred to in Directive 2009/08/EC and in Regulation (EC) No 124/2009. As the coccidiostats to be tested belong to Group B of Annex I to Directive 96/23/EC this method must be validated in accordance with Decision 2002/657/EC. The main parameters that were determined, are selectivity/specifi city, accuracy, precision, decision limit CCα and detection limit CCβ. The validation fi les and methods were submitted for accreditation to BELAC at the audit of 29 and 30 April 2009. The BELAC audit report was received on 18 May 2009. That report does not mention any A or B non compliances for the methods and validations. The procedure for validating the extension of the method to matrix “meat” has been started.

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Fig.: LC-MS equipment for determining coccidiostats

Eva Wevers, FLVVTeva.wevers@favv.be

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Screening method for the simultaneous determination of anabolics and corticosteroids in urine and feces using LC-MS

Anabolic steroids and corticosteroids

Anabolics are substances (hormones and substances with hormonal activity) with an anabolic eff ect, i.e. they have an infl uence on the metabolism, in which are formed components that are required for the constitution and the functioning of cells, tissues and organs. They are the opposite of catabolic substances, which play a part in the degradation of compounds. Anabolics are illegally used in stock farming as growth promotors. They are also known because of their abuse in sport. The use of anabolics in stock farming was given a lot of negative publicity following some incidents with the frequent use of DES (diethylstilbestrol). DES was not only used as a growth promotor in bovine animals. In the 1950s it was also used as a drug by pregnant women to prevent imminent abortion. In the 1970s it appeared that the daughters of these so-called DES-mothers showed an increased risk of vaginal and cervical cancer. In 1980, DES was found in baby food in Italy. Babies that had been given this food showed signs of early breast development. It is very likely that some of the ingredients of the baby food came from animals that had been illegally treated with DES. All such incidents make consumers feel suspicious of the use of growth promotors and, in a more general way, have given the word “hormone” a negative undertone.Anabolics may have a protein or a non-protein structure. The latter are mainly steroids, where a distinction has to be made between compounds with an androgenic, estrogenic or gestagenic eff ect. Growth hormones or soma-totropins are protein hormones that stimulate growth when they are present in the body of humans or animals. They are produced by the anterior lobe of the hypophysis.

Corticosteroids are frequently used in human and in veterinary medicine, often in combinations with other drugs, such as antimicrobial agents or ß-agonists. Two groups may be identifi ed, i.e. mineralocorticoids and glucocor-ticoids, naturally synthesized from the cholesterol in the cortex of the suprarenal gland. Mineralocorticoids are a group of steroid hormones with a structure that resembles that of aldosteron. They have an eff ect on the water and salt balance of the body (electrolyt homeostase) and hence, on the blood pressure, among other things. Their common feature is that they retain sodium and increase the excretion of potassium. Glucocorticoids are produced under the infl uence of ACTH (AdrenoCorticoTrope Hormone), that is released by the hypophysis. They stimulate the conversion of proteins and fats into glucoses, resulting in an increase of the blood sugar level. They play an important part in regulating the immune system activity and are released to a higher degree in stress situations. They suppress infl ammatory reactions.

Often, the activity of corticosteroids has to do with both groups. Cortisol and cortisone are examples of natural glucocorticoids. Dexamethasone and prednisolone are two well-known synthetic glucocorticosteroids.

The use of both groups of growth promotors is forbidden in the European Union (Directive 96/22/EC and Direc-tive 96/23/EC).

9

Developing the method

Formerly, the FLVVG fi rst carried out an extraction using diethylether before proceeding to the determination of anabolics and corticosteroids in urine and in feces. After evaporation of the ether the residue was distributed over a mixture of water/methanol and iso-octane in order to remove apolar substances and to remove the fat from feces. The extracts containing hormonal substances were then submitted to a Solid Phase Extraction. The primary extract was loaded onto the OASIS column, washed specifi cally several times and fi nally eluated. The eluate was then loaded onto an NH2 column, eluated and then chromatographed on a reversed phase HPLC column. The fractions surrounding the retention time of the substances to be determined were collected and further analysed using LC-MS and GC-MS following derivatisation. HPLC fractioning was required in order to protect the GC. Recently, a gradient U(H)PLC method was optimized in order to quantify more than 30 components, including both anabolics and corticosteroids. U(H)PLC/MS separation requires no more than a 10 minutes run. After enzy-matic hydrolysis of urine is performed an acetonitrile extraction according to the QuEChERS-principle. Further purifi cation on small SPE columns is required before injection into the LC/MS system. A similar extraction method has been developed for feces, using diethylether. After an SPE purifi cation an extra prepurifi cation step is perfor-med on a multi-immunoaffi nity chromatography (MIAC) gel developed by the CER (Centre d’économie rurale – Laboratory of Hormonology – Marloie, Belgium). The gel was prepared by mixing several individual gels, prepa-red from specifi c antibodies.

Fig. 1: Extraction on MIAC-columns Fig.2: Detection with LC-MS

All polyclonal antibodies against anabolics were prepared by hyperimmunisation of rabbits. Immunoglobulines (IgGs) were combined with cyanogen bromide-activated Sepharose 4B (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) according to the supplier’s instructions. The fi nal MIAC gel was obtained by mixing adequate amounts of the individual gels. The antibodies and the gels were produced by the Health department (CER groupe, Marloie, Belgium). These are available on the market, individually or as a combination. The MIAC gel may be used several times. Purifi cation on small immunoaffi nity columns increases the sensitivity by improving the signal-to-noise ratio as well as the specifi city.Purifi cation on the small SPE columns and the small multi immunoaffi nity columns was automated by means of an automatic 4 needle sample preparation system in order to increase eff ectiveness and throughput. The method was validated in accordance with Decision 2002/657/EC as a screening method : the decision limit (CCα), the detection capability (CCβ) and the specifi city were defi ned. The detection capability varied between 0.5 and 5 ng g−1, depending on the component and the matrix.

