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Page 1 NRL Contacts Antimicrobial Resistance Zoonoses (Salmonella) Dr R Slowey Listeria Staphylococci Milk & Milk Products Ms B Hickey E. coli (STEC) Dr M Gutierrez TSE’s Dr M McElroy Residues/Chemical Elements Dr S Earley Pesticide Residues Dr J Garvey Campylobacter, TB & Johnes Disease Dr J Egan Animal Proteins Dr S Nolan Exotic Animal Viral Diseases Dr R O’Neill & Dr E Ryan Parasites Dr W Byrne Contagious equine metritis Mr J Moriarty & Dr E Ryan Bovine Viral Diarrhoea Dr Jose Maria Lozano Volume 7, Issue 1 2016 DAFM LABORATORIES BACKWESTON Newsletter Activities of National Reference Laboratories Introduction In 2006, following the designation of a number of additional European Union Reference Laboratories (EURL’s), Member States were required under Article 33 of Regulation 882 / 2004 to designate one or more National Reference Laboratory (NRL) for each EURL. The Departments of Health and Children and Agriculture, Food and the Marine (DAFM), as the Irish Competent Authorities, assigned these NRL functions to a number of laboratories including those within the Backweston Laboratory Campus. The DAFM laboratories also host a number of NRL established under EU or national In this issue reports on: 10 th Workshop of the EURL for Listeria monocytogenes 21 st Workshop of the EURL for Antimicrobial and Dye Residues in Food Euroresidue VIII 10 th Workshop of the EURL Coagulase Positive Staphylococci 19 th Workshop of the EURL for Milk and Milk Products Annual Workshop of the EURL for Chemical Elements in food of animal origin. 11 th Workshop of the EURL Campylobacter 11 th Workshop of the EURL Escherichia coli 10 th Workshop of the EURL Animal Protein Also included a note on the Food Health and the Role of National Reference Laboratories Conference

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Page 1: DAFM NRL Newsletter Volume 6, Issue 1. - Agriculture · Exotic Animal Viral Diseases ... Parasites Dr W Byrne Contagious equine metritis Mr J Moriarty & Dr E Ryan Bovine Viral Diarrhoea

DAFM NRL Newsletter Volume 7, Issue 1.

Page 1

NRL Contacts

Antimicrobial Resistance

Zoonoses (Salmonella)

Dr R Slowey



Milk & Milk Products

Ms B Hickey

E. coli (STEC)

Dr M Gutierrez


Dr M McElroy

Residues/Chemical Elements

Dr S Earley

Pesticide Residues

Dr J Garvey

Campylobacter, TB & Johnes Disease

Dr J Egan

Animal Proteins

Dr S Nolan

Exotic Animal Viral Diseases

Dr R O’Neill & Dr E Ryan


Dr W Byrne

Contagious equine metritis

Mr J Moriarty & Dr E Ryan

Bovine Viral Diarrhoea

Dr Jose Maria Lozano

Volume 7, Issue 1 2016





Activities of National Reference Laboratories Introduction In 2006, following the designation of a number of additional European Union Reference Laboratories (EURL’s), Member States were required under Article 33 of Regulation 882 / 2004 to designate one or more National Reference Laboratory (NRL) for each EURL. The Departments of Health and Children and Agriculture, Food and the Marine (DAFM), as the Irish Competent Authorities, assigned these NRL functions to a number of laboratories including those within the Backweston Laboratory Campus. The DAFM laboratories also host a number of NRL established under EU or national legislation focusing specifically on animal health. In this issue reports on:

10th Workshop of the EURL for Listeria monocytogenes

21st Workshop of the EURL for Antimicrobial and Dye Residues in Food

Euroresidue VIII

10th Workshop of the EURL Coagulase Positive Staphylococci

19th Workshop of the EURL for Milk and Milk Products

Annual Workshop of the EURL for Chemical Elements in food of animal origin.

11th Workshop of the EURL Campylobacter

11th Workshop of the EURL Escherichia coli

10th Workshop of the EURL Animal Protein

Also included a note on the Food Health and the Role of National Reference Laboratories Conference

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Abbreviations: PT- Proficiency Test MS- Member States NGS- Next Generation Sequencing WGS- Whole Genome Sequencing FBO- Food Business Operator CA- Competent Authority CRM- Certified Reference Material


Workshop of the EURL for Listeria monocytogenes, ANSES, Paris, March 15-17

th 2016

NRL Representative: Bernadette Hickey (DSL)

Participants from 27 MS, Norway and Macedonia attended the meeting. Pamina Mika SUZUKI, DG Santé was present on 15

th and 16

th March. Invited

guests were Valentina Rizzi- EFSA, Johanna Takkinen -ECDC and Yi Chen, FDA.

A wide range of topics were discussed including Listeria monocytogenes outbreaks, updates on EFSA and ECDC activities, results on subsampling project and the temperature profile at retail from the EU baseline study on Listeria monocytogenes. Typing methods including the experience of MS in using WGS were also discussed. The meeting participants were briefed on the progress of the establishment of the database for Listeria monocytogenes using PFGE profiles from non-human sources. An update on ISO working groups relating to Listeria was provided.

Epidemiological Context

Valentina Rizzi (EFSA) and Johanna Takkinen(ECDC) presented data from the EFSA and ECDC 2014 Reports. In 2014 there were >2000 confirmed cases. This was an increase of 16% over 2013. The EU notification rate was 0.52 per 100,000 of the population. This increase is of great concern as the infection is acquired from consumption of contaminated RTE foods. Despite the rise in listerosis, Listeria monocytogenes was seldom detected above the levels set in the Regulation (EC) 2073/2005. RTE fishery products, mainly smoked fish are most frequently associated with non-compliances with 4.7% of single samples and 10.6% of batches non-compliant. There were 15 food borne outbreaks in 2015.

The highest rate of fatality was in the male population aged over 65. The relative risk is 100 for women over 65 and 160 for men over 65. There appears to be a higher incidence in the summer months. There is concern over the increasing level

of listerosis as the population is aging: in 2012, 17% of the population were over 65 but by 2060, 30% of the population will be over 65.

EFSA and ECDC have developed databases for the collection of typing information on Salmonella, Listeria monocytogenes and STEC. The ECDC will collect data from clinical isolates and the EFSA database from non-human isolates. The EFSA database will be piloted during 2016. Currently the database is designed to collect PFGE profiles and not sequence data from WGS. EFSA has urged MS moving towards WGS to continue to do PFGE during the transition phase so that data from MS can be compared.

Franceso Pomilio (NRL Italy) presented the use of WGS in tracking an outbreak in Italy. The Italians identified an epidemic PFGE pulsotype. Through the use of epidemiological data including questionnaires some risky foods were identified. WGS was performed on the human isolates and they contained 4 specific genomic islands, all the strains clustered closely and there was practically no differences in the accessory genes. A fast PCR targeting 2 of the genomic islands was developed and used to target isolates for WGS and PFGE typing.

Yi Chen (US FDA) gave an overview of the use of WGS in surveillance in the US. In the US, CDC and the FDA have developed a partnership to sequence every human isolate (CDC) and isolates from food, feed and the environment (FDA). They have a network of Genome TRACKR laboratories feeding sequences to the NCBI website. The kmer tree is updated daily and weekly discussions on clusters takes place between CDC and FDA. The US are using Single Nucleotide Polymorphisms (SNP’s) Cluster Analysis to analyse the data using freely available software. There were >30,000 sequences in the Genome TRACKR database by the end of quarter 2 in 2015 and the numbers are increasing rapidly. Approximately 4,000 are sequences from Listeria monocytogenes. When Listeria monocytogenes is detected, isolates obtained from premises before and after the cleaning are sequenced by FDA, and if the strains after clean-up match the strains obtained prior to clean-up the FDA takes action as cleaning is considered not effective.

Detection/ Enumeration

Lena Barre (EURL) presented results of trials carried out on the effect of the quantity of sample used for the primary dilution and its effect on reducing the measurement of uncertainty. The distribution of microorganisms is heterogeneous in solid matrices.

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The EURL asked for assistance from NRL’s to evaluate the results using 10g and 25g portions of naturally contaminated results. Two NRL’s returned results and the data were evaluated on 12 sets of results. The result obtained from this data set did not support that 25g gave a lower MU than 10g sample portion.

The recommendation is to take sub-samples at different places of the sample to cover heterogeneity; it seems to be more important than test portion size. In this regard the use of a larger subsample i.e. 25g would allow more subsampling from different portions of the product.

Shelf Life Studies

Helen Bergis (EURL) evaluated the temperature data collected from retail during the baseline study to see the temperature distribution in the MS. More than 10,000 temperature measurements were collected during the survey. The temperature of the retail cabinet and the surface temperature were recorded. The products checked in the survey were soft and semi soft cheese, packaged heat treated meat and packaged smoked or gravid fish. The results of the analysis showed that the mean temperature was 4.1°C for cheese and 3.5°C for fish and 3.7°C for meat. There was a 0.2% chance that the product would be outside 12°C. There was no difference in seasonality or geographic location. This analysis of the data was very beneficial and hopefully it can be used to amend the default temperatures in the “EURL Lm Technical guidance document for conducting shelf-life studies relating to Lm”

Characterisation and Epidemiology

This session was mainly devoted to presentations on EU funded projects to assess WGS that are currently on-going and the experience of the Austrian NRL in implementing WGS. Three projects currently in progress were presented. The purpose of all the projects is to harmonise protocols and interpretation.

Ariane Pietzka (AGES, NRL Austria) delivered a presentation on the approach to WGS taken in AGES in Austria. AGES with the cooperation of Munster University have developed and evaluated a typing scheme for Listeria monocytogenes based on core genome MLST (cgMLST). The genome wide gene by gene comparison is carried out on 1,700 core MLST targets. This typing scheme improves outbreak investigations and due to the availability

of cgMLST nomenclature interlaboratory exchange of data is possible.

ISO Updates

Updates on ISO working groups that the EURL are involved in and relating to Listeria monocytogenes were presented:

ISO 11290-1 and -2 Detection and

Enumeration of Listeria spp. including Listeria

monocytogenes. Draft under preparation for

final vote.

Working group on the revision of the ISO

21807:2004 on the Determination of Water

Activity in Foodstuffs.

Working group on Shelf life Studies. In 2014 a

working group under ISO/TC34/SC9/WG19

was set up to establish an ISO Guideline for

conducting challenge tests

A working group on Whole Genome

sequencing for typing and genomic

characterisation under ISO/TC34/SC9/WG25

was set up. The working group is focusing on

DNA and not RNA. The priority is for food

borne bacteria such as Salmonella spp,

Listeria monocytogenes, Shiga-toxin producing

strains of Escherichia coli, Campylobacter

jejuni and C. coli, Vibrio cholerae, V.

parahemolyticus and Chronobacter sazakii


st Workshop of the EURL for Antimicrobial and

Dye Residues in Food - Advances in LC-MS/MS

Methods for Screening and Confirming Antibiotics,

Fougeres, France October 6th

and 7th


NRL Representative: Seán Earley (VPHRL)

Pascal Sanders (EURL - ANSES) welcomed

participants to the annual EURL workshop. The 2-

day workshop was attended by 49 participants; 46

from EU member states, two from candidate

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countries and one from an EFTA country. Frank

Swartenbroux (DG SANTE) sent his apologies for his


Eric Verdon (EURL) gave an overview of the EURL

2015 and 2016 activities including:

Screening Methods

A survey update on micro/immunological

methods (Discussed in the 2015 Workshop in


2015 evaluation of the ‘Explorer' test in eggs.

The biosensor technology / high throughput

screening survey, which is ongoing. This was

partly presented at the 2015 Workshop.

Evaluation of new techniques for rapid

screening of Antibiotics in food -

immunoreceptor / electrochemical biosensors

this includes the Vantix potentiometric

method and an amperometric sensor

developed by Madrid University.

Evaluation of a new multiplex method -

Infiniplex in milk products.

Four rapid method kits for nitrofurans banned

in aquaculture products

Physicochemical Methods

Meat and fish: 70+ multi-analyte antibiotic


Meat and milk: aminoglycosides in kidney,

muscle and milk - discussed at the 2014


Poultry: LC-HRMS for critically important

antibiotics - C3G and C4G cephalosporins -

metabolic profiling of residues conducted in

poultry and eggs - presentation at the 2016 by

Murielle GAUGAIN.

Aquaculture multi-dye LCMS Method, 10 - 15

compounds plus LC-HRMS metabolic profiling

of contaminants in farmed fish.

Proficiency Tests

2015 and 2016 PTs - 2016 Honey and Shrimp.


2016 Workshop on the new multi-residue

screening method presented by Estelle


2017 Planned Visits

The EURL Anses representatives plan to visit the 3

NRLs in Ireland in 2017. Formal notification is

expected in December 2016.

Other Updates

Latest publications/posters/presentations

Updated Anses website was discussed/

presented. Any registered members of the old

website will have to re-register for the new

site (see the Closing Session also).

Publications/presentations/posters etc. from

the 2016 workshop will be available from the

member’s area of the Anses website.

Eric Verdon (EURL – ANSES) presented news from

the commission (in the absence of Frank

Swartenbroux (DG SANTE)) including discussion of

the Repeal of 96/23/EC and new Official Controls

Regulation. Note that the original annexes remain

valid until replaced by updated versions. For

Veterinary Medicinal Products (VMP) residues new

provisions are expected in 3-6 years with updates

to include MS residue monitoring and Third Country

import conditions. Technical discussion in this area

is to start as soon as possible.

