Labinfo No.5

SEMI-ANNUEL NEWSLETTER - N°5
DECEMBER 2010
Labinfo
Newsletter for the approved food safety laboratories
p. 4
Determining coccidiostats using LC-MS
p. 8
Screening method for the simultaneous
Responsible editor : Gil Houins
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 foodstuffs
p. 22
The importance of Norovirus
detection in foodborne outbreaks
p. 25
Milk and Milk Products
p. 30
Workshops & Symposia
FASFC
AC-Kruidtuin - Food Safety Center,
Kruidtuinlaan 55,
1000 Brussels
NRL
N AT I O N A L
REFERENCE
LABORATORIES
NRL
N AT I O N A L
REFERENCE
LABORATORIES
LabInfo
Newsletter for the approved food safety laboratories
Editors’ group
Dirk 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 issue
Geert 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 Denayer, Nadine Botteldoorn, Hadewig Werbrouck, Véronique Ninane, Koen De Reu and Jessy Claeys.
Translation
Translation Service of the Agency
Editors’ group
Photographs and illustrations
Supplied by the laboratories
Layout
Gert Van Kerckhove
Editor’s address
LabInfo
p.a. D. Courtheyn
FASFC
AC-Kruidtuin – Food Safety Center
4de verdieping, bureel K04/120218
Kruidtuinlaan 55
1000 Brussel
Tel.: 02.211.87.33
dirk.courtheyn@favv.be
2
Newsletter for the approved food safety laboratories
Editorial
Dear reader,
The end of the year is near, and generally it is a period of yearly review, talks, reflections
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 efficient public institution, able to realize its mission on a more than effective
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 fire is the Foodlims
module “Proficiency 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 Poorter
Director general Laboratories
3
Determining coccidiostats using LC-MS
Coccidiosis and coccidiostats
Coccidiosis is a very contagious infection disease in animals caused by eukaryotic single-celled parasitic organisms (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 fight coccidiosis do not have a common denominator. They may affect 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 effect. Whether an antibiotic is used for therapeutic purposes 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, coccidiostats may be classified into two groups: ionophore coccidiostats (semduramycin[-sodium], lasalocid[-sodium],
monensin[-sodium], salinomycin[-sodium], narasin and maduramicin[-ammonium]) and non-ionophore coccidiostats (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 Commission. Detailed rules for the assessment of the application and for issuing authorisations as well as the requirements 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 foodstuffs 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 specific 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 five (monensin, salinomycine, narasin, robenidine, diclazuril) of the abovementioned 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 different feedstuffs 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 effect that traces of coccidiostats are transferred to feedstuffs for “non target animals” that are manufactured
4
next. Carry-over may occur at all stages of the manufacturing and processing of feedstuffs as well as during the
storage and the transport of feedstuffs. 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 account the distinct authorised carry-over ratios that depend on the final 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 sensitive 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 feedstuffs are given directly to
the animals or when additional feed is given, the use of those feedstuffs 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 complete feedstuffs.
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 foodstuffs 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 foodstuffs 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 related to the possible carry-over of coccidiostats to non target feed. On the whole, the EFSA considers that the possible presence of coccidiostats that are authorised as additives in animal nutrition in non-target feed do not have
any negative effect on animal health if certain precautionary measures are taken. In this matter, the EFSA refers
to the conclusions of several scientific 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 feedstuffs 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 effect on 1 July 2009, established 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 classification regarding maximum residue limits in foodstuffs of
animal origin.
5
Determination of coccidiostats by the FLVVT (Federal Food Safety Laboratory in
Tervuren)
Some specific methods are available at the FLVVT for checking the maximum levels of coccidiostats in feedstuffs.
These methods are extremely appropriate to check the levels in terms of mg/kg but their analytical capacity is not
sufficient for detecting concentrations in terms of μg/kg. That is why LC-MS methods have already been developed before in order to detect coccidiostats residues in feedstuffs 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 feedstuffs 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 quadrupole 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 feedstuffs. 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 ionophore coccidiostats in eggs and meat, in addition to the method for feedstuffs. 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 appropriate for the matrices eggs and feedstuffs. 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 ‘feedstuffs’ 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/specificity, accuracy, precision, decision
limit CCα and detection limit CCβ. The validation files 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.