Mieke Van de Wiele (FLVVG, Gentbrugge)mieke.vandewiele@favv.be

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Report and ambiance of the IAG meeting 2010From 8 to 10 June 2010, the Tervuren Federal Laboratory for Food Safety (FLVVT) organised the annual IAG microscopy meeting. Founded in 1959, the IAG is an international association on feed materials microscopy with both offi cial control institutes and private laboratories among its members from a range of European countries. The annual meeting in June is organised by one of the members each time. In addition, the IAG is also engaged in method development, the organisation of ring tests and the organisation of workshops.

We welcomed 49 participants from 14 European countries. The programme comprised the following parts:

Day 1

- general subjects;

- specifi c microscopic parts;

- animal proteins part I

Day 2- discussion of ring tests organised by the IAG;

- discussion of methods set up by the IAG

Day 3- animal proteins part II

The complete programme can be read on the IAG website.

After the traditional presentation phase, whereby each participant presents themselves and explains the micros-copic activities of the past year, things could start properly. Geert De Poorter (FASFC, DG Laboratories) gave an introduction on the operation of the Belgian food safety system.

Picture: G. De Poorter (FASFC, DG Laboratories)

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This was followed by a more technical presentation on immunochemistry and microscopy in which among others the importance of the section preparation was explained along with the possible applications such as the tracing of mycotoxins.After a guided tour in the laboratory, the fi rst part of the presentations on animal proteins was proceeded.In a fi rst presentation, the safe use of animal proteins in fi sh food was presented. Now the BSE problem appears to be increasingly under control, there are more people in favour of using animal proteins in specifi c applications, subject to compliance with very strict control procedures.In a second presentation, a new analysis method for the identifi cation of animal proteins was presented which is a combination of a microscopic method and PCR analysis. When with the help of a microscope a suspect particle is identifi ed, a small piece can be cut off by a highly precise laser, whereupon it is ejected for PCR analysis.The third presentation was fi nally given by Camino Belinchon of the CRL animal proteins (CRA-W, Gembloux) regarding the databank with photos that they have developed. This databank can be consulted by the NRLs and the IAG members.After this, a visit to the Royal Museum for Middle Africa was on the programme.

The following day was started with a digital microscopy demo. The advantage of this technique is that you no longer have to sit behind a microscope and that you can follow all images directly on a screen.

This was followed by the presentation and discussion of the various ring tests that were organised by the IAG: the ring test regarding the microscopic analysis for the determination of the composition of animal feed, the tracing of ragwort (Senecio jacobea) in hay, the ring test for the tracing of Ambrosia and the ring test for the analysis of animal proteins. Presentations were also given regarding the appearance of poisonous weeds in feed and the identifi cation of ragwort and similar varieties with the help of a software program.

Visit to the Royal Museum for Middle Africa Digital microscopy

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In the afternoon, an additional number of analysis methods set up by the IAG were read and commented: the determination of palm stone shells, the tracing of poisonous plants in roughage and the determination of chaff from rice. After approval, these methods will be published on the IAG website. The programme was closed with a presentation about sustainable development with a few samples such as organic coff ee, organic fruit juice, yucca chips, southern rice pudding and gale beer. In the evening everyone was invited to the conference dinner in Leuven.

On the third day, the recognised laboratories were also present for the analysis of animal proteins. A general introduction was given by Christophe Keppens (FASFC, DG Control Policy) on the application of the feed ban in Belgium. After this, an overview was given by Koen Van Dyck (European Commission) regarding the current situa-tion and future perspectives of the feed ban.

Sustainable development activity Leuven conference dinner

C. Keppens (FAVV, DG Control policy) K. Van Dyck (European Commission)

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In addition to this, further presentations were given regarding the SAFEED-PAP project (Detection of presence of species-specifi c processed animal proteins in animal feed). A general project overview was given by Vincent Baeten (CRA-W, Gembloux). In this project, research is carried out in various areas into the species-specifi c de-tection of animal proteins. Included in this is the development of test kits (dipstick, PCR kit), the validation of PCR methods, the development of confi rmatory methods with LC-MS/MS based on collagen detection, the develop-ment of a quantitative NIRS/microscopy combination method, the observation of new markers for classic micros-copy and the development of an immuno-microscopic combination method. The state of play concerning the development of reference materials in the SAFEED-PAP was given by Jean Charoud-Got (JRC-IRMM, Geel). More information regarding the SAFEED-PAP project, in which the FLVVT participated, can be found at http://safeedpap.feedsafety.org.There was also a presentation regarding the validation of an ELISA kit (MELISA-TEK) for the tracing of animal nu-trients. Finally Pascal Veys of the CRL Animal Proteins gave another presentation regarding their activities over the past year (ring test, research into detection limit).There was also a request for membership of the ECCA Laboratory to join IAG.The meeting closed on Thursday afternoon. There will be a meeting next year from 7 to 9 June 2011 in Krefeld, Germany in the CVUA-RRW (Chemisches und Veterinäruntersuchungsamt Rhein-Ruhr-Wupper). More information can be found on the IAG website: www.iag-micro.org.

Jeroen Vancutsem (FLVVT, Tervuren)

Jeroen.vancutsem@favv.be

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NRL-GMOGMODetec research project (2007-2010)Authors: IPH: Barbau-Piednoir, E., Lievens, A., Leunda Casi, A., Roosens, N., Van den Bulcke, M., Sneyers, M. CRA-W: Debode, F., Jansens, E., Berben, G. ILVO: Ruttink, T., Taverniers, I., De Loose, M.

The GMODetec project (RT-06/6: ‘Ontwikkeling van een algemene strategie voor detectie, identifi catie en kwan-tifi cering van genetisch gemodifi ceerd materiaal in voedingsproducten en veevoeder’) is a collaborative project between the three labs of the NRL-GMO consortium (IPH, CRA-W and ILVO). The project is funded by the Federal Public Service (FPS) for Health, Food chain safety and Environment and coordinated by IPH.The overall objective of the project is to develop integrated strategies and models for detection, identifi cation and quantifi cation of GMOs in food and feed. The project focused on development of: • a strategy allowing development of a GMO detection model for all GM-events where suffi cient information

(especially on the DNA sequences) is available. The aim here is to develop PCR methods based on the com-parative analysis of the available offi cial DNA sequences. The analytical results will be integrated in a mathe-matical decision model allowing the identifi cation of the transgenic material present in the product.

