Part B:Sample collection and analysis for the Salmonella survey
1.Sample collection in the herds
1.1.Type and detail of the routine sample
The material collected for bacteriological analysis shall be freshly voided faeces representing the whole holding, which is the unit of interest. Since every holding is unique, it shall be decided, before starting the sampling, which pens, yards, or groups within the holding are sampled. The sample collected shall be placed in a separate container avoiding cross-contamination and sent to the laboratory.
Each pooled sample shall total at least 25 g and two approaches may be employed to collect these pooled faeces samples:
where there is an accumulation of mixed faeces within an area of a pen or yard, a large swab (e.g. 20 cm × 20 cm) can be used to pass through the faecal mass, ensuring that at least 25 g of mixed material is collected. This may be achieved by for example, moving the swab along a 2-metre zig-zag path such that it is well coated with faecal material. If necessary, for example due to hot weather or on slatted flooring, then the swab may be moistened with an appropriate liquid such as drinking water.
where there is no such accumulation, for example in a field, large yard, in a farrowing house, or pens or other accommodation with low numbers of pigs per group, then individual pinches shall be selected from individual fresh faecal masses or places so that a minimum of 10 individuals, contribute to a total sample volume of at least 25 g. The sites from which these pinches are collected should be distributed in a representative manner across the area concerned.
Approach 1 shall be preferred where practical. In this approach at least 10 individual pigs must contribute to each sample taken, otherwise approach 2 shall be applied.
1.2.Additional sampling for the within-holding prevalence study
Together, 10 holdings, selected at random from the overall sample of breeding holdings and of production holdings are subjected to more intensive sampling. On these holdings 10 routine samples shall be collected in the same manner as described previously (Section 2.1 of Part A). In addition, 10 individual samples of at least 30 g shall be collected in each selected pen and shall be identified in such a manner that these 10 individual samples can be associated with the routine sample from that pen. Thus in total, 10 routine samples and 100 (10 × 10) individual samples shall be collected from each of these 10 holdings. The processing of these samples is described in Section 2.3.1.
This sampling should be applied in Czech Republic, Denmark, Romania, Slovenia, Sweden and the United Kingdom.
1.3.Sample information
All relevant information available from the sample shall be recorded on a sampling form produced by the competent authority to enable the data requirements in Part D to be fulfilled.
Each sample and its sample form shall be labelled with a unique number which shall be used from sampling to testing, and with the code of the pen. The competent authority must arrange for the issue and use of a unique numbering system.
1.4.Transport of samples
Samples shall be preferably kept between + 2 and + 8 °C and free of external contamination during transportation. The samples shall be sent to the laboratory as quick as possible within 36 hours by fast mail or courier and shall reach the laboratory no later than 72 hours after sampling.
2.Laboratory analytical methods
2.1.Laboratories
Analysis and serotyping 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 control of Salmonella to perform the analyses. These laboratories shall have proven experience of using the required detection method and have a quality assurance system complying with ISO 17025 and be submitted to the supervision of the NRL.
2.2.Receipt of samples
At the laboratory, samples shall be kept refrigerated until bacteriological examination, which shall preferably be carried out within 24 hours after receipt but in any case no later than 96 hours after the sample was collected.
2.3.Sample analysis
Member States shall guarantee that all involved parties have been sufficiently trained to carry out the analyses.
2.3.1.Preparation
In the laboratory, routine samples shall be mixed carefully and thoroughly, before 25 g is collected for analysis.
For evaluation of the within-holding prevalence in accordance with paragraph 1.2, each of the individual collected samples (30 g) needs to be divided into two parts. One part, weighing at least 25 g shall be mixed carefully and thoroughly and subsequently cultured individually. The remaining second part is to be used to prepare an artificially pooled sample from the 10 individual samples in the selected pen, group or yard. This latter part shall be prepared by adding 10 times 2,5 g of the individual samples to create an artificially pooled sample of 25 g. The artificially pooled samples are mixed carefully and thoroughly before analysis. In total, 10 routine samples, 10 artificially pooled sample and 100 individual samples shall be analysed from each of the 10 holdings selected for the estimation of the within-holding prevalence.
2.3.2.Detection and identification methods
2.3.2.1.Detection of Salmonella
The method recommended by the Community Reference Laboratory (CRL) for Salmonella in Bilthoven, the Netherlands, shall be used. This method is described in the Annex D of ISO 6579: ‘Detection of Salmonella spp. in animal faeces and in samples of the primary production stage’. The latest version of Annex D shall be used.
2.3.2.2.Serotyping of Salmonella
All strains isolated and confirmed as Salmonella spp. shall be serotyped according to the Kaufmann-White scheme, by the NRL for Salmonella.
For quality assurance, 16 typable strains and 16 non-typable isolates shall be sent to the CRL for Salmonella. A proportion of these isolates shall be sent to the CRL on a quarterly basis. If fewer strains have been isolated, all shall be sent.
2.3.2.3.Phage typing of Salmonella
In case isolates of Salmonella Enteritidis and Salmonella Typhimurium are phage typed (optional), the methods described by the WHO reference centre for phage typing of Salmonella of the Health Protection Agency (HPA) Colindale, London, shall be used.