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Although deposition of Salmonella Enteritidis inside yolks is less common than deposition in albumen or on the vitelline (yolk) membrane in naturally contaminated eggs laid by infected hens, bacterial migration into the yolk to reach its nutrient-rich contents could lead to extensive multiplication. The present study used an in vitro egg contamination model to assess the ability of small initial numbers of Salmonella Enteritidis to penetrate the vitelline membrane and multiply inside yolks of eggs laid by 6 genetically distinct commercial lines of hens during 24 h of storage at 30 degrees C. Eggs from each line were tested at 4 different hen ages by inoculation of approximately 100 cfu of Salmonella Enteritidis onto the outside of the vitelline membranes of intact yolks in plastic centrifuge tubes and then adding back the albumen into each tube before incubation. Overall, the frequency of penetration of Salmonella Enteritidis into the yolk contents of eggs from individual lines of hens ranged from 30 to 58% and the mean concentration of Salmonella Enteritidis in yolk contents after incubation ranged from 0.8 to 2.0 log(10) cfu/mL. For both of these parameters, values for one hen line were significantly higher than for 2 other lines, but no other differences were observed. Hen age did not have a significant effect on egg yolk penetration by Salmonella Enteritidis. These results indicate that opportunities for the migration and growth of small initial numbers of Salmonella Enteritidis to attain more dangerous levels inside contaminated eggs during storage at warm temperatures can sometimes vary between different lines of laying hens.

Although Salmonella deposition inside yolks is uncommon in naturally contaminated eggs, migration through the vitelline membrane into the nutrient-rich yolk contents could enable rapid bacterial multiplication. Egg refrigeration restricts both penetration and growth, but a recently proposed national Salmonella Enteritidis control program would allow unrefrigerated ambient temperature storage of eggs on farms for up to 36 h. The present study used an in vitro egg contamination model to assess the ability of small numbers of 4 Salmonella Enteritidis strains and 4 Salmonella Heidelberg strains to penetrate the vitelline membrane and multiply inside yolks during 36 h of storage at either 20 or 30 degrees C. After inoculation onto the exterior surface of the vitelline membrane, all 8 Salmonella strains penetrated to the yolk contents (at a mean frequency of 45.1%), and most strains grew to significantly higher levels (with a mean (log)10 bacterial concentration of 2.2 cfu/mL) during incubation at 30 degrees C. Significant differences in penetration frequency and yolk multiplication were observed between individual strains and between serotypes (Salmonella Enteritidis > Salmonella Heidelberg for both parameters). Penetration and multiplication were significantly less frequent during incubation at 20 degrees C. These results demonstrate that controlling ambient temperatures during prerefrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness.

Eggs that harbor Salmonella in their edible contents pose a significant risk of transmitting disease to consumers. Although Salmonella deposition inside yolks does not usually occur at a high frequency in naturally contaminated eggs, bacterial penetration through the vitelline membrane could lead to rapid and extensive multiplication in the nutrient-rich yolk contents. The present study used an in vitro egg contamination model to assess the ability of Salmonella strains to penetrate the vitelline membrane and multiply inside yolks. An S. enteritidis strain and 2 Salmonella heidelberg strains, initially inoculated onto the outside of the vitelline membrane, were able to enter the yolk contents (at frequencies ranging from 10 to 25% of experimentally contaminated eggs) during 24 h of incubation at 30 degrees C. Variants of these parent strains, obtained by in vivo passage into eggs laid by infected hens, penetrated the yolk membrane at significantly higher frequencies. These results demonstrate that pathogens such as S. enteritidis and S. heidelberg can penetrate into and begin to multiply inside the yolks of contaminated eggs during the first day of storage at warm temperatures.

Identifying infected laying flocks is a critical component in efforts to prevent eggborne transmission of Salmonella enteritidis to humans. In the present study, egg yolk samples from experimentally infected chickens were tested for specific antibodies with a very rapid fluorescence polarization assay using tracers prepared from the O-polysaccharides of S. enteritidis and S. typhimurium and a conventional ELISA using an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with 106 or 10(8) cfu of S. enteritidis (phage type 13a) or with 10(8) cfu of S. typhimurium. Eggs were collected during five weekly postinoculation intervals. Both fluorescence polarization and ELISA detected the majority of hens infected with S. enteritidis at either dose level, although they also frequently cross-reacted with samples from hens infected with S. typhimurium. Fluorescence polarization with an S. typhimurium tracer was likewise able to consistently detect S. typhimurium infection but also tended to cross-react with samples from hens infected with S. enteritidis. Fluorescence polarization appears to offer a simple and rapid alternative to conventional serological methodology, although concerns about specificity may limit the usefulness of antibody testing data.