• a strategy allowing the development of a ‘GMO passport’ of a product by means of the methods employed in the ‘total genome analysis’ of an organism. By suitable choice of the reference points an image of all the transgenic sequences present in the product will be generated.

Further, the mathematical decision model for the authorized GMOs has to be extended to a model allowing the visualisation of the GM-composition of the DNA extracted from a product. In this case the presence of non-autho-rized GMOs can be traced by comparative analysis of the generated ‘fi ngerprints’ with the transgenic sequences present in authorized GMOs.The GMO passport strategy allows the development of a general GMO detection model in a uniform manner. This model will be less dependent on the available information of the respective GMOs and will be fl exible by the reference points that have to be taken in the analysis.

I. Development of qPCR methods - Screening

A. CoSYPS (IPH)

During the three year GMODetec Project, ISP worked on developing and improving CoSYPS (Combinatory SYBR®Green qPCR Screening). This decision support system, patented in 2008, allows the identifi cation of the potential presence of GMOs in food matrices1,2. This screening method is fl exible and can be adapted according to the requirements and the type of GMO being sought. At this moment in time, IPH has 14 SYBR®Green qPCR scree-ning methods (plants, soya, maize, oilseed rape, cotton, rice, beetroot, the 35S promoter and the nopaline syn-thase terminator 3, the CP4-EPSPS, PAT/pat and PAT/bar genes which confer tolerance to glyphosate (Roundup®) and glufosinate (Basta®, Liberty®..) herbicides respectively, the CryIAb gene which confers resistance to certain insects (lepidopteran pests) such as the European corn borer (Ostrinia nubilalis), and checking for the presence of “Caulifl ower Mosaic Virus”: CaMV in the sample. (Checking for the presence of p35S positive due to CaMV).These real time PCR methods use SYBR®Green technology. This is an intercalating agent which only fl uoresces

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when it is intercalated in double-stranded DNA ; this chemical agent allows the amplifi cation of the DNAg matrix to be monitored at each PCR cycle 4.

Figure 1: On the left: an example of amplifi cation measured in Real Time PCR. Fluorescence is measured on the Y-axis, the

PCR Cycle number on the abscissa. On the right: a Real Time PCR (ABI 7300)

The analysis of the results obtained from the SYBR®Green qPCR, based on a mathematical decision support sys-tem linked to CoSYPS, allows to establish a list of GMO events possibly present in the tested sample 2. During the second stage of the analysis, the presence of these GMO events will be tested through a transformation event-specifi c TaqMan® 4 method 5.As CoSYPS is a fl exible system, other methods can be developed and added to the system in order to allow the detection of future GMOs; ISP is in fact already involved in a European project called GMOSeek (2009-2012) in the course of which 5 new methods will be developed.During the GMODetec Project, a “Single Target Plasmid” (STP) was constructed for each of the methods developed. The use of a mixture of all these STPs was validated for being used as the sole positive control of all the screening methods.During the project’s third year, CoSYPS was evaluated through an inter-laboratory test which 13 European labora-tories took part in. The results of this inter-laboratory test revealed that CoSYPS could be transferred and used in other GMO detection analysis laboratories.We can conclude that CoSYPS is a versatile and operational screening method which can be transferred to other laboratories with a qPCR platform. The next step is to design and market ready-to-use plates (pre-fi lled and lyophi-lized) in order to simplify and reduce the cost of analysis preparation, thereby making this method accessible to as many laboratories as possible.

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B. Development of new screening elements for GMO detection using TaqMan® (CRA-W)

GMO detection through screening using TaqMan® methods was until recently almost solely based on searching for the 35S promoter and the NOS terminator 6-11. The ever-growing list of GMOs authorized in food requires new screening markers to be developed. The GMODetec project allowed CRA-W to develop a signifi cant number of screening methods based on TaqMan® targeting: • promoters: pFMV35S, pRice Actin, pSSuAra, pTA29, pNOS• terminators : t35S, tOCS, tG7• genes: gox, gus, EPSPS, bar, hsp70.

II. Searching for unknown GMO events

A. SELLUX (CRA-W) Technology

Technology called SELLUX was implemented to try to amplify unknown sequences close to sequences amplifi ed during screening and unexplained by known GMO events. SELLUX technology tries to amplify this area with the help of a “standard” length primer positioned on a known screening element and a short primer which must hybridize in a region whose sequence is unknown. Various types of technology were considered: the integration of LNA bases, the integration of inosine bases into diff erent places, tests with diff erent chemistries such as LUX primers, SYBR®Green and TaqMan® probes. Only the SYBR®Green method showed compatibility between the use of short primers and obtaining Real Time PCR signals. The use of inosine bases also had a positive eff ect on the signals. The conclusion of this work is that amplifi cation with short primers is possible but because of the lack of sensitivity generated by using short primers, it must be recognized that in the current state of knowledge, this technique is not yet suitable for creating profi les relating to the diff erent genetically modifi ed events which could be encountered.

Figure 2: The experiments were conducted to fi nd out if it was possible to generate an amplicon with very short primers

likely to hybridize in a region whose sequence is unknown. Inosine bases (bases which can pair up indiscriminately with

A, C, G or T) were used to increase the hybridization properties of short primers.