Review of 2002/657: Discussion/feedback

conducted at EURL WS in 2016/17 (Berlin, Rome &

Fougères) Member States/Competent Authorities

via NRL expert groups.

Reference Points for Action (RPAs): EFSA general

opinion in 2013; Opinion on Chloramphenicol (CAP)

in 2014; Opinion on Nitrofurans (NF) and Malachite

green (MG) in 2015/16. NFs marker residues to

include DNSH (for Nifursol) - AOZ, AND and DNSH ~

1ppb - health concerns unlikely. Note that Anses

has published articles on analysis of

Nitrofursol/DNSH. RPAs currently confirmed for

CAP 0.3µg/kg; MG 2.0 µg/kg (Sum of

MG/LeucoMG); NFs 1.0 µg/kg (for each AOZ, AMOZ

AHD, SEM and DNSH). Stakeholder consultation etc.

to be carried out before adoption.

Cascade Maximum Residues Levels (MRL):

Discussion on the draft proposal completed.

Although honey bees/honey production remain

sensitive, exclusion of honey from cascade/cascade

MRLs is not legally possible.

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Eric Verdon (EURL - ANSES) presented an update on

revision of 2002/657/EC giving a summary of the

timelines/work to date on the review. Also

discussed was the EURL Fougères questionnaire on

the fitness for purpose of the 2010 EURL Guidelines

for the Validation of Screening for Residues of

VMPs – Any simplification or additions deems

necessary by the NRL network? The outcome of the

questionnaire on the EURL 2010 guidelines was

summarized in the presentation - the majority of

the member states replies were in agreement with

the VPHRL opinion.

The inclusion of guidelines for Physicochemical

Methods and trends in LC-MS/MS, LC-HRMS for

screening was flagged. Additionally the use of a

‘Confirmatory-Only’ approach for screening of

samples was considered (e.g. LC-MS/MS approach)

for inclusion in the screening guidelines. The use of

Global Approaches (POD / Accuracy Profile) was

also considered in the questionnaire although the

majority of responses indicated no comment or no

expertise in applying these.

The following points were made with regard to the

2010 CRL Screening Guidelines:

It will be maintained as a separate guidance


Physico-chemical screening methods should

be included in the guidelines.

Revision every 3-4 years to include/address

new approaches & techniques.

Suggestions for simplification to be


The following points were made with regard to

Commission Decision/2002/657:

Main performance criteria will be kept, with a

more accurate definition of CCβ and reference

to the CRL guideline.

The possible inclusion of a list of suitable

screening methods – most likely to be in the

guideline as opposed to the CD.

Inclusion of ‘Global Approaches’ to be further


A workshop dedicated to consideration of

screening is proposed for 2017 to look at

simplification, the use of global approaches

and any other topics raised by the lab


Jens Hinge Andersen (National Food Institute, DTU

Food, Denmark) presented the building of Standard

Sample Description No . 2 explaining the origin and

overview of Standard Sample Description 2.0 (SSD

2.0). EFSA propose the use of data in the context of

Risk Management and Risk Assessment; this will

require a significant increase in the amount of data

required from laboratories/Competent Authorities.

The SSD 2.0 system is in an XML format and aims to

standardize the analytical data gathered in member

states to allow data mining for trends and potential

risks – the current data submissions are limited and

provide minimal information to EFSA e.g. Compliant

/ Non-Compliant. SSD 2.0 will expand this to looking

at the analysed levels of veterinary residues across

the EU, along with the inclusion of various other

sample data.

EFSA has been liaising with CAs with regard to

approaches to collecting the expanded lab

data and converting to the SSD 2.0 format.

The most common approach to generating the

required data from labs is to utilize LIMS

databases to export the detailed sample

information – there may be requirement to

modify LIMS to incorporate the required

information as per the SSD 2.0 format.

The experience of Denmark in producing lab

data for SSD 2.0 included the inclusion of

additional sample information at LIMS login

and in the sample results: differentiating

between screening/confirmatory LC-MS

methods, inclusion of various limits etc.

The possibility of demands for further sample

information by EFSA in future was also raised.

Eric Verdon (EURL - ANSES) presented a discussion

on possible changes to analytical methods in

response to SSD 2.0.

Current challenges include:

No LIMS in use in some labs – burden of

producing required sample data without LIMS.

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Need to include further sample information in

existing LIMS environments e.g. location,

sample size etc.

A spreadsheet should be made available to

CAs to collect data from LIMS and allow data-

mapping into the XML SSD 2.0 database.

SSD 2.0 will require the inclusion of the LOQ –

additional requirement in validation of

methods (typically CCα and CCβ required as

per 2002/657).

Only Negative Screening results will be

accepted in SSD 2.0, i.e. Positive Screens will

not be entered – the corresponding

confirmatory test results will be required for

any samples that screen positive.

There may be the possibility of amendments

to MRLs based on the data collated by SSD 2.0

with the possible need to extend the

validation from an MRL centered approach to

ALARA levels of detection – this may require

revalidation of methods.

Note that 2017 has been flagged as the

deadline for the inclusion of some results for

SSD 2.0 with 2019 being the target for full

rollout of the SSD 2.0 requirements.

Estelle Dubreille (EURL - ANSES) presented an LC-

MS/MS method for screening 70+ antibiotics in

meat and fish. A Workshop and lab tour were

conducted as part of this presentation. Note that

the screening method excludes aminoglycosides

which is performed using a separate method. A

single extraction approach with the same mobile

phase as the aminoglycosides method is used. The

Internal Standard used is sulfaphenazole; Extraction

entails - 2g of sample into ACN; LC acquisition time

= 15 min/sample; meat validated on AB-Sciex 4000

LC-MS/MS (Fish on ABN-Sciex 5500).

2 QC Spikes are used – Groups A & B; a 2 point

calibration is used (0 and Spike); Positive Criteria as

follows: MRM, S/N >3 and the estimated level of

analyte is > ¼ of the target MRL.

Regine Fuselier (EURL - ANSES) presented an

overview of PT programs 2015 – 2016 discussing

the outcomes from previous PT schemes and the

upcoming 2016 PT for Dyes, Nitroimidazoles and

Chloramphenicol in Prawn (Banned Substance sin

Aquaculture Products). The updated EURL website

and PT portal was also presented and an overview

given of the resources currently on the website –

information on PT schemes, methods, workshops,

standards etc.

Anton Kaufmann (Official Food Control Authority of

the Canton of Zurich, Switzerland) presented

ION_MOBILITY-HRMS based screening of residues

which introduced the radical approaches to

confirmatory analysis of veterinary residues

including the proposed elimination of the use of

physical reference standards in combination with

LC-HRMS methods – represents a significant cost

saving and overcomes difficulty of sourcing certain


Positive ID by accurate mass, relative retention time

predicted by Quantitative Structure Retentive

Activity software – ACD/ChromGenius and the use

of fragmentation patterns with prediction using ‘in-

silico’ approach (computer-based models).

The presentation highlights the approach to this

method using a Waters LC-IMS-Q-TOF (Vion

system). Note that the approach described in this

presentation is at an early stage and improvements

are needed before it can be used as a routine

technique (some issues observed with false

positives for large ions – high m/z ratio).

Andreia Freitas (INIAV, Instituto Nacional De

Investigacao Agraria e Veterinaria, Portugal)

presented screening of antibiotics in milk & muscle

by LC-TOF-MS describing a multiclass method for 36

antibiotics across the classes of Beta-Lactams,

Tetracyclines, Quinolones, Macrolides,

Trimethoprim and Sulphonamides. The method

uses a UHPLC system with AB Sciex Triple Time of

Flight 5600 mass spectrometer. The same

extraction solvent is used for milk and muscle with

an additional defatting step for milk following

extraction. The analysis utilises exact mass and

relative retention time for identification of

compounds with a semi-quantitative approach and

is used as a high throughput screening method.

Further confirmatory analysis is performed using


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Dominique Pessel (EURL - ANSES) presented a

proposal for a collaborative study on criteria for

screening/confirmatory residue control by HRMS

with NRLS equipped with LC-HRMS instruments

which will involve the following:

An inter-laboratory study for screening

veterinary drugs with high-resolution mass


The focus will be on targeted screening.

State-of-the art of HRMS screening methods

to be developed by NRLs.

The study will evaluate the False Negative and

False Positives rates, evaluate Detection Limits

and evaluate applied criteria using exact mass

data. The aim is to also identify/evaluate

trends or difficulties experienced during the

study. The following guidelines were given for

the participant labs:

1. Use your own LCHRMS method whatever the

acquisition mode.

2. Analysis of a set of extracted samples

3. Report the results

4. Report your acquisition method, criteria, and

automatic identification treatment.

The survey will start in December 2016 with

analysis of the samples in May-June 2017, Data

Evaluation over the following 2 months with results

& reports anticipated in November-December


Estelle Dubriel (EURL - ANSES) presented

occurrence of dye residues in aquaculture products

giving a general overview of issues with dyes in

aquaculture, approaches to regulatory controls,

analytical challenges and development and

optimisation of the method. A targeted screening

method using an AB-Sciex 5500 Q-Trap instrument

and the extraction method were detailed in the

presentation. Furthermore a non-selective

metabolomic study for compounds associated with

dye residues, using an LTQ-Orbitrap instrument was

described in the presentation. Work is ongoing in

identifying suitable biomarkers.

Rebecca Moffat (Marine Institute, Ireland)

presented minimizing analytical contamination of

dyes giving a brief introduction to dyes and use in

aquaculture. This presentation focused on a

contamination issue with Victoria Blue in the

Marine Institute and detailed the systematic

approaches used to eliminate the contamination

problem including steps to identify the

contamination source, changes to general lab

housekeeping and management of labcoat use, a

systematic approach to sample and standard

preparation and standardizing the associated

protocols, creating separate/ segregated work and

storage areas. Overall the contamination issue was

resolved using the approaches described above.

Murielle Gaugain (EURL - ANSES) presented non-

targeted analysis for search of biomarkers of 3G/4G

cephalosporins in poultry which looked at a non-

targeted metabolomic approach to determining

biomarkers for Cephalosporins (ceftiofur and

cefquinome) in dosed poultry (laying hens) versus a

negative group. This involved analysis of liver, eggs

and droppings. A generic method was utilised to

extract metabolites from the 3 matrices and the

samples were analysed using full scan high

resolution mass spectrometry. The presentation

detailed the scale of the chemometric and

predictive model approaches to analysis of data

from the control group and the dosed group in

identifying appropriate biomarkers (extracted

metabolites were in the order of 1398 from

droppings, 1734 from liver and 3633 from eggs).

Appropriate biomarkers were identified for

cefquinome and ceftiofur in droppings; however it

was only possible to identify suitable biomarkers for

cefquinome in eggs and liver. In discussion on the

biomarker approach a number of important

considerations were highlighted such as the lifetime

of metabolites following treatment in biological

systems and in test samples; the effect of

metabolites associated with other

antibiotics/veterinary drugs on the biomarkers of

interest; the importance of the matrix in the

availability of suitable biomarkers i.e. different

matrices can have different biomarkers associated

with a particular drug of interest; the need for a

rigorous approach that ensures the robustness of

the selected biomarkers and consider the above

issues in the approach and analysis of data.

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Sophie Mompelat (EURL - ANSES) presented honey

control for specific antimicrobial residues from

biosensors to LC-HRMS. This presentation dealt

with the analysis of the source of false positives for

quinolones seen with cross-reaction on

immunoassay screening by a quinolone-like

compound in the honey of chestnuts and of floral

origin. A large number of positive screens for

quinolones using the immunoassay were found to

be negative on confirmatory analysis by LC-MS,

leading to an investigation to characterize what

may be leading to this phenomenon.

The use of LC-HRMS and a non-targeted analytical

workflow to isolate and identify the compound of

interest in tandem was described. A quinolone

specific extraction method from honey was devised

to ensure extraction of the compound of interest

(the routine generic extraction was not effective in

extracting the compound of interest). Using

fingerprinting and metabolomics methodology, the

ions of interest were identified and the structure of

the candidate compound of interest was elucidated

and predicted to be Kyenuric Acid. Further

validation work involving samples spiked with

kyenuric acid is to be conducted in addition to

confirming the association of this compound with

chestnut or multifloral honeys.

Gitte Geertsen (Danish Veterinary and Food

Administration) presented multi antibiotic analysis

in honey by LC-MS/MS, describing a Multi-Class

Screening method. The scope of analytes covers 45

compounds in total and covers the following

antibiotic classes: Lincosamides, Quinolones,

Macrolides, Tetracyclines, Pleuoromutilins and

Sulphonamides. A specific route is used for

Sulphonamides (Acidic Extraction) and another for

the remaining antibiotic classes (McIlvaine Buffer).

The 2 extracts are combined on an Oasis HLB SPE

cartridge before washing & elution. The scope of

analytes covers 45 analytes in total and covers the

following classes: Lincosamides, Quinolones,

Macrolides, Tetracyclines, Pleuoromutilins and

Sulphonamides. LC-MS/MS analysis is conducted

with a Waters Acquity with TQD MS/MS.

The validation results were documented as part of

the presentation, including information on Cut-Offs

and proficiency test performance and the

prescribed other screening criteria: RT, RRT, Ion

ratio were demonstrated. Confirmatory analysis of

the analytes is performed using standard addition

with a compound/class specific method; this

approach is currently undergoing validation.

