ANNEX I

TECHNICAL SPECIFICATIONS REFERRED TO IN ARTICLE 4

PART A

Sampling frame

To avoid age-related effects the monitoring shall be carried out on slaughter batches at the slaughterhouse.

As prevalence of Campylobacter spp. has been shown to vary significantly depending on the season, this merits stratification. For that purpose a 12-month period must be divided in 12 periods of one month. In each of those periods 1/12th of the total sample size must be taken.

The sampling must otherwise be based on a random selection, both regarding slaughterhouses, sampling days each month and which batches are to be sampled on a selected sampling day. In particular, the randomisation scheme shall guarantee a selection of slaughter batches proportionate to the number of flocks fattened according to the different production types (conventional, free-range, organic). In addition, the Salmonella spp. or Campylobacter spp. status, if known at slaughter, must not bias the randomisation. The competent authority shall take responsibility for generating the randomization scheme and ensuring it is implemented correctly. An example of a randomisation procedure is provided in the Report of the EFSA Task Force on Monitoring of Zoonosis Data Collection on proposed technical specifications for a coordinated monitoring programme for Salmonella and Campylobacter in broiler meat in the EU. Details of the randomisation scheme shall be reported to the Commission.

PART B

Sample size

1.   Primary sample size

(a)	The primary sample size gives the number of slaughter batches to be tested.

(b)	At least 384 slaughter batches shall be sampled. Non-response shall be anticipated by sampling about 10 % more than the indicated numbers.

(c)	By way of derogation from point (b), the following numbers of slaughter batches (1) shall be sampled in Estonia, Latvia and Luxembourg: (i) in Estonia, at least 96 slaughter batches; (ii) in Latvia, at least 120 slaughter batches; (iii) in Luxembourg, at least 12 slaughter batches.

2.   Secondary sample size

The secondary sample size gives the number of individual broiler chickens per slaughter batch to be sampled. That number shall be 10 birds for the detection of Campylobacter in caeca and one bird for the detection of Campylobacter and Salmonella on carcasses. These caeca samples and carcass sample must be from the same slaughter batch.

PART C

Specimen collection, handling and analysis for the detection and antimicrobial testing of Campylobacter spp. in broiler flocks

1.   Collection and transport

Campylobacters are relatively fragile organisms, which die quickly outside the host gut. Therefore, care shall be taken to ensure that samples are taken appropriately and analysed quickly. Extreme temperatures have to be avoided and transport shall be as fast as possible.

Samples to be collected shall be intact caeca. Caecal samples shall be taken at the time of evisceration.

Only staff trained in standard sampling procedures shall collect samples. The main objective shall be to minimise external contamination from caecal content while sampling. That is best achieved by careful manual traction at the junction with the intestine. One intact caecum shall be taken per bird, and samplers shall verify that the caecum is full or it shall be disregarded. Birds shall preferably be sampled at random throughout the batch (avoiding the first part of the batch to be slaughtered), collecting samples from non-consecutive birds. The 10 caeca collected may be placed in a single sterile bag/pack for transport.

All relevant information available from the sample must be recorded on a sampling form produced by the competent authority to enable the reporting requirements in Part E to be fulfilled. Each sample and its sample form must be labelled with a unique number which must be used from sampling to testing. The competent authority must arrange for the issue and use of a unique numbering system. The same identifier of the slaughter batch shall be used as the one for the carcass sample.

Caecal samples shall be transported as intact caeca to the laboratory within 24 hours (i.e. overnight postage or courier) and analysed there immediately. In cases where that cannot be managed, the samples shall be kept refrigerated at least until transported to the laboratory and they shall be analysed no later than 72-80 hours after sampling. At the laboratory, samples that cannot be tested on the day of arrival shall be kept refrigerated until analysis.

At the laboratory, the caecal contents shall be aseptically removed and pooled to one composite sample.

2.   Diagnostic method

2.1.   Culture

Direct culture on a selective medium provides a good estimate of the prevalence of Campylobacters. Direct culture of the sample shall be carried out on a selective medium suitable for Campylobacter, (i.e. modified Campylobacter blood free selective medium (CCDA); Karmali; or Preston Agar).

The plates shall be incubated at 41,5 ± 1 °C, in a micro-aerobic atmosphere, for at least 48 +/– 2 hours. Growth may be detected after 24 hours.

The micro-aerobic atmosphere may be obtained in commercially available micro-aerobic incubators (gas mixture 10 % CO2/6 % O2). In the absence of such incubators, micro-aerobic culture systems can be used i.e. gas jars. Commercial gas pack systems providing the appropriate micro-aerobic atmosphere are available.

