Evaluation of alternative methodologies for the detection of Salmonella enteritidis in table eggs

Abstract

The following studies describe the use of enzymatic digestion and chemical reduction of albumen pools to allow for concentration of Salmonella enteritidis (SE) by centrifugation and detection of SE by culture and the polymerase chain reaction (PCR). In six experiments, eggs were inoculated with low numbers of viable SE. Albumen and yolk from five or six eggs in each group were separated, the albumen was pooled, treated with papain and N-Acetyl-L-cysteine, centrifuged, and the resulting pellet incubated in tetrathionate broth at 37 C for 24 h prior to plating on brilliant green agar (BGA). In three of the experiments, following concentration, centrifugation, and enrichment of albumen pools (centrifugation technique), a sample from each pool was subjected to DNA extraction and subsequent evaluation for SE by the PCR using primers specific for members of the genus Salmonella. For comparison to the centrifugation technique and the PCR, yolk and albumen from five or six eggs in each group were pooled, thoroughly mixed, and incubated at room temperature for 3 d prior to direct plating on BGA. Results indicated the three techniques were similar in sensitivity for detection of SE (range: 72 cfu SE/pool to .6 cfu SE/pool) in experimentally inoculated eggs. However, the centrifugation technique and the PCR allowed for more rapid (48 h or 72 h respectively) presumptive identification of SE than the room temperature protocol. In an additional five experiments, Single Comb White Leghorn hens were orally challenged with SE, and eggs were collected at selected times post-challenge and cultured for SE using the three culture techniques. Significant differences were not observed in the number of SE positive pools detected between the room temperature technique (6 of 35) and the PCR (4 of 36). There is no evidence from these experiments to suggest that the PCR will erroneously identify negative samples as positive. These experiments suggest that the PCR is comparably sensitive to room temperature culture for the detection of SE in eggs derived from experimentally infected hens, while providing presumptive identification of SE 72 h sooner.