Eric Verdon (EURL - ANSES) Closing session and

updated EURL Anses-Fougères website & workshop

conclusions. The closing session involved discussion

of the updated Anses website and reminded of the

need to re-register if previously registered with the

old website.

A mini tutorial for registration to Anses Fougeres

EU-RL New Website and an overview of the new

website were presented. The new website URL is:


A summary of conferences in 2015/2016 and

upcoming conferences/workshops was presented

to the attendees with upcoming events highlighted

as follows:


SMMAP 2017 (Spectrométrie de Masse,

Métabolomique et Analyse Protéomique 2017,

congrès), 2-5 October 2017, l’Hôtel New-York®,

(Disneyland® Paris, Marne-la-Vallée-Chessy),


Metabolomics 2017, 26-29 June, Brisbane,

Australia, http://metabolomics2017.org

Conference on Personalized Food and Nutritional

Metabolomics for Health, May 17& 18, 2017, The

Ohio Union, The Ohio State University




IHC (International Honey Commission 2016)

Antalya, Turkey - October 2016 postponed April


International Dairy Federation analytical week (IDF

2016) Madison (Wisconsin, USA) May 2017


5th International Conference on Biosensing

Technology Riva del Garda May 2017

7th Euro Biosensors & Bioelectronics Conference

Berlin July 10-12, 2017

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AOAC-Europe section symposium Barcelona (Spain)

postponed to 2-3 March 2017

EURACHEM / AOAC-Europe symposium:

“Uncertainty in Qualitative and Quantitative

Analysis” Nicosia (Cyprus) 29-30 May 2017

131st AOAC Int. Annual Meeting Atlanta (USA) 24-

27 September 2017

Eurachem 9th International Workshop on

Proficiency Testing Slovenia October 2017

8th International Symposium on Recent Advances

in Food Analysis (RAFA 2017) Prague November


Euroresidue VIII, Hotel Zuiderduin, Egmond aan

Zee, The Netherlands 22-25th

May 2016

NRL Representative: Seán Earley (VPHRL)

There was a large international attendance at the

Euroresidue VIII meeting (over 300 delegates) with

growing numbers of attendees from non-EU

countries/regions including The Philippines,

Thailand, Malaysia, Korea, the Middle East, New

Zealand, Australia and South America.

Overall there were 28 Oral Presentations and 6

Keynote lectures with an additional 206 Posters on

display over the course of the conference.

Presentations and Posters are available at the

Euroresidue website: www.euroresidue.nl

Introductory Workshop –presented by Dr Gaud

Dervilly-Pinel and Prof Dr Bruno Le Bizec.

This was designed as a practical session and

opportunity to get to know other attendees. It

included discussion of particular areas of interest at

the Euroresidue meeting and what attendees

hoped to gain from attendance.

The workshop covered the following topics:

Introduction to regulatory bodies, Food &

Feed Safety regulations.

EURL/NRL network.

Discussion of common analytical techniques

and emerging analytical strategies -

advantages & pitfalls and of new analytical

approaches (LC-TOF, HRMS, OMICS-

biomarkers as an alternative/complementary

approach to analysing for specific drug


Discussion of 2002/657 - current form and

points for review - proposed changes to RRT,

Spectrometric Criteria (SN & ID points) and

consideration for multi-residue methods.

Future Challenges in the area of residues analysis

were also discussed, these include:

New Natural Hormones.

Low Dose remedies - need for increasing

analytical sensitivity

Designer Drugs (compounds analogous to the

recently observed Novel Psychoactive

Substances/ 'Headshop Drugs' used for

recreational purposes).

Genetically Modified animals/feed.

Day 1 - Conference Presentations

The Sessions on Day One were:

S1. Antibiotics, Residues and Resistance

S2. Residues and the Environment

The potential analytical requirements and

expansion of matrices in light of these issues were

explored. There was some discussion on the

potential contribution of biocides and heavy metals

(arsenic, copper, silver) in the environment to

antibacterial (AB) resistance. Note that

tetracyclines were a recurring topic of discussion as

they are frequently detected in residues analysis.

Several studies were presented involving LC-MS

analysis of manure to determine AB levels and the

most effective approaches to reduction of ABs in

manure/waste, for example devising new

approaches to fermentation of waste.

Also presented was the potential use of High

Resolution Mass Spectrometry techniques to

replace current screening approaches i.e. 6 Plate

Test for antibacterials. The need for a harmonised

global approach to AB control was raised on several


Day 1 - Presentations of Note:

Keynote lecture 1: Prof Dr Dan Andersson, Uppsala

University Sweden - Evolution of antibiotic

resistance at very low antibiotic concentrations.

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Keynote lecture 4: Dr Jin-Wook Kwon -

Management of antimicrobials in the environment:

What have we learned and what should we prepare

for the future?

Oral 2: Dr Brigitte Roudaut – Contribution of

LC‐MS/MS methods to detection and identification

of antibiotic residues in meat – Application in

official control in France.

Oral 4: Dr A. Cannavan - Global perspectives on

antimicrobial resistance in the food chain.

Oral 7: Dr Steven Crooks - An investigation into the

sources of contamination of cattle with the

veterinary drug phenylbutazone.

Oral 8: Dr Tina Van Den Meersche - Quantification

of five different classes of veterinary antibiotics in

(processed) swine manure using a validated UHPLC-

MS/MS method.

Day 2 - Conference Presentations

The Sessions on Day Two were:

S3. New Analytical Techniques, Confirmatory


S4. Alternative Matrices

S5. Validation and Criteria Approaches

Presentations and discussion of several newer

approaches in the area of LC-MS were conducted

including - Supercritical Fluid Chromatography, Ion

Mobility/MS, DESI, Paper Spray and miniaturisation

of LC-MS instruments for field testing. Also

discussed were approaches to non-targeted

screening with the use of databases such as

Chemspider (www.chemspider.com) as a resource

to support non-targeted approach.

With regard to new matrices, the potential use of

hair and feathers for official controls was discussed.

This is due to presence of residues for significant

timeframes following withdrawal and these may be

of particularly use in the context of banned

substances. Also explored was the possibility of

insects as a growing protein source and the

potential regulatory/analytical implications. Also

discussed was the analysis of saliva for ABs and

chicken feet for tetracyclines.

On the subject of Validation and Criteria

approaches Keynote 6: Update to 2002/657/EC -

the Rikilt Collaborative Project, suggested changes

to 2002/657/EC to address multi-residue

approaches to include changes to Resolution, RRT

and Ion ratio criteria. It was also proposed that

2002/657/EC becomes a 'living' document with

regular updates (~biannual) to reflect analytical


Also of particular interest was Oral 19: Discussion

on Chloramphenicol PT 02256 - CAP stereoisomers

& ELISA method, which suggested an approach to

overcome issues observed with stereoisomers of


Day 2 ended with a Workshop/Panel Discussion on

Risk Based Approaches to Monitoring, the main

points being:

Possible criteria for Risk Analysis approaches

to monitoring based on production numbers;

quantities of imported food/feed; substances

for inclusion in control approach; sampling

frequency; screening approaches and future

testing programmes; consideration for imports

from third countries and developing countries;

changing/developing markets; consider

possible alternatives to using a ‘percentage’ of

production volume.

It was noted that a consensus needs to be

reached on the definition of a risk-based

approach and proper assessment of the

perceived risks.

Euroresidue will publish an article arising from

the Risk Assessment workshop.

Day 2 - Presentations of Note:

Keynote lecture 5: Prof. Dr Bruno Le Bizec, Laberca, Nantes, France - An overview of latest advanced technological options for residue analysis.

Keynote lecture 6: Dr Bjorn Berendsen, Rikilt Wageningen, the Netherlands - Time for an update! A unique collaborative study to assess confirmatory analysis performance criteria in veterinary drug residue analysis.

Oral 9: Dr Anton Kauffman, Zurich, Switzerland - Ion mobility coupled to high resolution mass spectrometry: The possibilities, the limitations.

Oral 11: Dr Roberta Galarini, Italy - Confirmatory multiclass method for residues of antimicrobials in milk by LC-HRMS/MS.

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Oral 12: Robin Wegh Msc, Rikilt Wageningen, the Netherlands - Hair and feathers: the matrix of choice for antedating the use of antibiotics, beta-agonists and steroid-esters?

Oral 13: Dr Wim Reybroeck - Testing of Saliva as ante-mortem screening for antimicrobials in pigs.

Oral 14: Dr Eline Kowalski – Insects on your plate: monitoring chemical contaminants and residues.

Oral 15: Dr Javiera Cornejo - Depletion study of oxytetracycline (OTC) and 4-epi-oxytetracycline (4-epi-OTC) residues in claws of broiler chickens by liquid chromatography tandem mass spectrometry.

Oral 18: Dr Katrin Kittler - Investigations on the influence of hydrolysis on the total amount of marker residue and the consequences.

Oral 19: Dr Mark Sykes - Chloramphenicol Proficiency Tests on a Global Scale – Unforeseen Consequences.

Day 3 - Conference Presentations

S6. & S7 – Both sessions covered the area of Broad

Screening with topics in the areas of:

Screening, Un-Targeted Screening and

OMICS/biomarker detection approaches.

Histopathological approaches to monitoring

growth promoters.

Approaches, challenges and a number of examples

of new screening applications were discussed over

the course of the oral presentations. The use of

accurate mass/full scan spectrometry technology

was highlighted as the main tool in untargeted

screening and OMICS/biomarker research. OMICS

approaches aim to identify unique biological

markers that are associated with the application of

banned or controlled veterinary

residues/medicines. These can include metabolites

of the compound or biological molecules/ markers

that are associated with exposure to the veterinary

residue, such as proteins, lipids or peptides.

Challenges associated with this approach include

the identification and isolation of these biomarkers

amongst the many chemical entities observed in

biological matrices and ascertaining that they are

uniquely linked to the veterinary residues in

question. The development of an appropriate

sample preparation procedure is also one of the

main challenges - an example includes enzymatic

hydrolysis of the sample followed by solid phase

extraction techniques to remove any interfering


The use of histopathology (Dr Mario Botta) as a

complementary tool to monitor the use of growth

promoted was discussed, indicating the approach

and histological criteria that may be associated with

administration of growth promoters an example of

which is an increased presence of lesions, i.e.

induced squamous metaplasia of glandular tissue

on administration of sex hormones (Estrogen,

Androgen and 17β-E2 + Androgen).

Of particular practical interest was the presentation

by Lieven Van Meulebroek on the use of a

biomarker to discriminate between semi-

endogenous and exogenous thiouracil (TU) in cattle,

which identified 4 potential biomarkers for

endogenous TU in calves and 2 potential

biomarkers in calves – note that further

investigation and validation is required with this

approach. In future this may prove useful for

overcoming the issue of discrimination of low levels

of TU (< 10ppb) observed in some samples that may

or may not be associated with the consumption of

rapeseed/cruciferous plants as opposed the illegal

administration of TU.

Day 3 - Presentations of Note:

Oral 21: Dr Kathrin Schmidt - Application of LC-QTOF technology for screening for hormonally active substances in matrices of animal origin.

Oral 22: Dr Roberto Stella - Targeted proteomics for

dexamethasone treatment in bovines.

Oral 23: Dr Mario Botta - The histopathological

approach for the monitoring of the illegal

administration of growth promoters in food

producing animals.

Oral 24: Dr Pilar Marco, Spain - Site-encoded DNA

strategies for residue analysis.

Oral 25: Jérémy Marchand, Laberca, Nantes, France

- Lipidomics: an alternative and complementary

tool to highlight biomarkers of growth promoting


Oral 26: Dr Marco H. Blokland, Rikilt, Wageningen,

the Netherlands - The Dutch approach for the

detection of (synthetic) natural steroids in the

Netherlands: A retrospective overview.

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Oral 27: Dr Gaud Dervilly-Pinel, Laberca, Nantes,

France - Are biomarkers universal and transferable?

Oral 28: Lieven Van Meulebroek - Discovery of a

urinary biomarker to discriminate between semi-

endogenous and exogenous thiouracil in cattle.

Parting Comments:

The International Symposium on Hormone and

Veterinary Drug Residue Analysis is to be held in

Ghent, Belgium was announced for May 2018.

A preliminary announcement for The 4th


International Workshop on Validation and

Regulatory Analysis of Residues in Foods in

Saskatoon, Canada was made for 2019.


Workshop of the EURL Coagulase Positive Staphylococci (CPS) dedicated to Staphylococcal Enterotoxins (SEs), ANSES Paris, France, 25



May 2016

NRL Representative: Regina Heslin (DSL)

43 participants from 27 NRLs were represented at

the workshop, including the members of the

working group on the use of Next Generation

Sequencing for bacterial/CPS typing and of Maldi-

tof for bacterial/CPS characterization and

identification. The DG SANTE representative Sylvie

Coulon was unable to attend. The workshop was

divided into sections by the following topics;

General, Recent Staphylococcal Food Poisoning

Outbreaks (SFPO’s), NRL Network and Analytical

works on Staphylococcal Enterotoxins (SE’s).

Recent Staphylococcal Food Poisoning Outbreaks

Table 1 Summary of the outbreaks presented by 4 MS Country

No. people ill/exposed population

Event where food served

Implicated Food

CPS/g SE Detected VIDAS/ Rida-screen

Genes Encoding for SE detected

Cyprus 2/10 Take away




SEA 2.15ng/g

Sea, seh

Cyprus 13/26


Pasta dish




Sea, seh

France School

Pork chop





Ireland 44 Flight Mousse dessert

>105 SEA


Sea,sed, seh,ser,seg,seisej


2 Street vendor

Roasted chicken legs

SEA 0.033ng/g


Giusi Amore EFSA also gave a presentation on

SFBOs and noted that a large proportion of them

are due to foods other than dairy products. EFSA

will start a discussion with the BIOHAZ panel on risk

assessment of CPS/SE’s in foods other than dairy


New Technologies

A working group met in advance of the workshop to

discuss new technologies for CPS characterisation

and identification. A summary of the advantages

and disadvantages of the NGS and Maldi TOF

technologies is presented in Table 2.