6
Fig.: LC-MS equipment for determining coccidiostats
Eva Wevers, FLVVT
eva.wevers@favv.be
7
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 effect, i.e. they
have an influence 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 effect. Growth hormones or somatotropins 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 identified, i.e. mineralocorticoids and glucocorticoids, 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 effect 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 influence 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 inflammatory 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 Directive 96/23/EC).
8
Developing the method
Formerly, the FLVVG first 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 specifically several times and finally 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 enzymatic hydrolysis of urine is performed an acetonitrile extraction according to the QuEChERS-principle. Further
purification 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 purification an extra prepurification step is performed on a multi-immunoaffinity 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, prepared from specific 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 final 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. Purification on small immunoaffinity columns increases the sensitivity by improving the signal-to-noise
ratio as well as the specificity.
Purification on the small SPE columns and the small multi immunoaffinity columns was automated by means of
an automatic 4 needle sample preparation system in order to increase effectiveness 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 specificity were defined. 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
9
Report and ambiance
of the IAG meeting 2010
From 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 official 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:
- general subjects;
Day 1
- specific microscopic parts;
- animal proteins part I
Day 2
Day 3
- discussion of ring tests organised by the IAG;
- discussion of methods set up by the IAG
- 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 microscopic 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)
10
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 first part of the presentations on animal proteins was proceeded.
In a first presentation, the safe use of animal proteins in fish food was presented. Now the BSE problem appears
to be increasingly under control, there are more people in favour of using animal proteins in specific applications,
subject to compliance with very strict control procedures.
In a second presentation, a new analysis method for the identification 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
identified, a small piece can be cut off by a highly precise laser, whereupon it is ejected for PCR analysis.
The third presentation was finally 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.
Visit to the Royal Museum for Middle Africa
Digital microscopy
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
identification of ragwort and similar varieties with the help of a software program.
11
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 coffee, organic fruit juice, yucca chips, southern rice pudding and gale beer. In the evening everyone was
invited to the conference dinner in Leuven.
Sustainable development activity
Leuven conference dinner
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 situation and future perspectives of the feed ban.
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-specific 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-specific detection of animal proteins. Included in this is the development of test kits (dipstick, PCR kit), the validation of PCR
methods, the development of confirmatory methods with LC-MS/MS based on collagen detection, the development of a quantitative NIRS/microscopy combination method, the observation of new markers for classic microscopy 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 nutrients. 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
13
NRL-GMO
GMODetec 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, identificatie en kwantificering van genetisch gemodificeerd 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, identification and
quantification 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 sufficient information
(especially on the DNA sequences) is available. The aim here is to develop PCR methods based on the comparative analysis of the available official DNA sequences. The analytical results will be integrated in a mathematical decision model allowing the identification 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-authorized GMOs can be traced by comparative analysis of the generated ‘fingerprints’ 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 flexible 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 identification of the
potential presence of GMOs in food matrices1,2. This screening method is flexible 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 screening methods (plants, soya, maize, oilseed rape, cotton, rice, beetroot, the 35S promoter and the nopaline synthase 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
“Cauliflower 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 fluoresces
14
when it is intercalated in double-stranded DNA ; this chemical agent allows the amplification of the DNAg matrix
to be monitored at each PCR cycle 4.
Figure 1: On the left: an example of amplification 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 system 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 eventspecific TaqMan® 4 method 5.
As CoSYPS is a flexible 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 laboratories 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-filled and lyophilized) in order to simplify and reduce the cost of analysis preparation, thereby making this method accessible to as
many laboratories as possible.
15
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 significant 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 amplified
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
different places, tests with different 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 effect on the signals. The conclusion of this work is that amplification
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 profiles relating to
the different genetically modified events which could be encountered.
5bp primer + inosin
Reverse primer
Unknown region
Known region
Figure 2: The experiments were conducted to find 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.
16
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 fingerprinting; on the other hand the developing
of integrated strategies for determining the composition of a GGO sample, including the detection and characterising of unauthorised GGOs. The second objective is in keeping with the final objective of the project, namely the
developing and optimising of a so-called ‘GGO passport’ identification technology.
The first objective within the project was the developing and further optimisation of a protocol for anchor-PCR
fingerprinting. A new fluorescent 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
refined 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 specific 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 fingerprinting,
which ultimately allows for a confirmation (detection) and identification 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 efficient 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 confirmation 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 fingerprinting 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 specific 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`
configuration (green, blue and black), each with another fluorescent label. The use of primers in a `nested` configuration
is directed towards an increasing of the specificity 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 amplicons allows for a unique identification of an event.