Suitable positive and negative controls shall be included for each batch of samples cultured.

2.2.   Confirmation and speciation of the genus Campylobacter

Isolation and confirmation of Campylobacter organisms should be undertaken as described in ISO 10272-1:2006(E). At least one Campylobacter isolate per batch must be speciated using phenotypic methods as described in ISO 10272-1:2006(E) or published molecular methods such as Polymerase Chain Reaction (PCR) techniques. The method used shall be indicated. The isolate speciated should be used for subsequent antimicrobial testing.

If a laboratory is less experienced in speciation, it shall store the isolate as set out in 2.4 pending additional training or send it to a more experience laboratory in consultation of the Community reference laboratory for Campylobacter.

2.3.   Quality control

For quality assurance, a proportion of Campylobacter spp. isolates with a maximum of eight isolates shall be sent to the Community reference laboratory for Campylobacter for confirmation and speciation.

A proportion of those isolates shall be sent to that laboratory either in one batch or on a quarterly basis. If isolates are to be transported between laboratories, appropriate conditions (for example charcoal swabs) shall be used.

2.4.   Storage

At least one isolate per positive sample shall be stored at the NRLs using the normal method for NRL culture collection, as long as it ensures viability of the strains for a minimum of two years.

2.5.   Antimicrobial resistance testing

The number of Campylobacter isolates to be included in the antimicrobial resistance monitoring per Member State shall be 170. Not more than one isolate per Campylobacter species from the same slaughter batch shall be included in the monitoring.

In those Member States where, in any given year, a lower number of isolates than the target sample size is available, all these isolates shall be included in the antimicrobial resistance monitoring.

In those Member States where a higher number of isolates is available all isolates, or a representative random selection equal or larger than the target sample size, shall be included.

Member States shall test at least the antimicrobials that are specified in Table 1, using the cut-off values given and an appropriate concentration range to determine the susceptibility of Campylobacter.

Table 1

	Antimicrobial	Cut-off value (mg/L) R >
Campylobacter Jejuni	Erythromycin	4
	Ciprofloxacin	1
	Tetracycline	2
	Streptomycin	2
	Gentamicin	1
Campylobacter Coli	Erythromycin	16
	Ciprofloxacin	1
	Tetracycline	2
	Streptomycin	4
	Gentamicin	2

Dilution methods shall be performed according to the methods described in CLSI guidelines M31-A3 — Third Edition, Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals and M100-S16, Performance Standards for Antimicrobial Susceptibility testing; Sixteenth International Supplement.

PART D

Specimen collection, handling and analysis for the detection of Campylobacter spp. and Salmonella Spp. in broiler carcasses

1.   Collection and transport

One whole carcass per slaughter batch shall be collected immediately after chilling but before further processing such as freezing, cutting or packaging. In some slaughterhouses this may mean that samples are taken after pre-chilling when this is the last step before further processing.

The sample collected shall be placed in a separate sterile plastic bag, avoiding cross-contamination and be sent to the laboratory where skin sampling shall be undertaken.

Cross-contamination by other carcasses or caeca samples must be avoided during the collection of the carcasses. Precautions must therefore be taken at all stages to ensure that the equipment used during sampling, transport and storage are not contaminated with the pathogens investigated in the survey.

All relevant information available from the sample shall be recorded on a sampling form produced by the competent authority to enable the recording requirements in Part E to be fulfilled.

Each sample and its sample form shall be labelled with a unique number which shall be used from sampling to testing. The competent authority must arrange for the issue and use of a unique numbering system. The same identifier of the slaughter batch shall be used as the one for the caeca samples.

The samples shall be kept at between + 2 to 8 °C and free of external contamination during transportation.

All samples shall ideally reach the laboratory within 24 hours of sampling. In exceptional situations (for example, long journeys, weekends and public holidays) that period may be extended to 80 hours.

In the case that different laboratories are used for Campylobacter and Salmonella testing then the laboratory testing Campylobacter should take preference in receipt of the sample.

2.   Sampling in the laboratory and the analytical methods

2.1.   Receipt of samples

On receipt of the samples, laboratories shall check the information recorded by the sampler and complete the relevant sections of the sample form.

Samples shall be held at + 2-8 °C in the laboratory and the laboratory sampling procedure shall begin as soon as possible after the arrival of the samples at the laboratory and in any case within 72-80 hours from the time of sampling.

2.2.   Sample preparation

All samples received shall be examined to ensure that the transport packaging is intact before testing.

Handlers must avoid cross-contamination between samples and from the surrounding environment at all stages.

