The cuticle covering the outer surface of an eggshell functions as both a physical and chemical barrier against invading microorganisms. Contamination of eggs by microbial pathogens progresses in four stages: bacterial attachment to the egg surface, penetration through the cuticle and eggshell, multiplication within the underlying membranes, and the final stage of contaminating the egg contents. Therefore, it is important to study bacterial count at the first point of contact, i.e., on the surface of the eggs. In this study, we have evaluated the impact of differences in cuticle quality (due to egg washing and hen age) on bacterial load. We compared bacterial adherence on the eggshell surface of white eggs which were either washed (graded) or unwashed (ungraded), collected from Lohmann laying hens of different ages: early (24–28 weeks), mid-lay (44–48 weeks), and late (66–70 weeks). We aimed to determine the impact of hen age and egg washing on differences in cuticle quality and bacterial adherence. Our results indicate that hen age (up to 70 weeks) and commercial egg washing do not significantly impact bacterial adherence on eggshell surfaces. We have developed a novel method using green fluorescent protein (GFP)-expressing Salmonella typhimurium to estimate adherence of bacteria to the eggshell surface, with independent measurement of autofluorescence to quantitate cuticle deposition. S. typhimurium were localized, adhering to cracks visible on the outer cuticle in ungraded eggs, indicating that egg-associated pathogens usually enter the egg interior either through respiratory pores in eggshells or through shell micro-cracks. The results of this study can be utilized to optimize innovative methods for predictive microbiology in order to achieve egg safety.

Real-time quaking-induced conversion (RT-QuIC) assays have become a common tool to detect chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for methods to optimize the RT-QuIC assay. In this study, receiver operating characteristic (ROC) analysis was applied to optimize the assay duration for CWD screening in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (T_stdev) and a max-point ratio (maximum/background fluorescence) threshold (T_MPR) to determine CWD positivity. The optimal assay duration was 33 h for obex and 30 h for RLN based on T_stdev, and 29 h for obex and 32 h for RLN based on T_MPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. At a replicate level, using the optimized assay durations with T_Stdev and T_MPR, the level of agreement with enzyme-linked immunosorbent assay (ELISA) was significantly higher (p < 0.05) than that when using a 40 h assay duration. These findings demonstrate that the optimization of assay duration via a ROC analysis can improve RT-QuIC assays for screening CWD in white-tailed deer.

Real-time quaking-induced conversion (RT-QuIC) assays have become common in the detection of chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for optimization of the RT-QuIC assay duration. In this study, receiver operating characteristic (ROC) analysis was applied to optimize assay duration for detection of CWD in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 27 h for both obex and RLN based on Tstdev, and 27 and 28 h for obex and RLN, respectively, based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. Results by RT-QuIC using optimized duration based on Tstdev and TMPR were in 100% and 92.3 % or higher agreement with those by the widely used screening assay, ELISA. In comparison, when using a 40 h assay duration, the agreement between RT-QuIC and ELISA reduced to 89.2% or higher, and the RT-QuIC results were significantly (p < 0.05) different from those using optimum durations. These findings demonstrated that the application of ROC analysis for the optimization of assay duration could improve the RT-QuIC assay for screening CWD in white-tailed deer.