17
Figure 4: Genome walking via anchor-PCR. Amplification of sequences which flank screening elements leads to an
identification 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 effectuated 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 difficult, 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.
18
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Van den Bulcke, M., et al Transgenic Plant Event Detection. PCT/EP2008/051059 [WO/2008/092866]. 2008.
Van den Bulcke M, et al. Anal Bioanal Chem 2009.
Barbau-Piednoir E,et al. European Food Research and Technology 2010;230:383-393.
Tse C, Capeau J. Ann Biol Clin (Paris) 2003;61:279-293.
Community Reference Laboratory (CRL) Status of dossier web-page.
http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm. 2004.
Waiblinger HU, et al. European Food Research and Technology 2008;226:1221-1228.
Reiting R, et al. Journal fur Verbraucherschutz und Lebensmittelsicherheit 2007;2:116-121.
Fernandez S, et al. J AOAC Int 2005;88:547-557.
Höhne M, et al. European Food Research and Technology 2002;215:59-64.
Corbisier P, et al. Anal Bioanal Chem 2005;383:282-290.
Pardigol A, et al. European Food Research and Technology 2003;216:412-420.
Ruttink T, et al. Anal Bioanal Chem 2010;396:1951-1959.
Ruttink T, et al. Analytical and Bioanalytical Chemistry 2010;396:2073-2089.
19
Benzene in foodstuffs
Benzene is a chemical compound used in the industry. It is also found in gasoline and in cigarettes. The International Agency for Research on Cancer (IARC) considers benzene as a substance that is carcinogenic for man.
Exposure to benzene is mainly environmental (traffic, industry, …). Yet, there are indications that certain foodstuffs
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 foodstuffs 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 effect on
the formation of benzene from benzoates. Benzene may also be transferred to foodstuffs as it leaks from packaging 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 concentrations 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 Scientific 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 foodstuffs 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 foodstuffs 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 foodstuffs.
20
COOH
-CO2
acide benzoïque
benzène
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
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 (Adenovirus, 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 specific procedures or methods available for an outbreak to be followed up and to demonstrate the epidemiological relation between infected persons and contaminated foodstuffs.
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 foodstuffs. However, for a number of matrices an optimisation is necessary in order to obtain
a better recovery.
Fig. : The Norovirus
22
Results
Thanks 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 identified 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 outbreak, 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 foodstuffs 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 foodstuffs but also of human samples (faecal and vomit) and can analyse environment samples. 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 sufficient 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 possible, 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 efficient 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 different concentrations (see table 2) for disinfection of the environment.
23
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 floors
*data - Centre for Disease Control – World Health Organisation
Discussion
In 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 foodstuffs were
detected. The transmission of Norovirus occurred mainly by secondary contaminated foodstuffs in which the food
handler is an important source of contamination, or through person-to-person transfer.
References
The 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 shellfish 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). Effectiveness 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
24
Milk and Milk Products
Workshop 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 represented 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 fields” 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 sufficiently pasteurized or not) in different species is now one of the priorities set by DGSANCO.
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 microflora in raw
milk and on the influence of stress conditions on this diversity. Milk that leaves the udder is free of micro-organisms 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 effect 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 microflora 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, psychrotrophic 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 different : bacteria are increasingly less capable of growing as
a group in specific environmental conditions (some groups are inevitably excluded), sometimes “dead” bacteria,
sometimes live bacteria, …
For the implementation of Regulation (EC) No 853/2004 laying down specific 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
25
obtained a good Z score against 82% in 2007. However, six European laboratories (not taken into account for assessing 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 flow cytometry methodology with detection of
bacteria by means of epifluorescence microscopy. In both cases the bacterial clusters are shattered into individual
bacteria, the bacteria are coloured by means of a fluorescent dye and then the coloured bacteria are transferred
to the reading system by means of a sheath liquid. The fluorescent 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 pulses 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
26
of the NRLs. Three European certified organisations specialised in that type of work were mentioned: Microval,
AFNOR certification 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 alternative method that is specific 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 specific standard and an addition to the ISO 16140 standard that may be applied 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 flora in milk, the EU-RL-MMP suggested to write a practical guide with a detailed description of the procedures (both standards ISO 16140 and 13297 combined).
