Molecular detection and serotyping of foodborne pathogens in poultry
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Salmonella and Campylobacter sp. are the leading causes of gastroenteritis in the United States and poultry is a significant vehicle for transmitting these foodborne pathogens to humans. Starting from 1996, the USDA Food Safety and Inspection Service (FSIS) mandated that all poultry processing plants implement hazard analysis critical control point (HACCP) programs and meet U.S. Department of Agriculture (USDA) -approved limits for Salmonella contamination of poultry and other meat products. To reduce the organisms from being introduced into the poultry flocks, on-farm control strategies such as probiotics, vaccines, and antibiotics were also applied. All of these actions necessitate reliable and rapid schemes to detect or type foodborne pathogens in order to trace back the source of contamination and at the same time supplement or replace the conventional culture-based detection methods, which are labor intensive and time-consuming when working with large sample size. In this study, we explored the possibility of using PCR-based O and H antigen detection scheme to type poultry-related Salmonella spp. and compared these results to the conventional serotyping method. O antigen biosynthesis rfb operons for Salmonella O serogroups D1, B, C1, C2, and E1 and the variable central regions of flagella fliC and fljB genes were analyzed to search for suitable multiplex PCR primers for specific detection of Salmonella O and flagella antigens. Based on statistical data, our PCR-based typing scheme had significant sensitivity (0.80~1.00) and specificity (0.95~1.00) and is correlated well (chi-square, p<0.001) with conventional serotyping method. We also extended Dauga’s fliC and fljB RFLP PCR serotyping scheme (Dauga et al., 1998) by including Sau 3A and Hha I restriction enzymes to serotype Salmonella. RFLP PCR generated distinct serotype specific DNA profiles. Finally, we developed a PCR-ELISA detection system to screen for Salmonella, Campylobacter jejuni, and C. coli directly from chicken carcasses and the poultry environment. The probes were designed using the Salmonella virulence gene invA, and Campylobacter ceuE gene, which encodes lipoprotein involved in siderophore transport. The system showed an excellent agreement with the culture method for detecting Salmonella from enrichment samples (kappa = 0.83; chi-square test: p<0.001) or directly from chicken carcass rinses (kappa = 0.63; chi-square test: p<0.001). PCR-ELISA also has an excellent relative sensitivity (0.97) to detect Campylobacter directly from carcass rinses. A standard curve was constructed by plotting OD values against bacterial cell count for different cycles of PCR to detect Campylobacter. The log-linear phase of the curve will enable us to semi-quantitate bacterial cell concentration (CFU/mL) in a sample by knowing its relevant OD in PCR-ELISA (Ferre, 1992). Overall, PCR-ELISA is a rapid and cost-effective approach to the detection of foodborne pathogens in poultry. Our results indicate that novel molecular techniques can be used as alternative tools to either type or detect foodborne pathogens.