Unknown region Known region

Reverse primer

5bp primer + inosin

17

B. GGO passport (ILVO)

The objectives of ILVO as a research partner within the GMODETEC project were bipartite: on the one hand the developing and further optimising of a protocol for anchor-PCR fi ngerprinting; on the other hand the developing of integrated strategies for determining the composition of a GGO sample, including the detection and characte-rising of unauthorised GGOs. The second objective is in keeping with the fi nal objective of the project, namely the developing and optimising of a so-called ‘GGO passport’ identifi cation technology. The fi rst objective within the project was the developing and further optimisation of a protocol for anchor-PCR fi ngerprinting. A new fl uorescent anchor-PCR protocol was drawn up and published utilising anchor-primers for the screening elements p35S and t-NOS12. Therefore a perfect link is possible with the matrix screening approach, which, amongst other things, makes use of these elements. In the second project year this protocol was further refi ned and extensively tested on wild types as well as GGO materials. The second objective of ILVO concerned the elaboration of alternative strategies for the tracing of unauthorised GGOs. Strategy 1 is based on molecular analytic detection and requires knowledge of the GGO DNA sequences for the design of specifi c analytical tests, but does not call for a knowledge of the GGO composition of products. It concerns a molecular toolbox consisting of various analytical techniques, including anchor-PCR fi ngerprinting, which ultimately allows for a confi rmation (detection) and identifi cation of a UGM. Strategy 2 is based on the systematic gathering of knowledge with respect to GGO product composition and authorisation status, so that an effi cient selection of potential unauthorised, suspect products can be made. This strategy employs the same technology as for strategy 1 (such as demonstrated for the UGM case study), but may lead to the optimisation of the monitoring through a targeted selection of products and analytical testing and shifts the use of analytical tools from screening to `blind samples` to a confi rmation of suspect products. Both strategies were put to a test and the proof-of-concept was demonstrated on a real life UGM case study. All was set out in two peer review publications. 12,13

Further research and additional tools were necessary for the development of a broad strategy, which integrates the PCR matrix screening model and the anchor-PCR fi ngerprinting model with, for example, whole genome analysis technologies, for the detection of GGOs including UGMs.

Figure 3: Depiction of anchor-PCR with use of a specifi c combination of restriction enzyme (NcoI, Mbo) and anchor

primer (red fragment), which renders a unique pattern. The anchor primers are used in sets of 3 primers in a `nested`

confi guration (green, blue and black), each with another fl uorescent label. The use of primers in a `nested` confi guration

is directed towards an increasing of the specifi city and sensitivity of the anchor-PCR. Anchor-PCR is followed by fragment

analysis through a separation of the products via capillary electrophoresis (CGE). The unique triplet of anchor-PCR ampli-

cons allows for a unique identifi cation of an event.

18

Figure 4: Genome walking via anchor-PCR. Amplifi cation of sequences which fl ank screening elements leads to an

identifi cation of all present GGOs and constitutes the positive proof for the presence of a UGM.

Figure 5: Depiction of 2 alternative approaches for the detection of UGMs. A standard analysis of GGOs is presently eff ec-

tuated on the basis of known sequences, to obtain information with respect to the product composition by use of various

analytical steps (above-left; below left triangle; left-hand arrow). This way of working is however becoming increasingly

more diffi cult, taking into account the increasing number of GGOs under development, and the increasing complexity

(which events to test, which not? Which tests/ analytes are to be applied, which not?). That’s why there is an alternative

procedure, starting from below, at product level (below right triangle; above-right; right-hand arrow). Product-based

UGM discovery becomes possible on the basis of documented evidence of the unauthorised presence of GGOs.

19

References:

1. Van den Bulcke, M., et al Transgenic Plant Event Detection. PCT/EP2008/051059 [WO/2008/092866]. 2008.2. Van den Bulcke M, et al. Anal Bioanal Chem 2009.3. Barbau-Piednoir E,et al. European Food Research and Technology 2010;230:383-393.4. Tse C, Capeau J. Ann Biol Clin (Paris) 2003;61:279-293.5. Community Reference Laboratory (CRL) Status of dossier web-page. http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm. 2004. 6. Waiblinger HU, et al. European Food Research and Technology 2008;226:1221-1228.7. Reiting R, et al. Journal fur Verbraucherschutz und Lebensmittelsicherheit 2007;2:116-121.8. Fernandez S, et al. J AOAC Int 2005;88:547-557.9. Höhne M, et al. European Food Research and Technology 2002;215:59-64.10. Corbisier P, et al. Anal Bioanal Chem 2005;383:282-290.11. Pardigol A, et al. European Food Research and Technology 2003;216:412-420.12. Ruttink T, et al. Anal Bioanal Chem 2010;396:1951-1959.13. Ruttink T, et al. Analytical and Bioanalytical Chemistry 2010;396:2073-2089.

20

Benzene in foodstuff sBenzene is a chemical compound used in the industry. It is also found in gasoline and in cigarettes. The Inter-national Agency for Research on Cancer (IARC) considers benzene as a substance that is carcinogenic for man. Exposure to benzene is mainly environmental (traffi c, industry, …). Yet, there are indications that certain foodstuff s contain increased concentrations of this and of similar components (e.g. toluene or ethylbenzene).

There are several possible sources of benzene in food. Benzene may be formed by decarboxilation of the benzoic acid salts (benzoates) in the presence of ascorbic acid (vitamin C) (Figure 1). Benzoic acid is added to many food-stuff s as a preserving agent and may be present either as a natural component or as a food additive. The presence of transition metal catalysts ( Cu (II) or Fe(III) ions ), the acidity, UV light and the temperature may have an eff ect on the formation of benzene from benzoates. Benzene may also be transferred to foodstuff s as it leaks from pac-kaging materials or the environment where the food is stored or by contaminated water. It may also be formed during irradiation processes. Contaminated carbon dioxide (CO2) has been described as a source of benzene in beer. Benzene contaminations of food were found more than 10 years ago in the USA in soft drinks at concentra-tions of more than 1 μg/kg. The European standard for benzene in drinking water is 1 μg/kg. That is also the limit that is suggested as an acceptable reference value for benzene in soft drinks by the Scientifi c Committee of the Belgian Federal Agency for the Safety of the Food Chain. For now, little is known about the presence of benzene in other foodstuff s at increased concentrations. However, it appears from recent literature on the subject, that benzene was found in carrot juice for infants, due to the heat treatment this product undergoes in order to rule out microbiological contamination (Lachenmeier, 2008). A systematic study of the exposure to benzene in the human food chain is not yet available, but research has been started.

What researchers want to know now is to what extent the intake of benzene contaminated food involves a risk for public health. To answer that question, they need reliable intake estimates. Hence, they need as much information as possible on the benzene concentrations in foodstuff s and must combine this information with the data on the consumption of these products.