The responsibility of the NRLs with regard to the appropriateness of the conversion relation for the alternative methods (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 final 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. significantly different climates). In practice, the establishment of a conversion relation
is in fact hampered by the difficulty to distribute the same milk samples among different laboratories when the
distance between laboratories is too great. An interesting approach that makes it possible to sidestep this problem 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 (CRAW-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
27
Developments in the field of standards and legislation
New IDF- FIL (International Dairy Federation – Fédération International de Laiterie) standards in 2010 (from 2 November 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 method
ISO 12081|IDF 036:2010 - Milk - Determination of calcium content - Titrimetric method
ISO 6091|IDF 086:2010 - Dried milk - Determination of titratable acidity (Reference method
ISO 6732|IDF 103:2010 - Milk and milk products - Determination of iron content - Spectrometric method (Reference 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 identity
ISO 11813|IDF 156:2010 - Milk and milk products – Determination of zinc content - Flame atomic absorption spectrometric method
ISO 17678|IDF 202:2010 - Milk and milk products – Determination of milk fat purity by gas chromatographic analysis of triglycerides (Reference method)
ISO 26462|IDF 214:2010 - Milk - Determination of lactose content - Enzymatic method using difference in pH
ISO/TS 27105|IDF/RM 216:2009 - Milk and milk products – Determination of hen’s egg white lysozyme by HPLC
ISO/TS 27106|IDF/RM 217:2009 - Cheese - Determination of nisin A content by LC-MS and LC-MS-MS
ISO 29981|IDF 220:2010 - Milk products - Enumeration of presumptive bifidobacteria - Colony count technique at
37 degrees C
ISO 10932|IDF 223:2010 - Milk and milk products – Determination of the minimal inhibitory concentration (MIC) of
antibiotics applicable to bifidobacteria
Other interesting IDF publications in 2009-2010:
Bulletin of the IDF No. 440/2009 - Interlaboratory Collaborative Study on the Kjeldahl Reference Method for Nitrogen Determination in Sheep and Goat Milk
Bulletin of the IDF No. 441/2009 - Monitoring success of paratuberculosis programs Proceedings of 2nd Paratuberculosis Forum, Minneapolis, August 2009
Bulletin of the IDF No. 442/2010 - Current situation & compilation of commercially available screening methods
for the detection of inhibitors/antibiotic residues in milk - E-Form
Bulletin of the IDF No. 443/2010 - Environmental issues at dairy farm level - E-Form
Bulletin of the IDF No. 444/2010 - Feed-associated Mycotoxins in the Dairy Chain: Occurrence and Control - EForm
IDF congresses:
2011_IDF Regional Conference on Domestic Milk Supply and Demand Systems : Lessons from Experience”, 26 - 29
April 2011, Seoul, Korea
2011_IDF International Symposium on Sheep, Goat and other non-Cow Milk, 16 - 18 May 2011, Athens, Greece
2011_IDF/ISO Analytical Week, 23-27 May 2011, Lyon, France
2011_IDF World Dairy Summit, 15-19 October 2011, Parma, Italy
2012_IDF International Symposium on Cheese Ripening and Technology, 20-24 May 2012, Madison, Wisconsin,
USA
28
2012_IDF/INRA International Symposium on Spray Dried Dairy Products, 19-21 June 2012, St. Malo, France
2012_IDF World Dairy Summit, 03-09 November 2012, Cape Town, South Africa
2013_IDF World Dairy Summit, October 2013, Yokohama, Japan
Koen De Reu (ILVO-T&V)
Jessy Claeys (ILVO-T&V)
Koen.Dereu@ilvo.vlaanderen.be
Jessy.Claeys@ilvo.vlaanderen.be
29
Workshops & Symposia
Date
Subject
Place
More information (website)
24-26/01/2011
Rapid Methods Europe 2011 – the 7th
conference
Noordwijkerhout,
The Netherlands
www.bastiaanse-communication.com/RME2011
24-27/02/2011
6th International Conference on
Emerging Zoonoses
Cancún, Mexico
http://www.zoonoses2011.com/
21-24/03/2011
Food Integrity and Traceability
Conference
Queen’s University
Belfast