Wearing disposable gloves, the chicken shall be removed from its sample bag, taking care not to contaminate the outer surface of the chicken.

Using a sterile instrument and aseptic technique, the neck skin shall be removed, if present, together with the skin from one side of the carcass avoiding any fat to make a 27 g test portion and placed into a stomacher bag (or pulsifier).

2.3.   Initial suspension

The 27 g test portion shall be transferred to NINE volumes (243 ml) buffered peptone water (BPW), brought to room temperature before adding. The mixture shall be treated in a stomacher or pulsifier for approximately ONE minute (27 g are needed to perform analyses for Salmonella spp. and Campylobacter spp. from one sample in parallel). Foaming shall be avoided by removing the air from the stomacher bag as much as possible.

This initial suspension shall be used as follows:

(a)	10 ml (~1g) shall be transferred to 90 ml enrichment medium for Campylobacter spp. detection;

(b)	10 ml (~1g) shall be transferred to an empty sterile tube; 1 ml is used for the enumeration of Campylobacter spp. on selective plates.

The rest of the initial suspension (250 ml ~ 25g) shall be used for the detection of Salmonella spp.

2.4.   Detection, identification methods for Salmonella spp.

2.4.1.   Detection of Salmonella spp.

The detection of Salmonella spp. shall be done according to ISO 6579-2002 (E). ‘Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Salmonella spp.’

2.4.2.   Serotyping of Salmonella spp.

At least one isolate from each positive sample shall be typed by the National Reference Laboratory for Salmonella, using the Kaufmann-White scheme.

For quality assurance, a proportion of the non-typeable isolates shall be sent to the Community Reference Laboratory for Salmonella, with a maximum of 16 non-typeable isolates. A proportion of those isolates shall be sent to that laboratory on a quarterly basis.

2.4.3.   Phage typing of Salmonella spp.

For S. Enteritidis and S. Typhimurium it is recommended that at least one isolate from each positive sample shall be phage typed, using the protocol defined by the Health Protection Agency (HPA), Colindale, London.

2.5.   Detection, identification and quantification methods for Campylobacter spp.

2.5.1.   Detection of Campylobacter spp.

Isolation and confirmation of Campylobacter organisms should be undertaken as described in ISO 10272-1:2006(E). At least one Campylobacter isolate per batch must be speciated using phenotypic methods as described in ISO 10272-1:2006(E) or published molecular methods such as Polymerase Chain Reaction (PCR) techniques. The method used shall be indicated.

For quality assurance, a proportion of Campylobacter spp. isolates with a maximum of eight isolates shall be sent to the Community reference laboratory for Campylobacter for confirmation and speciation.

A proportion of those isolates shall be sent to that laboratory on a quarterly basis. If isolates are to be transported between laboratories, appropriate conditions (for example charcoal swabs) shall be used.

2.5.2.   Quantification of Campylobacter spp.

The quantitative detection of Campylobacter spp. shall be carried out according to ISO/TS 10272-2:2006 ‘Microbiology of food and animal feeding stuffs — Horizontal method for detection and enumeration of Campylobacter spp. Part 2: Colony-count technique’. Starting with 10 ml initial suspension 0,1 ml of this initial suspension and of further dilutions thereof shall be examined to allow the enumeration of up to 106 cfu/g. In addition, 1 ml of the undiluted initial suspension shall be examined to obtain a limit of enumeration of 10 cfu/g. All plate determinations shall be done in duplicate.

To enable correct comparison and judgement of data (for future risk assessment) the measurement uncertainty (MU) of the quantitative determination method shall be estimated for each laboratory.

To estimate the MU the technical specification ISO/TS 19036:2006 shall be used with the exception that the parallel dilutions from the initial suspension is applied for estimation of the MU.

The MU is derived from the intra-laboratory standard deviation of reproducibility. Data on MU estimation shall be collected from May to September in order to ensure positive samples. A total of 12 positive samples shall be examined in duplicate and parallel dilutions prepared from the 10 ml initial suspension. Raw data on MU estimation shall be reported separately as part of the overall description on the implementation of the survey as set out in Part E.

3.   Storage of isolates

In order to allow for e.g. later testing for antimicrobial susceptibility storage of a representative subset of isolates is recommended. One isolate per positive sample shall be stored. The Campylobacter isolate obtained from the quantitative analysis must be preferred. The isolates must be stored at the NRLs using the normal method for NRL culture collection, as long as it ensures viability of the strains for a minimum of two years.

PART E

Reporting

Reports shall be made including at least the following information:

1.