A research is now being done on other possible sources of benzene in food. As it is, there are also products that may contain benzene in a natural way, e.g. mango and cranberry. Juices made from these fruits are also examined. Benzene may also be formed from certain precursors (ß-carotene, fenylalanine and certain terpenes) that may be present in food (Lachenmeier, 2010).In the next few years more information will become available. But, as yet, there are no indications that lead to believe that the health of consumers is at risk because of the presence of benzene in foodstuff s.

Figure 1: decarboxylation of benzoic acid into benzene

References:- Lachenmeier et al. (2008). Food Add. Contamin. 25, 1216-1224.- Lachenmeier et al. (2010). Food and Chemical Toxicology 48, 291-297.

Ilse Van Overmeire, Raquel Vinci and Joris Van Loco (IPH)Ilse.vanovermeire@iph.fgov.be; Joris.vanloco@iph.fgov.be

C O O H

-CO2

benzène acide benzoïque

22

The importance of Norovirus detection in foodborne outbreaks

Introduction Norovirus (NV) is one of the most important agents causing a viral gastro-enteritis in adults and regularly leads to outbreaks where food is at the origin of the infection. In Europe, as reported by EFSA, food related viruses (Ade-novirus, Norovirus, Enterovirus, Hepatitis A and Rotavirus) are responsible for 13.1 % of the reported outbreaks in 2008, this in comparison to 5.8 % in 2005. In Belgium, NV infections were strongly under-reported since there were no specifi c procedures or methods available for an outbreak to be followed up and to demonstrate the epidemio-logical relation between infected persons and contaminated foodstuff s.

Method Since 2006 an extraction and detection protocol for Norovirus genotypes GI and GII has been introduced in the food laboratory of the Institute of Public Health (IPH). Trizol reagent was used for the extraction (Baert et al., 2007). For the detection of Norovirus in bivalve molluscs based on real-time PCR, the procedure as set out by the CEN working group (CEN/TC 275/WG 06) is used, for which the accreditation was also obtained in July 2010. Since 2007, the extraction procedure has also been tested on other food matrices originating from foodborne outbreaks using a manually contaminated control sample with the Murine Norovirus, a virus closely related to the human Norovirus. This demonstrated that the extraction procedure can be applied to most of the matrices, except for highly fat-containing foodstuff s. However, for a number of matrices an optimisation is necessary in order to obtain a better recovery.

Fig. : The Norovirus

23

ResultsThanks to the introduction of the extraction and detection method for Norovirus, as well as the sensitisation of the inspectors of the Belgian Federal Agency for Safety of the Food Chain (FASFC) and the physicians of the Health Inspection, an increase in the number of reported outbreaks in which Norovirus was identifi ed as the causative agent is observed (see table 1).

Table 1 : Overview of the reported Norovirus outbreaks from 2004 to 2010.

2004 2005 2006 2007 2008 2009 2010*

Total reported outbreaks (>2 sick persons) 57 105 116 75 104 96 41

Total number of sick persons 531 673 1032 846 841 857 999

Number of Norovirus outbreaks 2 1 4 10 11 8 11

Number of sick persons caused by the Norovirus 33 65 154 392 439 95 368

* period 01/01/2010-31/08/2010

In Belgium, 54 outbreaks were reported to the National Reference Laboratory for foodborne outbreaks (NRL FBO) for the period January to August 2010 . In 11 outbreaks Norovirus was detected resulting in 368 sick persons and 21 hospitalisations. A person with an underlying pathology died during a Norovirus outbreak on a cruise ship. In two outbreaks with a co-infection, Norovirus was together with a bacterial germ at the origin of a general out-break, in which there were 360 sick persons and 31 hospitalisations. In these cases it is not possible to specify the number of sick persons who became ill due to a Norovirus infection. It is notable that during the summer months many youth camps in Flanders had to deal with Norovirus infections. It was mainly the environment which is used in common by the members of the youth camp which played an important role in the spread of Norovirus. Some of the outbreaks will therefore not be reported to the European Food Safety Agency (EFSA), since the dispersal did not take place via foodstuff s but from person-to-person or via the surroundings.It is thanks to the collaboration between all the actors (IPH, AFSCA and the Health Inspection) that the NRL VTI does not disposes of foodstuff s but also of human samples (faecal and vomit) and can analyse environment sam-ples. Environmental swabs of public areas and/or objects (toilets, washbasins, door handles, kitchen surfaces etc.) often point to the origin of the Norovirus infection, which can be present on a surface for several weeks. Moreover, only a few viral particles (~10) are probably suffi cient to cause an infection (http://www.cdc.gov/ncidod/dhqp/id_norovirusFS.html, 2006). Besides this, it is also of importance to identify infected persons as they can also be a source of transmission. Furthermore, up to 30% of the infections occur asymptomatically (http://www.cdc.gov/ncidod/dhqp/id_norovirusFS.html, 2006).The places of exposure are mainly common public areas such as hospitals, at work, recreation areas, at camps or during a visit to a restaurant.Thanks to a good diagnostics, an oriented approach towards Norovirus infections in a contaminated area is possi-ble, which helps to prevent the occurrence of secondary infections. For hygiene of the hands, washing the hands with liquid antibacterial toilet soap is recommended because this is more effi cient in inactivating the Norovirus as compared to alcohol-based sanitizers (Liu et al., 2010). Dependent on the nature of the surface to be cleaned, chlorine water (NaOCl) is used in diff erent concentrations (see table 2) for disinfection of the environment.

24

Table 2: Overview of the use of chlorine water for the disinfection of the Norovirus

Chlorine (ppm) Application

200 ppm Stainless Steel

Items with food/ mouth contact

Toys

1000 ppm Non-porous surfaces

Floor tiles

Counters

Sinks/ washbasins

Toilets

5000 ppm Porous surfaces

Wood fl oors

*data - Centre for Disease Control – World Health Organisation

DiscussionIn the past Norovirus was not often reported as causative agent of general food-borne infections as at that time there were no procedures for its extraction and detection. The application of the described procedures allows Norovirus detection in a remarkably high number of collective outbreaks and this is currently even the most important detected agent in these outbreaks. In none of the outbreaks primary contaminated foodstuff s were detected. The transmission of Norovirus occurred mainly by secondary contaminated foodstuff s in which the food handler is an important source of contamination, or through person-to-person transfer.