Table 2 Advantages and disadvantages of new technologies for CPS characterisation and identification

Technology Advantage Disadvantage

NGS Multiple results derived on a single experiment (AMR, Virulence genes, etc.)

Need of technical facilities, expertise and skilled personnel

Epidemiology (outbreak investigations, strains similarity, same origin)

DNA extraction (food samples)

Increased typing resolution & range of data

Data analysis: Need of standardization of data analysis protocols.

Access to "all" genomic data

Current lack of reference sequences.

Retrospective analysis for new targets

Storage (costly)

Portability of results Need of criteria to be used for differentiation of strains (outbreak investigation)

Comparison of data by different labs. Robustness and standardization of laboratory workflow

Only genotype available. Conventional microbiology still necessary => phenotype (e.g. biochemical properties, data on gene expression)

Maldi TOF Useful and accurate for strain identification (species)

Limitation of reference database (not all bacteria yet, some only

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to genus level, difficulties with rare species)

Good alternative when failure of identification with other techniques (biochemical galleries)

Expensive equipment (with dedicated room)

Time saving and Cost efficient analysis (when the instrument is available)

Skills for the sample prep (extraction step => standardization)

Possibility to process a large amount of samples

CPS Characterisation

The EURL CPS developed a multiplex real-time PCR

scheme for the detection of 11 se genes and a

target gene specific to S. aureus (nuc) and carried

out an in house validation and an interlaboratory

validation study in 2015. This method is very useful

for the investigation of SFPOs, as it is often

necessary to detect the presence of se genes in CPS

strains, in addition to SE detection in the suspected

food, in order to confirm the identity of the food

responsible for SFPO. However, a large variability

between the 16 participating labs was observed. As

unsatisfactory results were obtained for several

labs for control DNA samples & strains, the method

could not be validated.

Optimisation of the test method is required for

different manufacturers of instruments.

Another interlaboratory validation study will be

organised following training and test optimisation.

Study on the Prevalence of SE genes

Bacterial toxins are ranked after Salmonella as the

leading cause of food borne illness. Results of

studies from Italy and Poland were presented.


481 milk & cheese samples were studied in Italy.

SEA toxin was the toxin most frequently observed

followed by SED.

The most frequent SE gene found in the isolates

was SER, together with SED and SEJ, as these are

carried on the same plasmid. SEG together with SEI

were observed in 18% of isolates.


115 samples of raw milk were collected between

2009 and 2013 from 15 dairy farms and 15 dairies

located in the eastern part of Poland. CPS were

found in 71 of 115 (62%) analysed milk samples.

29% strains had enterotoxigenic properties with the

following genes identified in order of prevalence:

sed, ser, sej, sei, seg, sep, seh, sec and sea. The

genes encoding SEB and SEE toxins were not


57% isolates were susceptible to all 10

antimicrobial agents studied. The highest resistance

rate was found to Penicillin. No MRSA positive

strains were identified.

CPS Enumeration

Overview of recent PT trials for CPS

The EURL CPS organised an inter-laboratory trial on

CPS enumeration for evaluation of the ability of the

NRLs to apply satisfactorily the reference methods

for the analyses performed in the frame of controls

prescribed by EC Regulation 2073/2005.

The PT trial was performed according to the EURL

quality management system and allows them to

comply with ISO 13528 for statistical analyses.

There are conflicting requirements between

standards EN ISO 6888-1 maximum number of

colonies per plate is 150, while it is 100 in the

general Standard EN ISO 7218. The EURL will refer

this to the project leader for ISO 6888-1.

Update on ISO Standardisation

An amendment to ISO 6888-1 to permit the use of

RPFA for confirmation has progressed to DIS vote.

A query arose as to whether the target of EN ISO

6888 series is CPS or S. aureus. It was favoured to

maintain CPS as a hygienic parameter in Regulation

2073/2005 as confirmation for S. aureus would

involve PCR or Maldi TOF.



Overview of recent PT trials

NRLs have to implement the EURL Screening

method for the detection of SE’s in food matrices

and participate to the ILPT organized by EURL. A

total of 31 (29 in 2016) NRLs participated to 4 ILPTs

and analysed eight food matrices contaminated

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with four types of SE (SEA, SEC, SED, and SEE) at

different concentrations.

The rates of discrepancies identified decreased

from 2013 to 2016: NRL network for SE detection is

competent. 100% satisfactory results were

obtained in the last 2 PT’s.

EN ISO 19020 detection of SE in Food Matrices

Jacques Antoine Hennekinne (EURL CPS) was the

project leader on the development of a CEN / ISO

standard method for the Detection of SE in food

matrices. NRLs participated in interlaboratory

studies for the development of the method.

The Standard is expected to be published in June


Data was generated for 3 kits Vidas, Ridascreen and

Tecra. 3M have stopped the marketing of the Tecra

kit that was included in the studies.

Certified Reference Materials.

Certified reference material (CRM.s) are now

commercially available for Staphylococcal

Entertoxins from IRMM

Work Programme for 2017

The work programmes are now decided biannually

and EURL unsure if changes can be made to include

optimisation of real time PCR for the detection of

SE and optimisation of PFGE for CPS typing.


th workshop of the EURL for Milk and Milk

Products (EURL MMP) ANSES Laboratory for Food Safety, Maisons-Alfort, Paris 5-7

th October 2016

NRL Representative: Eoin O Brien (DSL)

General information

NRL’s from 27 Member States (MS) were

represented, Norway and Switzerland also

attended. 3 experts, Luisa Pellegrino (IT), Vesela

Tzeneva (NL) attended for sections. Sylvie Coulon

DG SANTE attended on the final day.

A wide range of topics were discussed including

updates on Irish Dairy Federation (IDF) / ISO

activities relating to the workshop, activities on

topics relating to total bacterial count and somatic

cell count in raw milk, and phosphatase activity.

Working group on a single European conversion equation for TBC in raw milk

Nathalie Gnanou-Besse

(ANSES) gave a presentation on the ANSES report

on the assessment of a unified European

conversion equation for TBC in raw milk.

To date most European countries have

implemented national equations for the conversion

of IBC readings from instrumental methods to TBC

(cfu/ml) for assessment of compliance with the

regulatory requirements for raw milk set down in

Regulation (EC) No 853/2004.

At the 2011 workshop it was agreed to form a WG

to look at the possibility of introducing a

harmonised European equation for the conversion

of IBC to TBC. Data was collected from MSs to allow

the comparison to take place and the EURL

produced a Technical Report on the outcome of the

study. A statistical analysis of all data by the EURL

lead them to conclude that a unified conversion

equation could be derived at EU level for cow’s

milk, irrespective of the type of flow cytometer.

Prior to the workshop MSs were asked to carry out

an assessment of the impact of the harmonised

European equation on the regulatory limits

established by Regulation (EC) No 853/2004 and

also its impact on the payment systems in

operation in MSs.

Switzerland and Italy made detailed presentations

on the impact at national level; Switzerland found

that there was not a big impact for them but noted

that payments were affected slightly. Italy noted

that the big impact was that at the higher levels but

no big difference at the legal limits.

Belgium noted big differences between the national

and harmonised European equations and predicted

a big impact at all levels. They made the point that

the impact of the use of preservatives by some MSs

needs to be fully evaluated.

Denmark concluded that it would have little impact

and commented that following consultation with

their largest milk purchaser (Arla) that company

would be implementing the harmonised equation

across all its operations to facilitate and simplify


Ireland noted increases of approximately 8 and 14%

for the BactoScan and Bactocount respectively at

around the legal limit of 100,000 cfu/ml. At high

levels there would be reduction in values using the

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harmonised equation. Based on the limited

information on milk payment systems available to

the NRL is could be seen that some milk producers

would move to higher penalty bands if the new

system was to be implemented. The NRL does not

have information on the quality (TBC) of Irish milk

and it is therefore difficult to make a detailed


It was agreed by the WG that the report could be

improved and the EURL are to circulate a corrected

version of the report to NRLs that will include a new

calculation tool to assist NRLs in examining the

impact of the harmonised equation.

Overall it was felt that given the feedback from MSs

and the fact that some MSs don’t have a single

national conversion factor it would be difficult to

require MSs to use the harmonised equation,

instead it could be implemented on a voluntary


ANSES opinion on French draft decree concerning the labelling requirements for raw milk intended for human consumption.

Pauline Kooh (ANSES) gave a presentation on an

ANSES opinion on a French draft decree concerning

the labelling requirements for raw milk intended for

human consumption.

In France the sale of raw milk for direct

consumption is possible only after authorisation by

the CA. The draft decree has set the following

criteria for milk:

Listeria monocytogenes – absent

Salmonella – absent

E. coli as process hygiene criteria

TBC < 50,000 cfu/ml

Max 4oC storage temperature

Shelf life maximum of 3 days

Boil before consumption for the at risk groups

The draft decree proposes the following labelling

statement: "Boil before consumption for children

under five years of age, pregnant women and

people whose immune system is weakened".

Information was provided that the latest EFSA

opinion on raw milk identifies that Campylobacter is

responsible for the highest number of outbreaks

concerning raw milk.

Overall ANSES concluded that the burden is difficult

to estimate but can be considered low, and while

severity can be high for at risk groups adequate

labelling can reduce the risk.

Therefore, the Agency considers it necessary to

inform consumers about the need to boil raw milk,

especially for vulnerable populations.

EURL presentation on the critical points for the

reference method for Somatic Cell Counts (SCC) /

SCC Reference methods critical points

EURL gave an update on a study of the critical

points that can affect the implementation of the

standard reference method EN ISO 13366-1

(Microscopic method), this followed on from a

request for such a study at the 2014 workshop.

The finding is that there is no significant deviation

where using 40X or 50X magnification. The use of

40X will be allowed in the next PT.

There will be no changes to the ISO method but

rather clarifications will be made on different

points. The conclusions regarding the magnification

levels will be transmitted to the IDF/ISO WG that

looks at this.

Vesela Tzeneva, QLIP Netherlands, Microval certification of flow cytometers for Total Flora and SCC in raw milk

There are two EURL guidelines on the validation of

the above instruments as the EN ISO 16140

protocol was unsuitable for evaluating these

instruments. Microval validations for total flora

were carried out on both the Bactoscan from FOSS

and Bactocount from Bently and comply with the

EURL documents and were awarded certification.

Microval validations for SCC instruments started in

2015 and it’s expected that certification will be

awarded at the beginning of 2017.

Reinhart Zeleny, IRMM, provided an update on the

project to develop certified reference materials

(CRM) for SCC in milk on behalf of the Institute for

Reference Materials and Measurements (IRMM)

Geel, Belgium. The Institute is still carrying out

studies on the best way to produce the material.

The main use of the CRMs will be to calibrate

secondary reference materials. Good progress is

being made and the freeze dried milk based

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materials developed so far have been found to be

stable over 17 months.

The goal of the project is to:

Enable calibration of measurements within range 50,000 – 1,000,000 cells/mL (cow milk) by making available two milks, 50,000 cells/mL (L) and 1,000,000 cells/mL (H), sufficient quantity to prepare calibration line samples by mixing L and H accordingly to provide 9 calibration levels

Improve comparability of measurement results

Link fluoro-opto-electronic results with microscopy results

Cross-calibrate secondary materials to the CRM

IRMM are in discussion with NIZO and Acatalia on

the best methods for producing large batches of the

reference material by spray drying and freeze


Hanene Ghezzal (EURL) on phosphatase in cheese and the publication of ISO 11816-2

The new ISO standard 11816-2 on alkaline

phosphatase in cheeses was published in August

2016, it is planned that EURL will now write to DG

SANTE and recommend the establishment of a legal

limit of 10 MU/g for ALP in cheese. DSL participated

in the inter laboratory trials related to the

establishment of this limit.

Luisa Pellegrino (University of Milan) on ALP in


An update was given on the work carried out to try

to optimise the flourophos method for use in

determining ALP in cream following on from a

request from DG SANTE raised by Ireland.

The experimental plan looked at the following areas

The ALP levels in different types of cream on the Italian market

Evaluation of some critical steps in ISO 11816-1/IDF 155-1 when applied to cream

Evaluation of ALP reactivation in cream during storage.

The following graphs were presented to the

meeting and show big increases in ALP levels during

storage at room temperature and at temperature

as low as 8 degrees C.

Graph of APL level vs. days to expiration

Graph of ALP level against holding time (H) at room

temperature after storage initially at 4 and 8oC

Due the reactivation issues encountered the

feasibility of this project is now in question and

EURL are to discuss the future of this work item

with DG SANTE.

Validation of an ALP Flourometric Microwell Assay

Thomas Berger of Agroscope Switzerland gave a presentation on the validation of a Flourometric Microwell method for the determination of alkaline phosphatase in milk and milk products.

The current reference method ISO 11816-1 is based on a proprietary method with the instrument and reagents supplied by Advanced Instruments.

This method has the following requirements:


Fluorescence microplate reader (e.g. Molecular Device, Biotek, Labtech, Millipore…) capable of temperature control (37°C) and running kinetic analysis mode.