The site of deposition of Salmonella enteritidis in eggs could influence the extent to which this pathogen multiplies before refrigeration achieves growth-inhibiting internal temperatures. The first part of this study sought to determine whether S. enteritidis inoculated onto the exterior (vitelline) membrane surface of egg yolks was able to penetrate into and multiply within the yolk contents. When 10(2) cfu of S. enteritidis was inoculated onto the exterior surface of intact egg yolks, multiplication within the interior yolk contents occurred in 10% of samples after 6 h of incubation and in 75% of samples after 24 h at 25 C (reaching mean levels of about 10(4) cfu/mL) but in only 20% of samples incubated for 72 h at 15 C. The second part of this study applied an oral infection model in laying hens to establish the relative proportions of contaminated eggs in which S. enteritidis deposition was associated with the yolk membrane or was found inside the yolk contents. Although approximately 4.3% of egg yolks were positive for S. enteritidis when both yolk contents and membranes were sampled, only about 0.5% of samples of yolk contents (without membranes) were positive. Although deposition of S. enteritidis within egg yolks appears to occur infrequently, rapid refrigeration of eggs is necessary to prevent the penetration of S. enteritidis into and multiplication within egg yolks.

Refrigeration of eggs is vital for restricting the multiplication of Salmonella enterica serotype Enteritidis contaminants, but differences between Salmonella Enteritidis strains or phage types in their survival and multiplication patterns in egg contents might influence the effectiveness of refrigeration standards. The present study compared the abilities of 12 Salmonella Enteritidis isolates of four phage types (4, 8, 13a, and 14b) to multiply rapidly in egg yolk and to survive for several days in egg albumen. The multiplication of very small numbers of Salmonella Enteritidis inoculated into yolk (approximately 10(1) CFU/ml) was monitored during 24 h of incubation at 25 degrees C, and the survival of much larger numbers of Salmonella Enteritidis inoculated into albumen (approximately 10(5) CFU/ml) was similarly evaluated during the first 3 days of incubation at the same temperature. In yolk, the inoculated Salmonella Enteritidis strains multiplied to mean levels of approximately 10(3) CFU/ml after 6 h of incubation and 10(8) CFU/ml after 24 h. In albumen, mean levels of approximately 10(4) CFU/ml or more of Salmonella Enteritidis were maintained through 72 h. Although a few differences in multiplication and survival were observed between individual isolates, the overall range of values was relatively narrow, and no significant differences (P < 0.05) were evident among phage types.

Serological surveillance can be an important component for egg quality assurance programs geared toward controlling problems with Salmonella enterica serovar Enteritidis (S. enteritidis) within a flock. Serum is the primary sample source for the procedures, although egg yolk antibody assays have become popularity in recent years. However, these assays tend to be labor intensive, requiring procedures for extracting antibodies from the yolk followed by assaying the samples. We describe an adaptation of the agar gel precipitin (AGP) test for use in detecting antibodies to S. enteritidis deposited in egg yolks of infected hens. Yolk or sera from infected birds were administered to wells cut into seven-well clusters in an agar gel plate, and detection antigen was added to the center well. The agar gels were incubated for 24 h and then examined for the presence of precipitin lines formed by the interaction of antibody with antigen. Three different antigens were tested: S. enteritidis flagella, SEF14 (a 14-kDa fimbrial protein produced ostensibly by S. enteritidis), and a sodium deoxycholate extract of whole S. enteritidis organism. Flagella and the organism extract detected antibodies to S. enteritidis in the yolk and sera, whereas SEF14 was not reactive. Positive reactions were observed in serum 1 wk postchallenge, whereas in yolks, this was further delayed by 1 wk. The sensitivity of the test was slightly less than the standard microagglutination assay, although specificity was slightly higher, as indicated by results from sera and yolks from birds infected with Salmonella enterica serovar Typhimurium. Simplicity and low labor requirements of the assay would allow for the potential testing of several hundred egg samples within a day, which would make up for test shortcomings due to sensitivity. The AGP test could be an important tool for individuals using serological testing to monitor the S. enteritidis situation within their flocks or as a rapid screen for vaccine responses. The assay could also be used in tandem with other AGP tests to screen for the presence of multiple avian pathogens.

