ANNEX I

Technical specifications in accordance with Article 2

1.   Sampling frame

A minimum number of pigs, kept during at least the preceding three months in the Member State, shall be sampled, at random, as follows:

Bulgaria: 192

Romania: 300

Bulgaria and Romania shall take a 10 % extra number of samples, to be analyzed in case some samples excluded from the study for various reasons.

Sampling shall be stratified by slaughterhouses that participate and proportional to slaughterhouse capacity. Each of those Member States shall rank all slaughterhouses according to their fattening pig throughput in the previous year. Thus, they shall each identify those plants that accounted for at least 80 % of all slaughtered fattening pigs.

The total number of pigs and carcasses to be sampled in each of the slaughterhouses included in the study shall be estimated by multiplying the sample size (for example, 2 400) by the proportion of fattening pigs processed in the previous year. For example, if one slaughterhouse accounted for 25 % of fattening pigs slaughtered in the selected slaughterhouses (those representing at least 80 % of all fattening pigs slaughtered in the Member State), then (2 400 × 0,25) 600 pigs shall be sampled. These shall be evenly divided so that 50 pigs sampled in every month, for 12 months. A further example is shown in Table 1.

However, if a slaughterhouse is no longer in production, if a new facility has been opened or there is predicted to be a significant change in plant throughput during the survey, then the estimated throughput shall be adjusted accordingly.

Table 1

Weighing of slaughterhouses for the purpose of allocating the number of fattening pigs to be sampled from each slaughterhouse; calculation of sampled animals per slaughterhouse

Slaughterhouse ID	Number of fattening pigs processed previous year	Percent of total slaughter included in the study	Number of samples per slaughterhouse	Samples per month (/12)
AXD	88 000	17,6	0,176 × 2 400 = 422,4	422,4 : 12 = 36
SVH	25 000	5,0
TPB	75 000	15,0
MLG	100 000	20,0
GHT	212 000	42,4
Total	500 000  (1)	100,0

For each slaughterhouse each month, a number between 1 and 31 shall be selected at random. If the randomly selected number is a slaughtering day, for that month, then that day is selected for sampling. If not, then a new number shall be selected randomly. This process shall be performed once a month and repeated as many times as there are samples to be collected at the slaughterhouse. For example, in the slaughterhouse AXD the process shall be repeated at least 36 times to select at least 36 working days randomly. Accordingly, there may be more than one carcass to be sampled on the same day.

As the number of animals slaughtered on a specific day may vary enormously, the random selection of the individual animal shall take place at the slaughterhouse at the date randomly selected for sampling. The given day, the total number of animals must be known, and the personnel of the slaughterhouse shall then randomly select a carcass or carcasses using the randomization sheet which has been provided to them and which has been generated using a maximum that exceeds the highest possible number of fattening pigs slaughtered on any given day in any slaughterhouse in the Member State.

A randomization table may then look as shown in Table 2.

Table 2

Randomization table.

Slaughterhouse	Day of the month	Identity of carcass (2)
AXD	19	5
	4	2
	12	124
	12	2
	8	59

The following animals shall be excluded from the baseline study:

—	animals with a live weight of less than 50 kg or more than 170 kg,

—	animals that have undergone emergency slaughter,

—	any carcass that is totally condemned.

2.   Samples

2.1.   Sampling in general

—	The aggregate of ileocaecal lymph nodes or at least five individual ileocaecal lymph nodes of all selected pigs shall be collected. If possible, at least 25 grams of lymph nodes without fat or connective tissues shall be collected.

—	Records shall be kept at the slaughterhouse on the date and time of sampling of each sample and the date and time and name of the courier that takes delivery of the samples.

2.2.   Details on sampling of ileocaecal lymph nodes

The mesenterium between the caecum and the part of the ileum that is closest to the caecum shall be torn and the ileocaecal lymph nodes are presented at the surface of the torn-open area. Without a knife, but with gloved fingers, the lymph nodes shall be bluntly ‘harvested’ from such opened mesenterium if individual lymph nodes are collected. The lymph nodes or the aggregate shall be placed in a plastic bag which is marked with date, time, slaughterhouse identification and sample identification code.

3.   Transport

Samples shall be sent within 36 hours by express mail or courier and shall reach the laboratory no later than 72 hours following sampling. Samples arriving more than 72 hours following sampling shall be discarded unless the analysis is initiated within 96 hours following sampling and the cold chain has not been interrupted.

4.   Analysis and serotyping of samples

Analysis and serotyping of samples shall take place at the national reference laboratory (NRL). Where the NRL does not have the capacity to perform all analyses or if it is not the laboratory that performs detection routinely, the competent authorities may decide to designate a limited number of other laboratories involved in official controls of Salmonella to perform the analyses.

Those laboratories must have proven experience of using the required detection method and have a quality assurance system complying with ISO standard 17025 and be subject to the supervision of the NRL.

At the laboratory, samples shall be kept refrigerated until bacteriological examination, which shall be carried out within 24 hours following receipt of the samples so that analysis is initiated no later than 96 hours following the time of collection of the samples.

4.1.   Sample preparation

Lymph nodes shall be surface de-contaminated before analysis by dipping into absolute alcohol and drying by air.

All lymph nodes shall be pooled and closed in a plastic bag and banged with a hammer or by similar means on the plastic bag smashing the lymph nodes.

The homogenized lymph nodes shall be weighed and placed in a sterile container with pre-warmed buffered peptone water (BPW) in dilution 1:10. Containers shall be incubated for a total of (18 ± 2) hours at (37 ± 1) °C.

4.2.   Detection method

The method recommended by the Community Reference Laboratory (CRL) for Salmonella in Bilthoven, the Netherlands, shall be used.

