National Microbiological Baseline Study in Broiler Chicken
December 2012 – December 2013

Laboratory analysis

Sample preparation

Whole carcasses and parts collected at abattoir and retail were aseptically removed from their packages or sample bags at the third-party laboratory and prepared for a rinsing procedure. All whole carcasses were rinsed according to the FSIS procedure for whole bird rinse whereby each carcass is rinsed inside and out with 400 mL of buffered peptone water (BPW) using a rocking motion for 1 min (CFIA, 2010). For chicken part samples, BPW was added to achieve a final meat to BPW ratio of 4.5 g per mL, and samples were manually massaged by kneading the stomacher bag for 2 min (FSIS, 2010a). The contents of each individual caecum from a sample of 20 caeca was aseptically collected to prevent possible cross-contamination from the external surface of caeca, and pooled to form one composite sample. The composite of caecal content was suspended in BPW in a 1:4 ratio (w/w) and stomached for 2 min. All prepared samples were refrigerated pending testing.

Analytical methods

All samples were screened for Salmonella by the BAX® PCR system using the MLG 4C.03 method (FSIS, 2011a). Depending on the sample type, presumptive positives were either confirmed by the culture method MLG 4.05 (FSIS, 2011b) or enumerated using a combination of MLG 4.05 and a 3-tube Most Probable Number (MPN) procedure as described in MLG Appendix 2.03 (FSIS, 2008). All MPN tubes were cultured and confirmed for Salmonella species according to MLG 4.05. For caeca samples, the concentration of Salmonella was determined in a limited number of positive samples set at a maximum of 36 samples per month. As the concentration of Salmonella in caeca samples was greater than the maximum MPN value in a large proportion of positive caeca samples, the number of dilutions was increased from three to five during the course of the study.

All samples were also tested by the direct agar plating method MLG 41.01 (FSIS, 2010b) for detection and enumeration of Campylobacter with the following modifications. For caeca samples, volumes of 0.1 mL of 10-4 to 10-6 dilutions of caecal contents were plated onto duplicate Campy-Cefex plates. The low dilutions of caecal contents (10-1 and 10-3) were not plated due to the observation of significant overgrowth on these plates during the pilot phase of the study resulting in plates that were not countable. For weep fluids, volumes of 0.1 mL of undiluted weep sample and of the 10-1 to 10-4 dilutions were plated onto duplicate Campy-Cefex plates. Regardless of sample type, five suspect colonies, proportional to all typical colony types observed from one or more plates were picked and confirmed for Campylobacter species. Each colony was examined for characteristic morphology and motility under phase-contrast microscopy. All presumptive positive isolates were pooled and confirmed by latex agglutination assay specific for C. jejuni, C. coli, and C. lari. Enumeration of Campylobacter was conducted for each confirmed pool whereby three colonies for each colony type were individually speciated using a multiplex PCR method specific for C. jejuni and C. coli (Health Canada, 2011). All colonies belonging to confirmed colony types were counted on the appropriate dilution plates. Thus, the Campylobacter counts reported herein are the total count of C. jejuni and C. coli. In addition to direct plating, all carcasses and parts were tested by a qualitative or broth enrichment culture method as described in MLG 41.01.

Detection and enumeration of generic E. coli was only performed on rinse fluids from whole carcasses and parts collected in abattoir according to the MFHPB-34 method (Health Canada, 2001), but using 1 mL of undiluted sample and four serial dilutions (10-1 to 10-4) prepared with BPW (Curiale, 1991).

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