ReferencesThe Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents, Antimicrobial Resistance and Foodborne Outbreaks in the European Union in 2008, EFSA Journal; 2010 8 (1): 1496

Baert, L., Uyttendaele, M & Debevere, J. (2007). Evaluation of two viral extraction methods for the detection of human noroviruses in shellfi sh with conventional and real-time reverse transcriptase PCR. Lett Appl Micriobiol 44 (1), 106-111

Liu, P., Yuen, Y., Hsiao, H-M, Jaykus, L-A & Moe, C. (2010). Eff ectiveness of liquid soap and hand sanitizer against Norwalk virus on contaminated hands. Appl Env Microbiol 76 (2), 394-399

Sarah Denayer and Nadine Botteldoorn (NRL Foodborne outbreaks and NRL Bacterial and Viral Food Pathogens, IPH)Sarah.Denayer@wiv-isp.be en Nadine.Botteldoorn@wiv-isp.be

25

Milk and Milk ProductsWorkshop Milk and Milk Products for the NRLs, Paris 2010

The European reference laboratory for Milk and Milk Products (EU-RL-MMP) held a workshop for national reference laboratories (NRLs) on 30 September and 1 October 2010. The Belgian national reference laboratory was represen-ted by Hadewig Werbrouck (ILVO-T&V) and Véronique Ninane (CRA-W-DVP).

Laurent Laloux (EU-RL-MMP) announced three changes regarding the EU-RL-MMP: the name “European reference laboratory Milk and Milk Products (EU-RL-MMP)” instead of “Community reference laboratory (CRL)”, the merger of the AFSSA and the AFSSET into ANSES (2) and the promotion of Véronique Deperrois who is now head of the Microbiology unit (3).

Paolo Caricato (DG-SANCO) reported on the activities of DG-SANCO within the European Commission. A review of the activities of the European reference Laboratories, in particular an extension to “all fi elds” related to milk such as Staphylococcus aureus and Listeria spp.; an agreement to be reached with the United States on a limitation of border inspections of exported (or imported) milk products and the issue of inspections of pasteurised milk obtained from other species than cows. The development of methods to determine alkaline phosphatase (check if a product has been suffi ciently pasteurized or not) in diff erent species is now one of the priorities set by DG-SANCO.

This workshop was dedicated to only one subject : the total bacterial count in raw milk. Véronique Deperrois (EU-RL-MMP) gave an introduction to this subject by means of a presentation on the diversity of microfl ora in raw milk and on the infl uence of stress conditions on this diversity. Milk that leaves the udder is free of micro-orga-nisms provided the cow does not show any signs of mastitis. The milk is contaminated only later by commensal microorganisms that are present in/on the teat of the cow, by man, by working tools, by the environment (e.g. litter and air), …. Maintenance, i.e. the practices adopted by the cattle farmer, of the “reservoirs” have an eff ect on the composition of raw milk; e.g. adding hay to the litter increases the number of lactobacilli in raw milk. Finally, one may say that the microfl ora in raw cow milk contains varying amounts of useful microorganisms (1) used e.g. in cheese making, such as leuconostocs, lactobacilli, lactococci, Propionibacteria, yeasts, fungi, streptococci, Enterococcus and “ripening” bacteria (cheese making); decay organisms (2) such as sporulated bacteria, psychro-trophic bacteria, coliforms, yeasts and fungi; and pathogenic microoganisms (3) such as staphylococci, Escherichia coli, Salmonella spp. and Listeria monocytogenes. This microbial diversity and the variability are the main factors that would possibly have to be taken into account when comparing methods used for determining the number of bacteria in raw milk since their targets may be diff erent : bacteria are increasingly less capable of growing as a group in specifi c environmental conditions (some groups are inevitably excluded), sometimes “dead” bacteria, sometimes live bacteria, …

For the implementation of Regulation (EC) No 853/2004 laying down specifi c hygiene rules for food of animal origin, the reference method for the plate count of raw milk is the method consisting of counting the colonies at 30°C, standardised as ISO 4833. The competence level of the European reference laboratories for this method was estimated by the EU-RL-MMP in ring tests carried out in 2007 and 2009. The results of these ring tests were presented by Alexandra Cauquil (EU-RL-MMP). She showed that the competence level of European laboratories was generally satisfactory. Moreover, that level had improved when compared to 2007: 88% of the participants

26

obtained a good Z score against 82% in 2007. However, six European laboratories (not taken into account for as-sessing the results) did not use the reference method as yet. Paolo Caricato, the representative of DG-SANCO, was not pleased to hear this.

Then, Rabid Miled (EU-RL-MMP) gave a complete view of the alternative methods for determining the bacterial count in raw milk. The alternative methods Bactoscan FC (Foss, Denmark) and Bactocount (Bentley, USA) were further discussed by representatives of the respective manufacturers: Berte Asmussen for Foss and Pierre Broutin for Bentley. The principle of these two methods is based upon the fl ow cytometry methodology with detection of bacteria by means of epifl uorescence microscopy. In both cases the bacterial clusters are shattered into individual bacteria, the bacteria are coloured by means of a fl uorescent dye and then the coloured bacteria are transferred to the reading system by means of a sheath liquid. The fl uorescent dye of each individual bacterium is excited by a laser and detected by a detector. The reading system converts the light beams into electronic pulses. These pul-ses are then converted into the number of colony forming units by means of a conversion table. There was also a demonstration of the Bactocount (Bentley).

Fig 1: Bactoscan FC and Bactocount IBC.