Excitation 365 nm and emission wavelength of 450 nm.

Black 96 well plates

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Substrate 4-methylumbelliferone-phosphate (4-MUP) dissolution in diethanolamine (DEA) buffer, pH 9.2

Standard 4-methylumbelliferone (4-MU) dissolution in 2-amino-2-methyl-1-propanol buffer (AMP), pH 10.1

Both the EURL and Agroscope are reporting good comparisons between the reference method and the microplate method

Activity Fluorophos = 0.5 x Activity Microtiter Assay

It is expected that a draft protocol will be available by end of 2016 and the EURL will seek expression from NRLs with the necessary equipment to participate in an inter laboratory trial.

Annual Workshop of the EURL for Chemical

Elements in Food of Animal Origin (EURL-CEFAO),

Rome, Italy, 28th September 2016

NRL Representative: Josephine Coloe (VPHRL)

Laura Ciaralli (Director of the EURL-CEFAO in

Rome) opened the 2016 workshop by welcoming all

participants, including the invited guest speakers.

The workshop was attended by 39 participants

which included 30 representatives of EU NRLs from

26 MSs, 6 representatives from laboratories outside

the EURL-CEFAO network, 9 staff from the EURL-

CEFAO, and 4 invited speakers, including a

representative from the European Commission. This

workshop covered a range of relevant topics

including developments in EU legislation, current

and future proficiency testing (PTs), selection and

use of reference materials, critical points in mercury

determination by Direct Mercury Analysis,

METROFOOD-RI for promoting metrology in food

and nutrition, determination of inorganic arsenic in

mussel tissues by HPLC-ICP-MS and a working group

on CD 2002/657 Revision led by Andrea Colabucci

(EURL-CEFAO in Rome).

Frank Swartenbroux, European Commission

There are ongoing discussions related to certain

heavy metals. The first is Chromium. The current

scientific opinion from EFSA on the risks to public

health from the presence of chromium in drinking

water concludes that overall the calculated MOEs

(Margin of Exposure) indicate low concern

regarding Cr(VI) intake via drinking water (water

intended for human consumption and natural

mineral waters) for all age groups when considering

the mean chronic exposure values with the

exception of infants at the upper bound (UB)

exposure estimates. No action is needed at the

moment for chromium in food due to

environmental contamination.

The second heavy metal discussed was Nickel.

Overall the CONTAM Panel (EFSA Panel on

Contaminants in the Food Chain) concluded that at

the current levels of acute dietary exposure to Ni,

there is a concern that Ni-sensitized individuals may

develop eczematous flare-up skin reactions. There

are discussions with MSs with regard to the

monitoring recommendation (EU) 2016/11 with a

potential time frame 2016-2018 to propose

possible risk management measures. The main food

groups that are recommended to be monitored for

Ni are cereals and cereal based products, infant

formula and baby food, food supplements,

legumes, oils and seeds, milk and dairy products,

alcoholic and non-alcoholic beverages, sugar and

confectionary, fruits and vegetables, dry tea leaves

and bivalve molluscs.

The third heavy metal discussed was Mercury. The

current scientific opinion from EFSA on the risks to

public health from the presence of mercury and

methylmercury in food is that high fish consumers,

which might include pregnant women, may exceed

the TWI (total weekly intake) by up to

approximately six-fold. Unborn children constitute

the most vulnerable group. Biomonitoring data

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from blood and hair indicate that methylmercury

exposure is generally below the TWI in Europe.

There may be changes to legislation on mercury in

the future to concentrate all legal standards in

mercury in a single legislative act. Current ML’s for

fish are 0.5mg/kg and 1mg/kg for predatory fish but

maybe changed to 0.3mg/kg for fish and 2mg/kg for

swordfish and shark. Food supplements are

maintained at 0.1mg/kg. The current state of play

with regard to mercury is technical discussions with

MSs and stakeholder consultations are finished and

this has been well received. Internal consultation

will start soon with some legal issues to be checked.

There is a current review of Commission Decision

(CD) 2002/657/EC and NRL input is via EURL

workshop circuit and Member States competent

authorities input via residues expert group.

With regard to the Official Controls Regulation (EC

882/2004) there is a political agreement with

adoption expected by end 2016, with a repeal of

96/23/EC with the timeframe for this expected to

be between 3 – 6 years.

Timo Kapp (Federal Office of Consumer Protection

and Food Safety, Berlin) Do we need fresh samples

for proficiency testing? Report on two PTs with pig


Timo Kapp discussed two PTs performed with pig

liver: The first exercise was done in 2011 with a

lyophilised sample, while in 2016 a deep-frozen

fresh material was used. While the first material

was easy to store and to work with, the second one

was assumed to be more representative for routine


The fresh material was prepared at the NRL from

liver of four individual animals. For some elements

(Cd, Cu, Hg, Mn, Mo, Se, Zn) the natural contents

were left unchanged, while for other elements (Ag,

As, Co, Pb, Tl, U) spiking was necessary to achieve

analytically reasonable values. The resulting

concentrations were between 0.020 mg/kg for U

and 42.7 mg/kg for Zn.

Twenty-two German official laboratories took part

in this PT. As only As, Cd, Hg and Pb were

mandatory analytes there were between 16 (for Ag)

and 22 (for Hg and Cd) data sets for statistical

evaluation. Reporting was done according to ISO

13528 using the participants’ data for calculating

the robust mean and the robust standard deviation.

The correlation with the standard deviation

calculated from the Horwitz equation (modified by

Thompson) showed HorRat-values between 0.28

for Co and 0.62 for Zn. The overall rate receiving a

satisfying score was nearly 95%, with no laboratory

producing clear outlier results.

Therefore the fresh material did not pose problems

to the participants. The ratios of repeatability to

reproducibility were between 1.9 (for Cu) and 3.8

(for Ag).

It is often stated that lyophilised materials are

easier to handle than fresh materials. Hence, the

recent data was compared to the data generated

with lyophilised pig liver in the same network in

2011. The data were normalised to lyophilised liver

and compared: For Pb and Se, the contents were

nearly identical. For Pb, the laboratory performance

expressed in terms of the reported standard

deviation was comparable, while for Se the recent

PT showed a significantly lower standard deviation

(8.5% vs. 16.3%). Even though for As, Cd, Hg and Zn

the concentrations in the recent PT were

significantly lower, the standard deviation was

nearly the same (for As) or even lower. The only

element with a higher content in the recent PT was

Cu. There the standard deviation was lower with

the higher content.

Even though some participants stated more

difficulties working with fresh material, the data did

not suggest a higher level of difficulty compared to

lyophilised material. Therefore, the use of

lyophilised materials as reference material is to be

preferred because of the better long-term stability.

However, fresh material could be a good alternative

in cases of matrices without stable lyophilisates,

e.g. fruit materials.

Angela Sorbo (EURL-CEFAO) 24th


Testing on Honey: Overview of Statistical analysis

and comments on Results

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The monitoring performed annually at national

level on the content of chemical elements in

different categories of food has highlighted the

presence of Pb in honey, sometimes at rather high

concentrations. In order to ensure the protection of

the EU market on one hand and the health of the

consumers on the other, the European Commission

published CR (EU) 2015/1005, which sets a

harmonized Maximum Level (ML) for Pb in honey.

The introduction of this new analyte/matrix

combination in the EU legislation forces the official

control laboratories to have adequate analytical

methods. Moreover, the methods performance

needs to be satisfactory and as uniform as possible

amongst the laboratories.

In view of providing suitable means for assessing

the performance of these methods and of assisting

laboratories in their improvement, the EURL-CEFAO

based the 24th

PT on the determination of Cd, total

Hg and Pb in honey. The analytes included in the

exercise had been selected on the basis of the

regulation in force, the possible inclusion in new or

existing regulations and analytical considerations.

All the NRLs belonging to the EURL-CEFAO network

joined the PT.

The statistical evaluation of the results was

performed in accordance with ISO 13528:2015. As

for Cd and Pb, the statistical analysis was

straightforward: data distribution was roughly

symmetric and unimodal without significant

differences between results obtained by different

analytical techniques. Robust mean was set as the

assigned value for both analytes (0.0072 mg/kg and

0.140 mg/kg for Cd and Pb, respectively).

With regard to Hg, the criteria on robust standard

deviation and uncertainty of the assigned value

were not fulfilled. This was due to the wide data

dispersion. The general performance of the

network was extremely satisfactory: almost all NRLs

obtained a z-score lower than 2 for all analytes.

Laura Ciaralli (EURL-CEFAO) Planning of 2017 PTs

PTs are provided using combinations of matrices

and elements of interest in order to check the

general performance of the network and maintain

this at a steady level. The 2017 PTs have been

planned with this specific view. In particular, the


PT will be based on the determination of Cd,

Cu, Pb and total Hg in freeze dried meat.

The 27th

PT will be based on the determination of

total As, Cd and Pb in powdered and liquid Infant

Formula based on animal proteins. This choice was

influenced by the publication of CR EU 2015/1005,

amending Regulation 1881/2006, as regards

maximum levels of lead in certain foodstuffs, which

entered in force on 1 January 2016.

Marina Patriarca (Department of Food Safety and

Veterinary Public Health, Istituto Superiore di

Sanità, Rome, Italy) TrainMic - Selection and use of

Reference Materials

TrainMiC is based on national TrainMiC teams

sharing common training materials (lectures,

examples, and exercises) and in turn contributing to

its production. TrainMiC material covers all aspects

of the technical requirements of ISO/IEC 17025.

Over the last few years, a process of revision,

update and extension of the guidance from

ISO/REMCO on reference materials has been in

place, aiming to cover quality control materials

prepared in-house as well as setting the basis for

formal accreditation of reference material

producers. However, using a reference material

does not automatically provides better results and

it is of the utmost importance that the criticalities

associated with appropriate selection and use of

reference materials are fully understood by all

those involved.

Reference materials are materials sufficiently

homogenous and stable with reference to its

specific properties which have been established to

be fit for its intended use. A CRM is a reference

material characterized by a metrologically valid

procedure for one or more specified properties,

accompanied by a certificate that provides the

value of the specified property, its associated

uncertainty, and a statement of metrological

traceability. A CRM should state traceability,

provide uncertainty and demonstrate traceability of

certified value. The main types of CRM are – Pure

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substances for calibration, pure substances for

matrix matching, matrix CRM, and physic-chemical

standards. CRM can be used for calibration as part

of a measurement procedure, validation of

measurement procedure, quality control, and

comparison with in house materials. The

preparation of CRM is described in general in ISO

Guide 34 and in more detail in ISO Guide 35.

General steps required in production of a certified

reference material typically include:

Collection or synthesis of material

Sample preparation (including homogenization,

stabilization, bottling etc.)

Homogeneity testing

Stability assessment

Value assignment

Maria Ciprotti (EURL-CEFAO) Critical Points in

Mercury Determination by Direct Mercury Analysis

Maria Ciprotti spoke on the characteristics of

mercury including its high volatility, absorption on

container walls and suspended particles and

colloids, and inclusion in stable complex and


Methods for determination are neutron activation,

Cold Vapour Atomic Absorption Spectroscopy, Cold

Vapour Atomic Fluorescence Spectroscopy, and ICP-

MS plus direct mercury analysis (COMB-AAS).

The main advantages of direct mercury analysis are:

No sample treatment

Time saving

No purchase of high purity acid

No acid wasted

Reduced possibility of sample contamination

Increased stability of calibration curve

The main disadvantages of direct mercury analysis


Tools contamination

Environmental contamination

Time consuming calibration curve

Correction for dry weight

Claudia Zoani (Italian National Agency for New

Technologies, Energy and Sustainable Economic

Development, ENEA - C.R. Casaccia, Rome, Italy)

Metrofood-Ri: A New Pan-EU Research

Infrastructure for Promoting Metrology in Food

and Nutrition

METROFOOD-RI “Infrastructure for promoting

Metrology in Food and Nutrition” is a European

Research Infrastructure project listed as “Emerging”

on the 2016 ESFRI Roadmap aiming to carry out

different activities supporting data collection and

measurement reliability, as well as basic and

frontier research in food and nutrition. Its general

objective is to enhance scientific cooperation and

encourage interaction between the various

stakeholders, as well as the creation of a common

and shared base of data, information and


A network of plants, laboratories and experimental

fields/farms will be realized (Physical-RI) and an e-RI

will be developed. The Physical-RI will enable to

carry out different research activities supporting

data collection and measurement reliability; quality

& safety and traceability of food production, as well

as basic and frontier research in food and nutrition.

The e-RI will provide a new useful, free access web

platform to share and integrate information and

data on availability of metrological tools for food

analysis. It will deal with integration of existing

databases on food, focusing on emerging needs and

collection of data on food composition, nutritional

contents and levels of contaminants in foods

produced in different geographic regions by

applying to different technologies.

METROFOOD-RI is supported by the economic

endorsement of 3 MSs and the political

endorsement of 13 countries. Currently, 35

partners from 17 countries, together with an

international partner (FAO) are involved. Ireland is

not involved in this. Each partner brings its wide

and consolidated network of international

collaboration, which will ensure a very broad range

of action, open also to developing countries and

new markets and able to meet the needs of the

scientific community and all stakeholders at a

global level.

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Andrea Colabucci (EURL-CEFAO) Annual update

As announced in 2015 EURL-CEFAO annual

workshop, the PT results submission has changed.

As for the 24th

PT on honey, an intermediate

procedure has been chosen: participants were

required to submit the official results by e-mail, as

usual, but also to test the online submission system.

