Detection and Characterisation of Salmonella in Animal Feed Samples by PCR-Based Methods

Detta är en avhandling från Applied Microbiology (LTH)

Sammanfattning: Animal feed is a recognised source of Salmonella enterica for farm livestock and may also indirectly cause infection in people consuming foods of animal origin. It is therefore important to have rapid, reproducible and specific methods for the detection of Salmonella in feed, and for the characterisation of strains for further epidemiological investigations or to trace the source of contamination in a production facility. This study focuses on the development and validation of PCR-based methods for the detection and characterisation of Salmonella in the farm-to-fork chain and, particularly, in animal feed samples. The PCR performance of a 5' nuclease real-time PCR assay was studied to optimise the detection of pre-enriched Salmonella cells in buffered peptone water (BPW). Using rTth instead of AmpliTaq Gold resulted in an earlier detection during enrichment. A simple pre-PCR processing strategy to overcome inhibition by substances in the feed was developed, based on enrichment in BPW followed by PCR using Tth DNA polymerase, which was found to exhibit resistance to PCR-inhibitory feed samples. No DNA extraction or cell lysis was included in the pre-treatment. The probability of detecting Salmonella in feed samples was found to follow a logistic regression model and the probability of detecting 1 CFU/25 g feed in artificially contaminated soya samples was 0.81. The use of the PCR method for routine analysis of feed was validated in a study on 250 feed samples where no significant difference could be observed in the results obtained by the PCR method and the culture-based standard method (Nordic Committee on Food Analysis, NMKL). By applying the PCR method the analysis time can be decreased from at least three days to 24 h. The PCR method was found to be superior to the NMKL method when analysing Salmonella in acidified feed samples due to failure to detect living but stressed Salmonella cells by NMKL while they were detected by PCR. The three genotyping methods automated ribotyping, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were evaluated for the analysis of S. Senftenberg strains originating from long-term contamination in a feed mill, and in tracing the origin of an outbreak of salmonellosis connected to consumption of a fish gratin. It was found that the reproducibility of RAPD could be improved by the use of Tth DNA polymerase and that RAPD could be used as a screening method to select the isolates that should be further studied by the more expensive and time-consuming PFGE. PFGE was useful both in finding the source of contamination in the feed factory and in investigating the epidemiology behind the outbreak. Animal feed was suggested as the source of contamination in the outbreak associated with the consumption of fish gratin. In conclusion, the implementation of PCR-based methods for the detection and characterisation of Salmonella in the food chain from farm to fork can help improve food safety.