These methods may be used within the context of Regulation 853/2004 provided that they have been validated in accordance with the ISO 16140 standard and that their conversion relation has been established in accordance with the ISO 21187 standard. Betrand Lombard (EU-RL-MMP) gave a short review of the main rules. It was said, in concrete, that none of the alternative methods have already been validated but that a provisional approval had been issued for Bactoscan FC given the fact that part of the validation assessment is documented and to the fact that the Bactoscan FC equipment has been used for more than 10 years in most European countries. Yet, the manufacturer (Foss) will have to have the equipment validated in accordance with the ISO 16140 standard; if not, it will not be allowed to keep on using the equipment for the purpose of Regulation 853/2004. The EU-RL-MMP did not set a period of time but it is expected that the validation of the BactoscanFC will take up some two years. With a view to validation within the context of a ring test, EU-RL-MMP asked the manufacturers, including those of the Bactoscan FC, to call on the help of a specialised organisation. That aspect does not come under the authority

27

of the NRLs. Three European certifi ed organisations specialised in that type of work were mentioned: Microval, AFNOR certifi cation and Nord Val. As for the evaluation intralaboratory tests have been performed for both the bacterial count and the somatic cells count by the EU-RL-MMP with a view to validating the equipment. The tests have been completed and will be published shortly.

Bertand Lombard (EU-RL-MMP) mentioned some changes brought on by the review of the ISO 16140 standard (standardisation). These changes relate to the statistical processing, the introduction of acceptation criteria, the improved workability (praticability) and precision of the scope. As it is, the standard does not apply to an alterna-tive method that is specifi c for the laboratory (in this case an interlaboratory test is useless) but it does apply to commercial methods.

The issue of standardization, with respect to the ISO 16297/IDF 161 standard, was concluded by Harrie Van den Bijgaart (Qlip, Nederland). This is a specifi c standard and an addition to the ISO 16140 standard that may be ap-plied to the evaluation part, i.e. the intralaboratory tests of the alternative methods for the bacterial count in milk.

Given the complexity of standardisation with respect to the evaluation of alternative methods for determining the total fl ora in milk, the EU-RL-MMP suggested to write a practical guide with a detailed description of the procedu-res (both standards ISO 16140 and 13297 combined).

The responsibility of the NRLs with regard to the appropriateness of the conversion relation for the alternative me-thods (preparation and control according to ISO 21187 standard) was pointed out. The “checklist” drawn up by the EU-RL-MMP, mainly with the help of Koen De Reu (ILVO-T&V), was distributed among the NRLs to help them judge the quality of the work done by routine laboratories. Jolanta Rola (PIWET, Poland) who used the “checklist” also made some suggestions for the improvement of the list and for focusing the list on the fi nal purpose. In addition was set up a working group that is to re-examine the list taking into account the remarks made.

Summing up, one might say that the general rule is that one conversion relation may be established per type of equipment and per country. Exceptionally, several conversion relations are possible but a motivation will be required in that case (e.g. signifi cantly diff erent climates). In practice, the establishment of a conversion relation is in fact hampered by the diffi culty to distribute the same milk samples among diff erent laboratories when the distance between laboratories is too great. An interesting approach that makes it possible to sidestep this pro-blem was illustrated by Giusseppe Bolzoni (IZLER, Italy). His approach (details of statistical processing are available on www.izler.it ß reference material) suggests that there is a possibility to have one single conversion relation in Europe. A working group was set up that must examine the feasibility of that ambition. Véronique Ninane (CRA-W-DVP) is a member of this working group.

Hadewig Werbrouck (ILVO-T&V, Melle) and Véronique Ninane (CRA-W-DVP, Gembloux) hadewig.werbrouck@ilvo.vlaanderen.be and ninane@cra.wallonie.be

28

Developments in the fi eld of standards and legislationNew IDF- FIL (International Dairy Federation – Fédération International de Laiterie) standards in 2010 (from 2 No-vember 2009 up to 8 October 2010):

Standards:ISO 1211|IDF 001:2010 - Milk - Determination of fat content - Gravimetric method (Reference method)ISO 5536|IDF 023:2009 - Milkfat products - Determination of Water content - Karl Fischer methodISO 12081|IDF 036:2010 - Milk - Determination of calcium content - Titrimetric methodISO 6091|IDF 086:2010 - Dried milk - Determination of titratable acidity (Reference methodISO 6732|IDF 103:2010 - Milk and milk products - Determination of iron content - Spectrometric method (Refe-rence method)ISO 5546|IDF 115:2010 - Caseins and caseinates - Determination of pH (Reference method)ISO 13366-1|IDF 148-1:2008 - Milk - Enumeration of somatic cells - Part 1: Microscopic method (Reference method) + Technical Corrigendum 1 (2009)ISO 27205|IDF 149:2010 - Fermented milk products – Bacterial starter cultures - Standard of identityISO 11813|IDF 156:2010 - Milk and milk products – Determination of zinc content - Flame atomic absorption spec-trometric methodISO 17678|IDF 202:2010 - Milk and milk products – Determination of milk fat purity by gas chromatographic analy-sis of triglycerides (Reference method)ISO 26462|IDF 214:2010 - Milk - Determination of lactose content - Enzymatic method using diff erence in pHISO/TS 27105|IDF/RM 216:2009 - Milk and milk products – Determination of hen’s egg white lysozyme by HPLCISO/TS 27106|IDF/RM 217:2009 - Cheese - Determination of nisin A content by LC-MS and LC-MS-MSISO 29981|IDF 220:2010 - Milk products - Enumeration of presumptive bifi dobacteria - Colony count technique at 37 degrees CISO 10932|IDF 223:2010 - Milk and milk products – Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifi dobacteria

Other interesting IDF publications in 2009-2010:Bulletin of the IDF No. 440/2009 - Interlaboratory Collaborative Study on the Kjeldahl Reference Method for Nitro-gen Determination in Sheep and Goat MilkBulletin of the IDF No. 441/2009 - Monitoring success of paratuberculosis programs Proceedings of 2nd Paratuber-culosis Forum, Minneapolis, August 2009Bulletin of the IDF No. 442/2010 - Current situation & compilation of commercially available screening methods for the detection of inhibitors/antibiotic residues in milk - E-FormBulletin of the IDF No. 443/2010 - Environmental issues at dairy farm level - E-FormBulletin of the IDF No. 444/2010 - Feed-associated Mycotoxins in the Dairy Chain: Occurrence and Control - E-Form

IDF congresses:2011_IDF Regional Conference on Domestic Milk Supply and Demand Systems : Lessons from Experience”, 26 - 29 April 2011, Seoul, Korea2011_IDF International Symposium on Sheep, Goat and other non-Cow Milk, 16 - 18 May 2011, Athens, Greece2011_IDF/ISO Analytical Week, 23-27 May 2011, Lyon, France2011_IDF World Dairy Summit, 15-19 October 2011, Parma, Italy2012_IDF International Symposium on Cheese Ripening and Technology, 20-24 May 2012, Madison, Wisconsin, USA