Starting from the 25th

PT on milk, results are

submitted only in the on-line restricted area.

The second issue is related to changes in the

European legislation. In fact, the entering into force

of the Commission Regulation (CR) 582/2016

amending CR 333/2007 has changed the definitions

of Limit of Detection and Limit of Quantification. In

particular, one of the main issues he highlighted is

that not all the EU MSs take into account the

Maximum Residue Limits set for Hg and Cu in the

pesticides legislation (Regulation 396/2005).

National Monitoring Residues Programs (NRCPs)

results for 2015 were also discussed. The high

number of non-compliant samples found in some

specific matrix/element combinations (e.g. Cd in

kidney) was highlighted as well as the relevant

follow-up action reported by the MSs in the

“Questionnaire on the actions taken as a

consequence of non-compliant results”. Finally, the

representatives of the NRLs were reminded of the

opportunity to include As and Ni in their 2016-2018

NRCPs at least for the matrices prescribed in the

Commission Recommendations 1381/2015 and


Andrea Colabucci (EURL for Chemical Elements in

Food of Animal Origin) Applicability of the CD

2002/657 to the Analysis of Chemical Elements in

Food of Animal Origin

The urgent need for a review of the CD

2002/657/EC was highlighted in the expert

committee on residues of veterinary medicinal

products meeting, held in June 2015. For this

purpose, the four EURLs for residues (BVL, RIKILT,

ANSES, and ISS) were asked by the European

Commission for technical assistance including the

revision of CD 2002/657/EC in their work

programmes for 2016/2017.

In July 2015, a questionnaire was prepared by the

three “organic residue” EURLs (BVL, ANSES and

RIKILT) asking their NRLs network to rate for each

article of the Decision the need for changing from 0

(no changes required) to 5 (changes urgently


For group B3c (Chemical Elements), the field of

competence of the EURL-CEFAO, more specific

provisions are reported in the Commission

Regulation (CR) 333/2007 that, however, does not

indicate any mandatory document to be preferred

for method validation.

The EURL-CEFAO sent a questionnaire to the NRLs

of its network asking them whether and how this

decision is applied. 23 out of 31 were collected. The

main outcome from the questionnaires was that

there is a need for revision of validation

procedures, to simplify the classification of

analytical methods by performance characteristics,

and more detailed information should be given on

whether CRMs are available or not.

The review could lead to – full CD 2002/657 revision

or ‘light’ version plus technical guidance in EURL


Marilena D’Amato, EURL-CEFAO Determination of

Inorganic Arsenic in Mussel Tissues by HPLC-ICP-


Marilena D’Amato presented on how analytical

methods for arsenic speciation in food of animal

origin can still be demanding for implementation as

a part of the routine activities of those laboratories

not primarily focused on research. Most works

reported in the literature deal with the

identification of the variety of As species typically

found in marine samples, whereas the interest in

developing robust analytical methods focusing on

inorganic As as the most toxic fraction has

increased only recently. Therefore, the

implementation of a relatively cheap, fast, and

easy-to-use method directly applicable to incurred

samples can represent an added value for routine


Due to the rising interest in As speciation at the

level of control laboratories and in compliance with

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its tasks, the EURL-CEFAO has developed a method

for inorganic As determination in fresh mussels

using water bath extraction and anion exchange

chromatography-inductively coupled plasma mass

spectrometry (HPLC-ICP-MS). This method might be

applicable to incurred samples of animal origin. In

particular, an effective procedure for extracting,

separating and quantifying inorganic As was

developed in order to reduce sample preparation,

e.g. avoiding freeze-drying. The proposed method

will be considered only as a general guideline for

inorganic As determination in fresh mussels and as

a support to NRLs when approaching analytical

issues related to inorganic arsenic speciation.

During the method development, the optimization

of an HPLC-ICP-MS method for selective inorganic

As determination was investigated and the results

obtained with anion-exchange chromatography by

using different extraction mixtures and columns

were compared and evaluated. The method

consists of extraction with a HNO3/H2O2 mixture in

a shaking water bath and elution with ammonium

phosphate and ammonium nitrate in 2% methanol

(pH=5.5) on Hamilton PRPX-100 column. Following

good preliminary results obtained, the method was

considered for validation.

The method was validated using samples of

Mediterranean mussels containing roughly 3.8

mg/kg of total As. With the proposed extraction

procedure using HNO3/H2O2 mixture, the complete

oxidation of As(III) into As(V) was achieved, which

resulted in a well-separated peak in the anion

exchange HPLC-ICP-MS chromatogram. Then, the

inorganic arsenic was easily quantified as As(V). In

the validation procedure LoD, LoQ, repeatability,

within-laboratory reproducibility and accuracy were

assessed. A provisional value of expanded

uncertainty was also estimated.


Workshop EURL Campylobacter, Uppsala,

Sweden, 4th


October 2016

NRL Representative: Tony O’Brien (Bacteriology

and Parasitology Division)

The workshop was opened by Dr Hanna Skarin of

EURL Campylobacter who outlined the topics to be

covered in the workshop and reviewed the work

carried out by the EURL in 2016

The main issues to be covered in the workshop


Campylobacter activities at EU level

Molecular typing and surveillance

Epidemiology of Campylobacter

Quality controls and standards for WGS

National presentations

PT results and performance

ISO updates

Group discussions on three topics ( subtyping,

detection and enumeration and MALDI-TOF )


The 2016 work program for EURL Campylobacter

included the following

PT17 and PT18 sent out in March

Two questionnaires

Update of website, new learning materials

Training courses (detection, enumeration and


Tests of analytical methods ( detection of

Campylobacter in water, freeze drying of

Campylobacter, tested PCR’s, comparisons of

WGS subtyping methods and pipelines )

Participation in revision of ISO for


Participation in meetings / conferences

The presentation on subtyping focussed on the

various methods used by the different labs. These

were mainly PFGE, MLST, PCR-based and WGS-

based. Most labs used subtyping for research

reasons followed by outbreak investigations and

then surveillance. Very few laboratories had

embarked on WGS. Those that had were using the

Illumina equipment with the MiSeq being the most

popular. Some countries (including Ireland) had

purchased equipment and were ready to proceed

to training and implementation of protocols. There

was a consensus that it would be very helpful if the

various EURLs could coordinate training and give

advice on the best protocols and pipelines available

to laboratories about to embark on WGS. There was

also discussion on the creation of a Campylobacter

WGS-network which will be considered at a later


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Some laboratories expressed an interest in

obtaining training in the use of MLST subtyping.

This is being planned to occur in late 2017 and it is

hoped that an analyst from NRL-IRE will attend this


The presentation on PTs for WGS discussed the

objectives of any future PT. The preference would

be to include two components, one would be using

lyophilised culture and the other using purified

DNA. The aim would be to quantify differences

among laboratories in order to facilitate the

development of reliable laboratory results of

consistently good quality within the areas of DNA

preparation, sequencing and analysis. Also to

facilitate harmonisation and standardisation in WGS

and data analysis. On the latter point the

participants were agreed that this is crucial as the

various labs take the steps to implement WGS as a

diagnostic and research tool.

Global Microbial Identifier is involved in setting up

PTs in these areas with the support of Compare,

Genome Trakr and Microbiologics.



Ingrid Hansson and Ann Nyman (EURL-

Campylobacter) proficiency tests update

PT 17 included detection and enumeration of

Campylobacter in 10 samples consisting of broiler

carcass skin. The skin was analysed together with

10 vials each containing one capsule with or

without Campylobacter.

PT 18 involved detection and species identification

of Campylobacter in avian caeca samples. 18 caeca

samples from a Campylobacter negative flock were

inoculated with Campylobacter of various species

and concentrations. Some caeca were inoculated

with non-Campylobacter organisms.

Results among participants (%)

Excellent Good Acceptable

PT 17


95 4 4

PT 17


74 17 3

PT 17


88 8 4

PT 18


67 30 3

PT 18


60 23 10

NRL-IRL achieved excellent results in all

components of PT17 and PT18. Overall both PT17

and PT18 showed improved results compared to

the previous two PTs.

Ingrid Hansson (EURL) led a discussion on drying

times of mCCDA selective agar plates. This was

based on feedback from a questionnaire circulated

to the NRL’s by EURL.

Campylobacter has a tendency to swarm over agar

particularly when it is wet. This means it is very

difficult to enumerate the colonies. Most

laboratories either dry out the plates at room

temperature for several days or for a few hours in

an incubator prior to use. It is the practice in the

NRL-IRL to dry the plates at room temperature for

up to three days. This is essential to achieve

accurate and reproducible enumeration of


Elisabeth Reperant (NRL-FR) gave a presentation

on whether using different selective media

influence Campylobacter detection. They carried

out comparisons of four commercially available

selective agars with the mandatory mCCDA.

The summary was that two of the agars were more

efficient than mCCDA in detecting Campylobacter

and more selective for negative samples.

Also mCCDA was less efficient in detecting samples

in cross contaminated samples. It was mentioned

that NRL-IRL had done some work examining the

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effect of adding tazobactam to mCCDA based on

work done by colleagues in UCD which showed a

decrease in the amount of background flora

growing in the presence of the Campylobacter.

Miriam Koene (NRL-NL) gave a very comprehensive

presentation on the use of MALDI-TOF in

identification of Campylobacter species. They use

the Bruker instrument. All of the NRLs using MALDI-

TOF (including NRL-IRL) use the Bruker. Miriam

described the principles and practicalities of using

the instrument and also went into detail on how

they validated the test prior to accreditation. This

was particularly interesting for NRL-IRL as we are in

the process of validating MALDI-TOF in our


Miriam concluded that although the instrument

was very expensive, it produced fast and accurate

results and the cost per tests were very low.

Hanna Skarin and Ingrid Hansson (EURL) gave an

update on the proposed changes to ISO 10272:

Horizontal Method for the Detection and

Enumeration of Campylobacter Species. Although

the preparation and discussion of the changes to

the ISO method have been going on for several

years, it is envisaged that they will be implemented

in 2017.

The main changes to the detection method include

Samples from the primary production stage

have been added to the scope

Option of using another enrichment broth

(Preston), primarily to overcome problems

with background flora resistant to 3rd

generation beta lactams

Option of direct plating onto mCCDA agar

The use of closed containers with a reduced

headspace as an alternative to incubation in a

microaerobic environment has been deleted

Confirmation tests on based on microaerobic

growth at 25oC and aerobic growth at 41.5


will be replaced by the study of aerobic

growth at 25oC

Performance testing for the quality assurance

of the culture media has been added to Annex


Performance characteristics have been added

to Annex C

The main changes to the enumeration method


Samples from the primary production stage

have been added to the scope

Serial dilutions will be plated singularly rather

than in duplicate, to be comparable with ISO


Confirmation tests on based on microaerobic

growth at 25oC and aerobic growth at 41.5


will be replaced by the study of aerobic

growth at 25oC

Publication of the final standards is expected in

March 2017.


Workshop EURL E. coli, Rome, Italy, 10-11th

November 2016

NRL Representative: Regina Heslin (DSL)

The 11th

EURL E. coli workshop was attended by 33

NRLs representing 24 EU countries, plus the NRLs of

Norway, Iceland, Switzerland, FYROM, Serbia,

Turkey and Russia; Commission, EFSA, ECDC and

WHO Collaborating Centre for Reference and

Research on Escherichia and Klebsiella

Session 1: Update of surveillance and monitoring

activities on STEC in the EU and the development

of a database on molecular typing of foodborne


1. Karin Johansson from ECDC spoke on the role of

EPIS -FWD (Epidemic Intelligence Information

System for Food and waterborne diseases). It has

been updated to manage non-clinical and joint

clinical/non clinical clusters that can be detected

through the joint ECDC- EFSA molecular typing

database. This updated version is due for release in

early Dec 2016. Training for the nominated users

from the food safety and veterinary sector is being

planned with dates to be confirmed and invitations

to be sent out by EFSA.

The importance of data being referred to them was

stressed. The main EPIS-FWD features discussed

were Molecular Typing Cluster Investigation (MTCI)

and Urgent Inquiry (UI). MTCI is a forum for

exchange of information about a specific cluster /

molecular type. This can only be assessed by MS

that have isolates in the cluster. UI is a forum for

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exchange of information about an unusual event

with the potential for international spread and is

accessible to representatives of all MS and

international partners. If the information provided

in the MTCI leads to suspect that more countries

may be involved, and/or there is value in informing

both the food safety and veterinary expert

networks, the MTCI can be escalated to an Urgent

Inquiry (UI).

2. Valentina Rizzi, EFSA, gave an update on the

annual reporting of STEC in the EU and on EFSA

activities for molecular typing data collection for

food and animal isolates. The EU summary report of

2014 includes the different analytical methods used

as well as data reported for certain food categories

and animal species. In total 12 MSs provided

information on STEC serogroups in 226 STEC

isolates. In food the main serogroups are ranked as

follows and are also the most commonly reported

serogroups in human infections in the EU/EEA.

Serogroup Frequency %

Main source

VTEC O157 33.5 Bovine meat, raw milk

O26 8.7 Milk & dairy products

O103 6.9 Meat and Milk & dairy products

O113 6.4 Meat Products

O146 4.6 Meat Products

O174 4.6 Meat Products

O91 4.0 Meat Products

O145 2.9 Meat and Milk & dairy products

An increasing trend of reporting in food was

observed for STEC O26 and STEC O103 two

serogroups strongly associated with severe human

infections in the EU. Contamination was rare in

ready-to-eat food of vegetable origin. No STEC

positive samples reported for spices and herbs or

for sprouted seeds, the sole food category for

which microbiological criteria for STEC have been

established in the EU.