http://www.conffidence.eu/img/event/event_ASSET_leaflet.pdf
7-10/05/2011
21st ECCMID (European Society of
Clinical Microbiology and Infectious
Diseases)
Milan, Italy
http://www.escmid.org/dates_events/calendar/
8-11/05/2011
LAPRW2011, 3rd Latin American
Pesticide Residue Workshop
Montevideo,
Uruguay
Contact: Prof. Dr. Horacio Heinzen; e-mail: heinzen@
fq.edu.uy
18-20/05/2011
First International Conference on Organic Food Quality and Health Research
Prague,
Czech Republic
http://www.fqh2011.org/
20-24/05/2011
111th General Meeting of the American New Orleans,
Society for Microbiology
Louisiana
gm.asm.org/
24/05/2011
Mycotoxins: Challenges and
Perspectives
Ghent, Belgium
www.mytox.be
19-22/06/2011
4th International IUPAC Symposium for
Trace Elements in Food (TEF-4)
King’s College in
Aberdeen, Scotland
http://www.abdn.ac.uk/tef-4/
24/05/2011
63rd International Symposium on Crop
Protection
Ghent, Belgium
http://www.iscp.ugent.be/
12-17/06/2011
15th Gordon Research Conference on
Mycotoxins & Phycotoxins
Waterville, Maine,
USA
http://www.grc.org/
19-22/06/2011
Second Saskatoon International Valida- Saskatoon,
tion Workshop for Regulatory Analyses Saskatchewan,
of Residues in Foods (SaskVal Workshop) Canada
www.SaskVal.ca
19-22/06/2011
SafePork Conference 2011
9th International Conference on the
Maastricht,
Epidemiology and Control of biological,
The Netherlands
chemical and physical hazards in pigs
and pork
http://www.safepork.org/
20-24/06/2011
2nd Global conference on GMO analysis Como, Italy
http://mbg.jrc.ec.europa.eu
24-25/07/2011
International Food Microbiology
Conference
Chipping Campden,
UK
26-30/06/2011
The 4th Congress of European
Microbiologists, FEMS 2011
Geneva, Switzerland
www.kenes.com/fems/
6-8/07/2011
Euro Food Chem XVI
Translating food chemistry to health
benefit
Gdansk, Poland
http://www.eurofoodchemxvi.eu/
24-29/07/2011
10th International Conference on
Mercury as a Global Pollutant,
Halifax, Nova Scotia,
Canada
http://mercury2011.org/com/
1-6/08/2011
13th 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. China
http://www.ict13.org/
30
07-12/08/2011
57th ICoMST
International Congress of Meat Science
and Technology
21-25/08/2011
23rd. International Conference of the
Buenos Aires,
World Association for the Advancement
Argentina
of Veterinary Parasitology (WAAVP)
http://www.waavp2011-argentina.com.ar/
21-25/08/2011
DIOXIN 2011,
31st International Symposium on Halogenated Persistent Organic Pollutants
POPs’ Science in the Heart of Europe
Brussels, Belgium
http://www.dioxin2011.org/
JF.Focant@ulg.ac.be
Vancouver, Canada
http://www.chro2011.com/Contact_Us.php
New Orleans,
Louisiana
http://www.aoac.org/meetings1/125th_annual_
mtg/main_2.htm
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 developments of MoniQA as an association and will also
provide ample room for discussion with stakeholders. Related EU projects will be invited to contribute and a special session will focus on food safety
in Bulgaria. Including poster presentations and exhibitions, the conference will give an in-depth look
at the following thematic areas: Mycotoxins/Phycotoxins, Food Allergens, Chemical Contaminants,
Microbiological Contaminants, Food Additives and
Processing Toxicants, Emerging Issues.
http://varna2011.moniqa.org/
16th International Workshop on
28/08-1/09/2011 Campylobacter, Helicobacter & Related
Organisms
18-21/09/2011
125th AOAC Annual Meeting &
Exposition
Ghent, Belgium
http://www.icomst2011.be/
27-29/09/2011
3rd MONIQA International Conference
1-4/11/2011
5th International Symposium on Recent Prague,
Advances in Food Analysis (RAFA 2011) Czech Republic
www.rafa2011.eu
1-3/02/2012
Twelfth International Symposium on
Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12)
Bruges, Belgium
http://www.ordibo.be/htc/
Bruges, Belgium
http://www.ordibo.be/htc/
Las Vegas, NV
http://www.aoac.org
Second International Symposium on
31/01-1/02/2012 Hyphenated Techniques for Sample
Preparation (HTSP-2)
30/09-3/10/2012
126th AOAC Annual Meeting &
Exposition
31
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