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Abstract
Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission; thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monocytogenes) is a vital food safety issue. Recently, third generation PCR technology, known as droplet digital PCR (ddPCR) has been developed to be more accurate and sensitive to detect genetic targets than current quantification methods, but this technology has not been tested within an industrial setting. There is an on-going study within our laboratory is investigating the effects of sampling times and sampling methods on the cultural and molecular (via qPCR) quantification of dominant zoonotic pathogens within a poultry processing facility. This presents a unique opportunity to compare the quantification resulted from this emerging, third generation technology to traditional quantification methods currently employed by the poultry industry. The results show that ddPCR detected pathogen-specific genes from more pathogen:sampling time combinations than either the qPCR or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End). In fact, both ddPCR and qPCR substantially outperformed culture methods commonly used in poultry processing food safety-related studies, with Salmonella recovered only from the Mid and End sampling times from the scalder tank. While neither C. jejuni nor L. monocytogenes were recovered culturally, ddPCR was able to detect their respective genes commonly throughout the processing day in both the scalder and chiller water samples. Additionally, the use of unfiltered processing water provided significantly greater detection of bacterial and pathogen-specific gene abundances than did an analysis of larger volumes of filtered water. Considering the ddPCR-derived concentrations of the bacterial pathogens were consistent with what was previously found culturally in commercial poultry processing operations, ddPCR represented a significant advancement in poultry processing zoonotic pathogen quantification.
Highlights
The handling and consumption of poultry or poultry products have been repeatedly associated with the transmission of bacterial pathogens to the human population
The results show that droplet digital PCR (ddPCR) detected pathogen-specific genes from more pathogen:sampling time combinations than either the quantitative PCR (qPCR) or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End)
Considering the DNA extraction process claims to remove contaminants such as chlorine, and ddPCR was the most robust pathogen quantification technique in the chiller water samples, ddPCR represents a powerful new tool effectively detect and quantify zoonotic pathogens within chiller water. These findings represent the first report of the use of third generation PCR technology to detect zoonotic bacterial pathogen signatures in environmental samples along the poultry production continuum
Summary
The handling and consumption of poultry or poultry products have been repeatedly associated with the transmission of bacterial pathogens to the human population. Considering the high environmental, economic, and public health costs of these zoonoses, a comprehensive understanding of the poultry production and processing parameters that allow for the survival/transmission of these bacterial pathogens is essential. The quantification of foodborne pathogens in food production systems was based either on cultures or quantitative PCR (qPCR). While these methods have been used successfully, both come with caveats; either being time consuming and too ineffectively selective (culture-based) or dependent upon the proper standards and assay efficiencies (qPCR-based). To circumvent these issues, third generation PCR technology, known as droplet digital PCR (ddPCR) was introduced
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