Overall description on the implementation of the survey: — slaughterhouses: total number per country and the number that were sampled, — primary sample size realised, — description of the stratification and randomisation procedures, — description of the quality control activities, inclusive a report on the 12 MU estimations per laboratory for Campylobacter quantification, — overall results.

2.

Specific information with regard to prevalence data Member States shall submit the results of the investigation in the form of raw data using a data dictionary and data collection forms provided by the Commission. That data shall include at least the following information: — name/code of the slaughterhouse, — identifier of the slaughter batch, — name/code of the holding (farm) of origin of the slaughter batch, — holding size if known, — Salmonella vaccination status of the flock if known, — age of the broilers at sampling (slaughter), — information regarding if this was the first or a subsequent batch to be slaughtered from the flock (preceding thinning or not), — production type (i.e. conventional, free-range, organic), — results of previous Salmonella and Campylobacter testing in the same flock, — date of sampling, — number of birds slaughtered per year in that slaughterhouse, — type of chilling method used (air, immersion, spray), — details of transport protocol (as specified: Y/N), — date received at laboratory, — date of testing, — identification of the laboratory, — type of sample, — description of the culture methods used, in particular the selective medium/media, — Campylobacter isolate: Method used for speciation, — Campylobacter: result of the bacteriological testing, including speciation from the caecal sample, — Campylobacter: result of the bacteriological testing, including speciation and quantification from the carcass sample, — Salmonella: result of bacteriological testing and serotyping, — time between sampling and analysis (per 12 hour period).

3.

Specific information with regard to antimicrobial resistance testing of Campylobacter isolates of caecal samples. The results of the antimicrobial resistance monitoring shall be assessed and reported, in accordance with Article 9 of Directive 2003/99/EC, in the yearly report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance. Without prejudice to the provisions of Annex IV of Directive 2003/99/EC the following information shall be reported: — origin of isolates i.e. baseline study, control programme, passive surveillance, — number of isolates susceptibility tested per Campylobacter species, — number of isolates found to be resistant per antimicrobial per Campylobacter species, and — number of fully-susceptible isolates and number of isolates resistant to 1, 2, 3, 4 and > 4 antimicrobials listed in Table 1 per Campylobacter species.

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(1)  Estimation: number of holdings (four in Estonia, five in Latvia) × two flocks per holding × two slaughter batches per flock × six rounds per year. In Luxembourg, only broilers from three small flocks are slaughtered. A slaughter batch from each of them will be sampled each trimester.

ANNEX II

Maximum Community financial contribution to the Member States

(EUR)
Member State	Maximum total amount for co-financing of sampling and analyses
Belgium — BE	58 092
Bulgaria — BG	58 092
Czech Republic — CZ	58 092
Denmark — DK	58 092
Germany — DE	58 092
Estonia — EE	14 688
Ireland — IE	58 092
Greece — EL	58 092
Spain — ES	58 092
France — FR	58 092
Italy — IT	58 092
Cyprus — CY	58 092
Latvia — LV	18 360
Lithuania — LT	58 092
Luxembourg — LU	1 836
Hungary — HU	58 092
Malta — MT	58 092
Netherlands — NL	58 092
Austria — AT	58 092
Poland — PL	58 092
Portugal — PT	58 092
Romania — RO	58 092
Slovenia — SI	58 092
Slovakia — SK	58 092
Finland — FI	58 092
Sweden — SE	58 092
United Kingdom — UK	58 092
Total	1 429 092

ANNEX III

Certified financial report on the implementation of a survey on the prevalence of Campylobacter spp. in broiler flocks and their antimicrobial resistance and on the prevalence of Campylobacter spp. and Salmonella spp. in broiler carcasses

Reporting period: … to …

Statement on costs incurred on the survey and eligible for Community financial contribution:

Reference number of Commission Decision providing Community financial contribution:

…

…

Costs incurred related to functions at/by	Number of tests	Total costs of testing incurred during reporting period (national currency)
Bacteriological detection of Campylobacter spp.
Bacteriological detection of Salmonella spp.
Confirmation of Campylobacter spp.
Speciation of Campylobacter isolates
Enumeration of Campylobacter isolates
Serotyping of Salmonella isolates
Antimicrobial resistance testing of Campylobacter isolates

Declaration by the beneficiary

We certify that:

—	the above costs are genuine and have been incurred in carrying out the tasks laid down in this Decision and were essential for the proper performance of those tasks;

—	all supporting documents supporting for the costs are available for audit purposes;

—	no other Community contribution was requested for this programme.

Date: …

Person financially responsible: …

Signature: …