3. Antonella Maugliani gave an update on the STEC

Network preparedness for molecular typing data

collection. The EURLs for STEC, L. monocytogenes

and Salmonella have all produced PFGE SOPs for

the production, interpretation and curation of PFGE

profiles. The STEC EURL has distributed EQAs on

PFGE annually between 2012 and 2015. They have

also provided training sessions on PFGE (9 courses

to date) and basic courses on BioNumerics software

(2 courses to date). Training on the 3rd

edition of

BioNumerics will be organised jointly by all 3 EURLs

in 2017 with the 4th

expected to be organised by

the EURL Salmonella in 2018 in the Netherlands.

EQAs on PFGE are valuable in assessing the labs

ability to produce good quality profiles. An outline

on the NRLs performance in PTs showed vast

improvement year on year. The main areas labs

have problems with are image acquisition and

staining and compressed central or fuzzy bands.

42% of NRLs improved their performance after

attending training. The EURL will focus training on

the areas that proved problematic for the


Session 2: E. coli Genomics.

1. Valeria Michelacci EURL gave a talk on

preparedness towards the use of NGS for

characterization and typing of pathogenic E. coli. In

2014 all NRLs expressed the need for training in

genomics data analysis. 8 NRLs had access to

bench-top sequencers with 8 planning to outsource

NGS data production. The EURL has provided

training on Bioinfomatics for NGS since 2015. ARIES,

a Galaxy based workspace for intensive data

analyses was demonstrated.

2. Federica Gigliucci EURL presented the use of

metagenomics to study STEC infections. The

metagenomics approach was used to investigate

the changes in the microbiota in patients with STEC

infections as compared to healthy individuals. The

cases were from an O26:H11 outbreak in Rome. 10

faecal samples were analysed: 3 from patients (1

bloody diarrhoea, 2 diarrhoea), 3 from recovered

patients and 4 from healthy individuals. DNA was

extracted from 0.20g of faecal sample. Sequencing

was by Ion Torrent and Illumina Miseq platforms.

Bioinfomatics analysis was through ARIES and

MEGAN software. A comparision of the E. coli

virulence genes was made from the three groups

with 20% more genes present in the group with

diarrohea than the other two groups. In terms of

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Taxonomic abundance, Clostridia were more

abundant in negative samples while Bacilli

predominated in recovered or clinical cases,

particularly Bifidobacteria. This different

microbiological distribution between cases and

healthy controls may be related to the clinical

demonstration of the patient. Prevalence of

Clostridia may indicate a protective role in the gut.

When colonisation by STEC occurs the reduction in

Clostridia allows Bacilli to increase. Faecal samples

from patients with Crohns disease (with and

without evidence of infection) were also studied.

Two of three samples had genes associated with

aEPEC (escV), and the third with stx2f gene (STEC).

In summary, on STEC infection, the balance

between species composing the intestinal

microflora is altered.

3. Sabine Schlager, NRL Austria presented on a

study of STEC O26:H11/HNM isolated in Austria in

2009-2014 by NGS analysis. Incidence of STEC

cases are increasing year on year. Human isolates in

order of occurrence O157 (22%): O26 (14%): O103,

O111, O145 (<10%), others (44%). In terms of HUS

cases the occurrence is as follows: O157 (22%),

other (22%), O26 (18%), no isolate (16%), O145

(9%), O111 (7%), O103 & O104 (3%). Of 109 E. coli

O26 strains studied (MLST) 11% belonged to ST29

with the remaining belonging to ST21, ST591 and

ST1566. However geographically throughout

Austria ST21 predominates. In clinical outcomes,

42% stx2a genes from ST21 caused HUS versus 18%

from ST29.

4. Invited speaker Flemming Scheutz from the

International Escherichia Centre of the WHO talked

on the EQA schemes for Public Health laboratories.

The results of the 7th

EQA scheme for typing of STEC

in 2015-2016 were presented. EQA-7 includes the

following methods:

Molecular typing: PFGE

Serotyping O and H

Virulence determination: genotyping of STEC

virulence genes: vtx1, vtx2, eae, aaiC and aggR

& subtyping of vtx1 (vtx1a, vtx1c and vtx1d)

and vtx2 (vtx2a to vtx2g)

Phenotypic testing: production of:

Verocytotoxin/Shiga toxin, Extended Spectrum

Beta Lactamases (ESBL), β-glucuronidase,

Enterohaemolysin & fermentation of sorbitol.

30 participants took part. Over 90% of participants

submitted results for serotyping, genotyping and

Phenotyping while PFGE were submitted by 63% of


Session 3: Laboratory methods and Proficiency


Rosangela Tozzoli EURL discussed the results of

recent Proficiency Tests: PT 16, detection of STEC in

sprout irrigation water and PT17, the inter-

laboratory study on the detection of STEC in ground

beef. The objective of PT 16 was to allow the

evaluation of a procedure for the pre-treatment of

spent irrigation water to be added to ISO/TS 13136:

2012. The water used had been obtained from a

sprout producer. 3 samples were spiked with STEC

O157 (vtx1+, vtx2+, eae+) at high (500cfu/ml), low

(200cfu/ml) and 0 levels. 50 labs responded. 44/50

successfully isolated the high sample and 42/50 the

low sample. The results confirmed the suitability of

the added centrifugation step for processing spent

irrigation water. 36 labs reported results for PT 17

on ground beef. The strain used to spike samples

was ED 76, STEC O91 (vtx1+, vtx2+, eae-) at high

(50cfu/g) sample C, low (5cfu/g) sample B and 0

levels sample A. 35 labs (97%) correctly identified

STEC O91 virulence genes in both samples. Four

labs picked up O26 in sample B and six labs picked

up eae in sample C. However these were not

considered penalties as the raw material may have

been naturally contaminated with these genes. At

isolation stage 4/25 labs did not determine the O91

serogroup (ONT). However isolation was correctly

achieved by 78% of labs. In conclusion ISO/TS

13136:2012 method is suitable for the detection of

all the serogroups analysed so far in foods most

regarded as vehicles of human infection.

2. Rosangela Tozzoli EURL gave an update of

Revision of ISO/TS 13136:2012. There are 8 main

areas for revision. (i) Enrichment: data from EURL

shows that most non-O157 STEC of the top 5 O-

groups show inhibition when grown in the presence

of supplements. Therefore BPW is considered the

most reliable enrichment media. (ii) Revision of stx-

gene sub types detected: possibility of adding stx2f.

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NRLs for E. coli in the EU were surveyed on the

presence of this stx sub type in E. coli isolated from

food. 3 MSs perform routine testing of stx2f. No

positive findings so far. Therefore it is unlikely that

this will be added to the revised standard. (iii)

Inclusion of Enteroaggregative E. coli (EAEC)

virulence genes: EFSA opinion “Current evidence

indicates that in EU MSs EAEC are primarily non

zoonotic in origin and that transmission mainly

occurs by person to person spread and by the

contamination of foods by asymptomatic carriers”

Points (iv-vii) Inclusion of protocol for spent

irrigation water, Reconsideration of serogroups,

Expression of results and Performance

characteristics will be adopted as per evaluation of

data from EU-RL PT programs 2009-2015. (viii)

Isolation: Acid treatment (pH 2 for 1h) to reduce

background flora without addition of supplements.

Not going to be added to the revision as it is a

complex procedure and not verified for all STEC

types. Dilution of the enrichment broth before

plating is recommended instead of acid wash.

Lina Cavaco EURL AMR: Overview of activities.

Information can be obtained at www.eurl-ar.eu

Session 4: Presentations from the NRLs and other


1. Pathogenic E. coli in Czech Republic overview by

Ivana Kolackova. Reported occurrence of

STEC/EHEC in humans in the Czech Republic is rare

with O26 as the dominant serogroup and stx2

subtype A as the most often identified virulence

factor. Monitoring of STEC in carcasses at

slaughterhouses shows higher prevalence of STEC

in cattle (8.3%) than in pigs (2.8%). O91, O146 &

O157 predominate in pig swabs while O91 and

O113 predominate in cattle swabs. 90% of STEC in

pigs carry the gene for stx2 subtype E (42% in

cattle). The spectrum of detected O- serogroups is

varied but the O26 was not detected in any of


2. Estelle Loukadis Enterohaemorrhagic E. coli

Hybrid Pathotype O80:H2 an emerging clone in

France. In 2012 an atypical EHEC O80:H2 was

isolated from a HUS patients’ blood culture. From

2005 to 2015 five foodborne outbreaks leading to

HUS were detected with O157:H7 & O177:H25

found in frozen and fresh beef, O104:H4 in

Fenugreek and O26:H1& O80:H2 found in raw milk

cheese. There appears to be a shift in STEC

serogroups causing HUS in France. Clinical cases of

O157 have gone from 66% in 2005 to 23% in 2015.

O26 has remained stable at 13% and 11% in the

same time period. O80 has risen from 3% in 2010 to

35% in 2015 from stools of paediatric HUS cases.

3. Shona Neal from UK NRL spoke on their activities

and responsibilities regarding UK –NRL structure,

dissemination of information and training for

Official Control labs and NRL colleagues. She

focused on how they handled the implementation

of regulation 208/2013.

4. Rosangela Tozzoli EURL presented a study of

Stx2f-producing E. coli and HUS from a

collaboration with NRLs in Austria, The Netherlands

and the Faculty of Veterinary Medicine, Garmsar,

Iran. Pigeons are known to carry STEC producing

stx2f. Prior to 2004 few human infections were

related to stxf2. From 2008 it has been identified in

human diarrhoeagenic E. coli strains formally

classified as atypical EPEC. In 2013 the first HUS

associated cases implicating stx2f emerged. The

majority of human cases caused by stxf2 have been

reported as uncomplicated diarrhoea and may

therefore have been overlooked. Stxf2 in HUS cases

are different from those isolated from the pigeon

reservoir and from other diarrohea cases leading to

the conclusion that stxf2 toxin can move from

pigeon strains to E. coli strains with a genetic

background of aEPEC (not found in pigeons).

Therefore stxf2 is a fully functional toxin and its

ability to cause HUS depends on the strains genetic

receptor for host colonisation.

Session 5: Investigation of a multistate outbreak of

STEC O26 infections

1. Ciupescu Laurentiu NRL Romania presented a

Romanian O26 STEC outbreak in February 2016

involving pasteurised cow’s milk cheese and 15

paediatric cases, resulting in three deaths. The

common STEC genotype was stx2+, eae+ O26. Initial

food consumption surveys of the cases involved did

not target one food in particular leading to 574

different suspect food samples being tested, 40% of

which were dairy products, by the ISO method. Milk

fat caused interference requiring two to three extra

washing steps at the extraction stage. The results

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showed that from 16 types of food (five dairy

products, ten poultry and one mixed meat (beef

and pork), 86 samples were positive by RT-PCR for

one or more of the investigated genes (stx1, stx2,

eae, wzx-O26). The target genes were detected in a

batch of soft cheese linked to the cases so the

investigation focused on this product. O26 culture

could not be isolated.

2. Valentina Rizzi, EFSA spoke on the assessment of

this outbreak and general background of O26

involvement in STEC infections. Among STEC,

serogroup O26 is the second most commonly

reported in Europe after O157. In the period 2010-

2014, 19 EU/EEA reported 2,356 confirmed cases of

STEC O26 with most cases during this period being

reported from Ireland (32%) followed by Germany

(15%) and Sweden (12%). These cases were

predominantly in young children; the median age

two years. Symptom onset for STEC O26 cases was

distributed over the year with a peak during the

period June-September. 88% of the cases were

acquired within the reporting country. An increase

in STEC O26 cases has been reported from 2012

onwards, most likely due to the increased use of

methods capable of identifying serogroups other

than O157. As of 23 March 2016, 463 cases of STEC

O26 have been reported to the European

Surveillance System (TESSy) for 2015. Since

November 2012, ECDC has been collecting PFGE

molecular typing data for STEC isolates from MSs

on a voluntary basis. As of 23 March 2016, 1,610

STEC isolates have been submitted to TESSy, 167 of

them reported to be of O26. Of these, 130 isolates

from eight MSs have approved and curated PFGE

profiles, involving 106 PFGE pulsotypes, 13 of which

are represented in the database by more than one

isolate. Two of these 13 patterns include isolates

from more than one country. Similar to the data for

human infections, O26 was the second most

frequently reported serogroup in food and animal

samples in Europe from 2005, with an increasing

trend between 2011 and 2014. Overall, a wide

range of STEC serogroups was reported, with STEC

O157 being the most frequent in food and animal

samples. However, many of the MS surveillance and

monitoring programmes are focused on STEC O157,

potentially biasing the estimates of the frequency

of non-O157 serogroups. An analysis of the 2014

STEC data indicated that serogroups O26 and O103

were reported more frequently than O157 in the

food samples (mainly from bovine meat, raw milk

and dairy products) tested using the ISO/TS

13136:2012 standard method which is able to

detect STEC regardless of its serogroup.