29

2012_IDF/INRA International Symposium on Spray Dried Dairy Products, 19-21 June 2012, St. Malo, France2012_IDF World Dairy Summit, 03-09 November 2012, Cape Town, South Africa2013_IDF World Dairy Summit, October 2013, Yokohama, Japan

Koen De Reu (ILVO-T&V) Koen.Dereu@ilvo.vlaanderen.be Jessy Claeys (ILVO-T&V) Jessy.Claeys@ilvo.vlaanderen.be

30

Date Subject Place More information (website)

24-26/01/2011Rapid Methods Europe 2011 – the 7th conference

Noordwijkerhout,The Netherlands

www.bastiaanse-communication.com/RME2011

24-27/02/20116th International Conference on Emerging Zoonoses

Cancún, Mexico http://www.zoonoses2011.com/

21-24/03/2011Food Integrity and Traceability Conference

Queen’s University Belfast

http://www.conffi dence.eu/img/event/event_AS-SET_leafl et.pdf

7-10/05/201121st ECCMID (European Society of Clinical Microbiology and Infectious Diseases)

Milan, Italy http://www.escmid.org/dates_events/calendar/

8-11/05/2011LAPRW2011, 3rd Latin American Pesticide Residue Workshop

Montevideo, Uruguay

Contact: Prof. Dr. Horacio Heinzen; e-mail: heinzen@fq.edu.uy

18-20/05/2011First International Conference on Orga-nic Food Quality and Health Research

Prague, Czech Republic

http://www.fqh2011.org/

20-24/05/2011111th General Meeting of the American Society for Microbiology

New Orleans, Louisiana

gm.asm.org/

24/05/2011Mycotoxins: Challenges and Perspectives

Ghent, Belgium www.mytox.be

19-22/06/20114th International IUPAC Symposium for Trace Elements in Food (TEF-4)

King’s College in Aberdeen, Scotland

http://www.abdn.ac.uk/tef-4/

24/05/201163rd International Symposium on Crop Protection

Ghent, Belgium http://www.iscp.ugent.be/

12-17/06/201115th Gordon Research Conference on Mycotoxins & Phycotoxins

Waterville, Maine, USA

http://www.grc.org/

19-22/06/2011Second Saskatoon International Valida-tion Workshop for Regulatory Analyses of Residues in Foods (SaskVal Workshop)

Saskatoon, Saskatchewan, Canada

www.SaskVal.ca

19-22/06/2011

SafePork Conference 20119th International Conference on the Epidemiology and Control of biological, chemical and physical hazards in pigs and pork

Maastricht, The Netherlands

http://www.safepork.org/

20-24/06/2011 2nd Global conference on GMO analysis Como, Italy http://mbg.jrc.ec.europa.eu

24-25/07/2011International Food Microbiology Conference

Chipping Campden, UK

26-30/06/2011The 4th Congress of European Microbiologists, FEMS 2011

Geneva, Switzerland www.kenes.com/fems/

6-8/07/2011Euro Food Chem XVITranslating food chemistry to health benefi t

Gdansk, Poland http://www.eurofoodchemxvi.eu/

24-29/07/201110th International Conference on Mercury as a Global Pollutant,

Halifax, Nova Scotia, Canada

http://mercury2011.org/com/

1-6/08/201113th International Conference on Trichinellosis

Changchun, P. R. China

International Commission on trichinellosis (ICT); will be organized by Pr Liu Mingyuan in Jilin University, Changchun, P. R. Chinahttp://www.ict13.org/

Workshops & Symposia

31

07-12/08/201157th ICoMSTInternational Congress of Meat Science and Technology

Ghent, Belgium http://www.icomst2011.be/

21-25/08/201123rd. International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP)

Buenos Aires, Argentina

http://www.waavp2011-argentina.com.ar/

21-25/08/2011

DIOXIN 2011, 31st International Symposium on Halo-genated Persistent Organic PollutantsPOPs’ Science in the Heart of Europe

Brussels, Belgiumhttp://www.dioxin2011.org/JF.Focant@ulg.ac.be

28/08-1/09/201116th International Workshop on Campylobacter, Helicobacter & Related Organisms

Vancouver, Canada http://www.chro2011.com/Contact_Us.php

18-21/09/2011 125th AOAC Annual Meeting & Exposition

New Orleans, Louisiana

http://www.aoac.org/meetings1/125th_annual_mtg/main_2.htm

27-29/09/2011 3rd MONIQA International Conference Varna, Bulgaria

The theme “food safety and consumer protection” will form the overarching focus of the conference, bringing together food scientists from research and industry, socio-economists and policy makers. The conference will allow the MoniQA project to present its results and the plans for further deve-lopments of MoniQA as an association and will also provide ample room for discussion with stakehol-ders. Related EU projects will be invited to contri-bute and a special session will focus on food safety in Bulgaria. Including poster presentations and ex-hibitions, the conference will give an in-depth look at the following thematic areas: Mycotoxins/Phy-cotoxins, Food Allergens, Chemical Contaminants, Microbiological Contaminants, Food Additives and Processing Toxicants, Emerging Issues. http://varna2011.moniqa.org/

1-4/11/20115th International Symposium on Recent Advances in Food Analysis (RAFA 2011)

Prague, Czech Republic

www.rafa2011.eu

1-3/02/2012

Twelfth International Symposium on Hyphenated Techniques in Chroma-tography and Hyphenated Chromato-graphic Analyzers (HTC-12)

Bruges, Belgium http://www.ordibo.be/htc/

31/01-1/02/2012Second International Symposium on Hyphenated Techniques for Sample Preparation (HTSP-2)

Bruges, Belgium http://www.ordibo.be/htc/

30/09-3/10/2012 126th AOAC Annual Meeting & Exposition

Las Vegas, NV http://www.aoac.org

N A T I O N A LR E F E R E N C ELABORATORIESNRL