That method is described in the current version of draft Annex D of ISO 6579:2002: ‘Detection of Salmonella spp. in animal faeces and in samples of the primary production stage’. In this method, the modified semi-solid Rappaport-Vassiladis (MSRV) medium shall be used as the single selective enrichment medium.

4.3.   Serotyping

All strains isolated and confirmed as Salmonella spp. shall be serotyped according to the Kaufmann-White scheme.

For quality assurance, 16 typable strains and 16 non-typable isolates shall be sent to the CRL. If less strains have been isolated, all shall be sent.

4.4.   Phage typing

In the case where isolates of Salmonella serovar Typhimurium and Salmonella serovar Enteritidis are phage typed (optional), the methods described by WHO reference centre for phage typing of Salmonella of the Health Protection Agency (HPA), Colindale, UK, shall be used.

4.5.   Testing of anti-microbial susceptibility

In the case of testing for anti-microbial susceptibility (optional), a validated and controlled method for testing shall be used, such as those recommended by the National Committee for Clinical Laboratory Standards (NCCLS, since 1st of January 2005: ‘Clinical and Laboratory Standards Institute’ — CLSI).

Both agar diffusion and broth dilution methods shall be acceptable. Results shall be reported both as quantitative data (MIC for dilution methods and inhibition zone diameter for diffusion methods) and as qualitative data (proportion resistant isolates).

Qualitative data shall be based on interpretation according to epidemiological cut-off values presented by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) at: http://www.eucast.org

The isolates shall be tested for the susceptibility to the antimicrobial substances listed below:

—	Ampicillin or Amoxicillin

—	Tetracycline

—	Chloramphenicol

—	Florfenicol

—	Nalidixic acid

—	Ciprofloxacin (preferably) or Enrofloxacin

—	Sulphonamide (preferably Sulfametoxazole)

—	Sulphonamide/Trimethoprim or Trimethoprim

—	Gentamicin

—	Streptomycin

—	Kanamycin (preferably) or Neomycin

—	3rd generation cephalosporin, (preferably cefotaxime)

—	Colistin (optional)

Before initiation of the study the two Member States shall organise training for the involved parties.

5.   Records and sample storage

Records of bacteriology shall be kept on all samples processed in a format in accordance with or comparable to the example given in Table 3.

All strains isolated shall be stored at the NRLs of the two Member States as long as it ensures integrity of the strains for a minimum of five years.

All samples of meat juice for serology shall be stored frozen for two years.

Table 3

Example of records to be taken on all processed samples

Sample

Receipt

Analysis

Sample ID + type

Slaughterhouse ID

Name

Date

Time

Name

Date

Time

Pos or Neg

Serovar

Phagetype

Antibiogram

Storage ID

1 S

EU012

PW

3-10-06

12:00

AB

3-10

14:00

Neg

2 L

EU023

PW

4-10

12:30

AB

4-10

14:00

Pos

Typh

DT104

ASTSu

(IDnr)

3 L

EU083

PW

8-10

16:30

AB

9-10

9:00

Pos

Agona

n.a.

ASTE

(IDnr)

Etc

6.   Reporting from Bulgaria and Romania

The competent authority responsible for the preparation of the yearly national report on the monitoring of Salmonella in animals pursuant to Article 9 of Directive 2003/99/EC shall collect and evaluate the results and report to the Commission.

Those reports shall include at least the following information:

6.1.   Overall description on the implementation of the survey programme

—	description of the population under study stratified according to slaughterhouses capacity,

—	description of randomization procedure, including notification system,

—	sample size calculated,

—	details of authorities and laboratories involved in sampling/testing/typing,

—	overall results of the study (samples analyzed by bacteriology, number of positive, serovar, phage type and antibiotic resistance testing).

6.2.   Complete data on each animal sampled and corresponding tests results

The Member States shall submit the results of the survey in the form of raw data using a data dictionary and data collection forms provided by the Commission.

That dictionary and forms shall be established by the Commission and include at least the following:

—	reference of the slaughterhouse,

—	capacity of the slaughterhouse,

—	date and time of sampling,

—	reference of the samples (the number),

—	type of samples taken: lymph nodes,

—	date of dispatch to the laboratory.

The following information shall be collected in the Member States for each sample sent to the laboratory:

—	ID of the laboratory (in case several laboratories are involved),

—	means of transport of samples,

—	date of reception by the laboratory,

—	when testing lymph nodes, weight of the specimen,

—	results for the individual samples tested: ‘negative’ or in case positive for Salmonella spp., also the results of serotyping ‘ Salmonella serovar’ or ‘untypable’,

—	results for strains subject to antimicrobial susceptibility testing and/or phagetyping results.

---

(1)  This number must represent at least 80 % of slaughtered fattening pigs in a Member State.

(2)  The 5th carcass to be processed on the 19th day of that month should be sampled for the survey.

ANNEX II

Maximum Community financial contribution to Bulgaria and Romania

(EUR)
Member State	Amount
Bulgaria	4 992
Romania	7 800

ANNEX III

Certified financial report on the implementation of a baseline survey on the prevalence of Salmonella spp. in herds of slaughter pigs

Reporting period: 1 April 2007 to 30 September 2007

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)
Bacteriology for Salmonella spp.
Serotyping Salmonella isolates

Declaration by the beneficiary

We certify that

—	the costs set out in the statement on costs are genuine and have been incurred in carrying out the tasks laid down in Commission Decision 2007/219/EC and were essential for the proper performance of those tasks;

—	all supporting documents for those costs are available for audit purposes.

Date: …

Person financially responsible: …

Signature: …