3. The story was taken up by Gaia Scavia of the

EURL who initially became aware of the outbreak

from the TV and newspapers. The information

relayed from EPIS FWD described it as an Urgent

Inquiry: unusual cluster of severe diarrhoea with

HUS. In March a HUS case was reported through

the Early Warning and Response System (EWRS) in

Italy from a Romanian child living in Tuscany. The

same cheese as in the Romanian RASFF had been

consumed. STEC O26 isolated from the cheese

upgrading the RASFF to an ALERT. There were a

further 10 epidemic cases, 5 HUS, caused by STEC

O26 in Italy from April to August 2016 with a link to

Romania, both food and travel related. Person to

person contact as well as consuming the Romanian

cheese were the cause of illness. Romanian

nationals are one of the highest immigration

populations in Italy. PFGE was performed for five

STEC O26 isolates obtained from confirmed cases.

Two showed indistinguishable PFGE patterns

whereas the other three all showed non-identical

PFGE profiles (86-93% similarity). The three cases

with non-identical PFGE patterns are from different

districts, had onset of symptoms in early to mid-

February and all have different virulence gene

profiles (one case is PCR-positive to both stx1 and

stx2, one case is PCR positive only to stx1 and one

case is PCR positive only to stx2). The two cases

with indistinguishable PFGE patterns are from the

same district, had onset of symptoms in late

February to early March and share the same

virulence gene profile (PCR-positive to both stx1

and stx2). PFGE data from more cases would be

highly desirable to be able to further characterise

the outbreak strain(s). WGS is awaited on the

isolates from the Romanian outbreak.

4. Details of a recent outbreak, August 2016,

associated with rucola in Helsinki, Finland was also

presented. The rucola was supplied to a catering

company and wholesalers. The product was grown

in Denmark and packaged in Sweden. 200 people

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were reported ill. Of 80 hospitalised cases STEC was

isolated in 30 cases and EPEC in 50. No HUS were

reported. Chicken salad where rucola was used as a

garnish was analysed. 1 out of 5 samples was stx2,

eae and O111 positive. The laboratory carried out

acid treatment for 1h following IMS isolation. They

found that the selective agars did not grow the

outbreak strains and used culture from non-

selective TBX and SHIBAM (STEC Heart Infusion

blood agar with Mitomycin).


Annual EURL-Animal Protein Workshop,

Namur, Belgium September 2016

NRL Representative: Oliver Burke and PJ Conway

1. Introduction

This meeting is held every year to inform the

National Reference Laboratories for Animal Protein

(NRL-AP) network of developments in the area of

feedingstuff analysis and to discuss issues such as

new or revised scientific protocols. The meeting

also celebrated the 10th

anniversary of the EURL-AP.

The meeting was attended by 50 delegates from 27

countries. Ireland was represented by Oliver Burke

(AAI) and PJ Conway (Lab Analyst).

2. Presentations

As with other workshops in recent years, the

programme was dominatd by the implementation

of PCR for the detection and differentiation of

animal protein from different species in

feedingstuffs.The newest item on the agenda was

insect meal detection: possible methods.

EURL-AP activity report 2015

The activities of the EURL-AP were outlined with

reference to its functions as detailed in legislation.

Among the activities highlighted were:

ISO17043 accreditation and conformity


NRL proficiency for qualitative testing –

Network performance;

Preparation for legal extension to feed ban

( Pig- Poultry);

Transfer of the EURL-AP micrograph

collection to the public site;

Parameters around Official Lab interaction

with EURL-AP;

PAP detection in liquid samples;

Training in light microscopy and PCR;

Effect of grinding on bone detection by

light microscopy- Study Results;

Production of pDNA calibrants for the

detection of poultry in processed animal

proteins; and

Implementation study for pig PCR

Production of pDNA calibrants for the detection of

poultry in processed animal proteins

The production of a poultry calibrant was initiated

in 2015 by the JRC in Geel with a validation study of

a chicken/turkey PCR method. Currently, the short-

term stability study and the determination of the

copy numbers by chamber and droplet digital PCR

are finalised. The long-term stability study to obtain

a certified reference material is still pending and

will end in January 2017. A report and the ERM-

AD484 should be available in February 2017.

The EURL- AP concluded that the short term

stability studies already highlighted the high

susceptibility of the poultry plasmid to

frozen/thawing cycles. For that reason, it was

recommended to thaw only once and to store it at

4°C during the few days of the cut-off

determination of the PCR platform.

Implementation study for pig PCR

An interlaboratory study was organised to run

jointly with the proficiency test 2015 to assess the

use of the validated pig PCR assay by the NRLs. The

implementation of the method was already

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initiated in August 2015 with the distribution of all

necessary information and material. The DNA

extraction protocol to use is the same as for the

ruminant DNA detection currently being operated

by the NRL network. A dedicated Excel file has to be

used for the determination of the cut-off at 5

copies and a PCR protocol was distributed by EURL-

AP. The sample set was composed of a total of 9

samples (3 samples in triplicate). The method can

now be considered as validated including the DNA

extraction step and successfully implemented in the

majority of the NRLs. Four labs did not submitted

results in time and are asked to continue the

implementation. The implementation study

attracted detailed discussion among the attendees.

Some of the points raised by the representatives

included; the report for the implementation of the

test pig PCR which is not yet available, the

sensitivity of the test?. The pig PCR method is less

sensitive than the ruminant method. The

mitochondrial target is present in a lower copy

number. For that reason, the cut-off was adapted

to be determined at a level of 5 copies and the

dedicated Excel file must be used. The Danish NRL

observed high differences between CT values when

the dilution steps are repeated. No other NRL

observed this. EURL-AP will investigate the

repeatability of the test.

New NRL: Republic of Serbia - Control of feed for

the presence of PAPs in Serbia (by Ksenija Nesic,

Serbian NRL)

Mrs Ksenija Nesic presented the activities of the

different departments of the Institute of Veterinary

Medicine of Serbia. She drew attention on the

difference between European and Serbian

legislation. A total feed ban has been applied in

Serbia since 2011.

Ksenija also presented the results of their 10 years

monitoring. Since 2005, the number of positive

samples has decreased significantly.

Combined proficiency test 2015 “Microscopy + PCR

(by O. Fumière and P. Veys EURL-AP )

For the first time, EURL-AP organised a proficiency

test combining microscopy and PCR. Moreover, the

study was conducted in agreement with ISO 17043

standard. The sample set was constituted of 12

samples: 4 to analyse by microscopy, 5 to analyse

by PCR and the 3 remaining samples to analyse by

both methods. The performances of the NRL

network was reported as good whatever the

method. The cases of underperformances (1 NRL

for both methods) were resolved and corrective

action completed.

Results of the study were presented; no special

remarks or comments were brought up by the

audience. The performance of the Irish NRL was

good and in line with the other NRL participants.

Mass spectrometry – updates (by M.C. Lecrenier


The transfer of the mass spectrometry method to

routine testing was presented. This transfer is firstly

focused on the haemoglobin powder and blood

meal detection using the haemoglobin peptides

biomarkers identified in high resolution mass

spectrometry (Q-TOF). After collision energy

optimisation and best transitions selection,

retention times for each MRM transitions were

determined. The limit of detection was evaluated in

various matrices using blood meal or haemoglobin

powder. The first estimation of the LOD for bovine

and porcine blood detection is around 0.05 % w/w

which is below the LOD imposed by the European

commission. A prospective study also started on

aquafeed known to contain porcine blood derived

products. Using PCR, all samples gave positive

results for porcine DNA but one gave a positive

result for ruminant DNA. The Porcine biomarker

was clearly detected. Regarding the bovine

biomarkers, a weak signal was detected for the

positive aquafeed for ruminant DNA though it was

below the internal threshold. Additional feeds and

bloods samples will be analysed. Secondly, the

suggestion to analysis milk powders and whey

powder was made. Evaluation of milk biomarkers is

underway is already underway in the EURL-AP


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Insect meal detection: possible methods (by

P.Veys, A.Marien, M.C. Lecrenier EURL-AP)

A review of the preliminary test on different

analytical methods was presented: light

microscopy, PCR and mass spectrometry.

Requirements for future insect PAP detection were

highlighted (modification of existing protocols, new

target development). The possible methods for

insect meal detection were discussed in detail with

the NRL representatives. Points raised included the

possibility of natural insect contamination of feed.

The EURL- AP responded by stating that the

implementation of the methods is not yet fully

developed. Regarding the setting of a specific

concentration procedure for microscopic

observation, it is reminded to look at the work

performed in the STRATFEED and SAFEED-PAP

projects where different procedures were tested to

concentrate muscles in the light fraction of the

sample being tested. As for some tested procedure,

the fraction of sample taken into account is low

(c.200 mg), therefore the sample preparation will

be a key issue. The protocol will need to take into

account the sampling of the material. Some of the

legislation surrounding this has yet to be clarified by

the Commission. The final point raised on this topic

was asking about the perspective offered by

fluorescence microscopy and immunology.

Effect of sample grinding on PAPs detection by

light microscopy (by P. Veys EURL-AP)

This study was completed by the Irish NRL. The

study demonstrated that the different grinding

process used did not significantly impact the bone

detection at level of 0.01% and 0.05% PAP in the

feed matrix used. Although variations were

observed in bone numbers those variations (on the

exception of a combined grinding with a rotor mill

operating with a 5 mm sieve and an Alizarin red

colouring) were negligible. Unexpectedly no

correlation could be found between the amount of

sediment used and the number of bones found.

Update on DG SANTE’s TSE activities (by L.

Carrouée, DG-Sante)

The lecture started with a presentation of the EFSA

recent reports and opinions: Zoonotic potential of

scrapie, Croatia's BSE monitoring regime and

Insects as food and feed. The ongoing and future

mandates of EFSA were also listed. The second

point presented by L. Carrouee, was about the

regulatory updates regarding the feed ban

(reintroduction of non- ruminant PAPs in aquafeed,

use of starfish, export of PAPs and use of insect PAP

in aquafeed) and the SRM. Last point presented

was about a retrospective exercise on BSE

surveillance and CWD. Items discussed in response

to the presentation were the clarification between

feed and substrate. Substrate has to be considered

as feed. PAP detection by light microscopy should

probably not be affected by the presence of insect

material. Light microscopy has some potential to

detect the presence of illegal addition of insect

material. The method still need some development

and has to be widely tested.

Pig feed and poultry feed survey (by P Veys EURL-AP) Based on commercial samples being collected and analysed by the EURL-AP, the lecture attempted to inform the delegates on the risk of authorising terrestrial PAP (other than prohibited ruminant PAPs) to be added to pig feed and to poultry feed. The key point is to sort the source of ruminant and pork DNA signals in presence of those authorised terrestrial PAPs. For poultry feed it seems that casein could likely cause issues since it is used in the composition of feed additives. There was a discussion on the test results presented. Positive PCR results for poultry feed are clearly below the cut-off value. It is mentioned that casein is used as carrier. Combination of PCR and ELISA results should be able to conclude if the positive result is coming from the use of an additive. This has to be investigated by EULR-AP and will be reported at a future date.

Page 32: DAFM NRL Newsletter Volume 6, Issue 1. - Agriculture · Exotic Animal Viral Diseases ... Parasites Dr W Byrne Contagious equine metritis Mr J Moriarty & Dr E Ryan Bovine Viral Diarrhoea

DAFM NRL Newsletter Volume 7, Issue 1.

Page 32

Food Health and the Role of National Reference

Laboratories Conference Backweston 3-4th

February 2016

A national NRL conference was organised in

February 2016, entitled “Food, Health and the Role

of National Reference Laboratories”. The

conference was attended by 140 delegates and

included representatives from all of Ireland’s NRLs,

policy makers and academics.

The conference was addressed by a range of

international and national speakers. This report

details the presentations given and presents

conclusions on future actions to support Ireland’s

NRL network.

Among the speakers were:

Pamela Byrne, CEO, FSAI - The role of NRLs in

ensuring safe and trustworthy food for


Jan Von Kietzell, Head of Sector, Pesticides

and Import Controls, FVO - FVO perspective on

official laboratories and NRLs

Lucie Carrouée-Pook, Legislative Officer, Food

Safety & Zoonoses EURLs, DG-Sante -The EU

approach to reference laboratories

Leen Van Ginkel, Director EURL for Residues,

RIKILT, Wageningen - A view on the changing

role of EURLs

Bertrand Lombard, L. monocytogenes,

Staphylococci and Milk EURL, ANSES -

Experiences and potential of food

microbiology laboratories – Illustration with

EURLs/NRL networks for L. monocytogenes,

Staphylococci, Milk & Milk Products

Donald King, Foot and Mouth Disease EURL,

The Pirbright Institute - Examples of the

coordination, advice, training and surveillance

activities undertaken by the EURL for foot and

mouth disease

Enikő Varga, Scientific Officer, Evidence

Management Unit, EFSA - Usage of data in

EFSA’s work and risk assessments

Gail Carroll, Services Contract Manger, FSAI -

Impact of the new Official Controls Regulation

on laboratories.

Adrienne Duff, Director of Irish National

Accreditation Board - Accreditation of Flexible


Martin Maiden, Prof Molecular Epidemiology,

University of Oxford - Whole genome data for

the analysis of food borne Infections

Baldissera Giovanni, Co-ordinator, European

Plant Health Research Co-ordination, EPPO -

EUPHRESCO and plant health research

David Butler, Director of Sustainable Food

Systems Ireland - Applying Ireland’s agri-food

expertise in international markets

Lance O’Brien Head of Strategy and

International Relations, Teagasc -Teagasc

Technology Foresight 2035

Richard Howell, Head of Research and Codex

Division, DAFM - DAFM strategic planning and

competitive funding for research; and role in

Codex Alimentarius Commission

A full report on this conference can be read at Food Health and the Role of NRL's, Final Report