Because egg yolk and albumen differ substantially in their abilities to support bacterial growth, the initial level and location of Salmonella enteritidis deposition are critical for determining whether proposed standards for refrigerating eggs are likely to protect public health by preventing extensive microbial multiplication. In the present study, three groups of laying hens were infected with oral doses of approximately 10(9) cells of different S. enteritidis strains (two were phage type 4 and one was phage type 13a) in two replicate trials. For all three S. enteritidis strains, the incidence of yolk contamination (approximately 2.5% overall) was significantly greater than the incidence of albumen contamination (approximately 0.5% overall). The phage type 13a strain was less often isolated from fecal samples at 2 wk post-inoculation than were the phage type 4 strains, but no significant differences between strains were observed in the incidence of egg contamination. Most freshly laid contaminated eggs contained fewer than 1 S. enteritidis cell/ml of egg yolk or albumen, and no sample contained more than 67 S. enteritidis cells/ml.

The identification of infected commercial poultry flocks has become a pivotal component of efforts to reduce the incidence of egg-associated transmission of Salmonella enteritidis to humans. To assess the sensitivity with which testing for specific antibodies in egg yolks can be applied to detect S. enteritidis infection in laying chickens, groups of hens were orally inoculated with either 10(3), 10(5), or 10(7) cfu of a phage type 13a strain of S. enteritidis. Eggs from these hens were collected for 4 wk after inoculation and yolk samples were tested for antibodies to S. enteritidis flagella by ELISA. All hens that were inoculated with 10(7) cfu of S. enteritidis were detected as infected by the egg yolk ELISA when eggs were tested individually, as were up to 66 and 35% of hens inoculated with 10(5) or 10(3) cfu, respectively. Even when yolks from infected hens were diluted 1:10 in yolk from uninfected hens, specific antibodies could still be found in eggs from 31% of hens given 10(7) cfu of S. enteritidis and 13% of hens given 10(3) cfu. These results demonstrate that egg yolk antibody testing can provide a highly sensitive indication of prior exposure to S. enteritidis, and should accordingly be useful for verifying the effectiveness of programs designed to reduce the incidence of S. enteritidis infection in poultry.

Detecting Salmonella enteritidis contamination in eggs has become the cornerstone of many programs for reducing egg-borne disease transmission, but egg culturing is time consuming and laborious. Preliminary screening tests are thus generally applied to minimize the number of flocks from which eggs must be cultured. The usefulness of such tests is directly proportional to both their detection sensitivity and their ability to predict the likelihood of egg contamination. In the present study, samples were collected for 24 days after groups of laying hens were orally inoculated with S. enteritidis. Eggs from each hen were cultured for S. enteritidis in the contents and samples of egg yolk were diluted and tested for specific antibodies to S. enteritidis flagella using both experimental and commercially available enzyme-linked immunosorbent assay (ELISA) methods. Samples of voided feces were also collected regularly from each bird and cultured for S. enteritidis. Although fecal shedding and egg yolk antibody production followed opposite patterns over time (fecal shedding was decreasing as egg yolk antibody titers were increasing), tests for both parameters were effective in predicting whether particular hens would lay contaminated eggs. Among hens that laid at least one egg contaminated by S. enteritidis, 82% were detected as infected by fecal culturing and 96% by the experimental egg yolk ELISA test. Using easily collected samples, egg yolk antibody testing offers a rapid and effective screening method for identifying S. enteritidis-infected laying flocks that might lay contaminated eggs.

Eggs laid by hens experimentally infected with Salmonella enteritidis were assayed for the presence of Serogroup D-specific yolk antibodies. Yolk antibodies were detected with S. enteritidis and Salmonella pullorum antigens in the microantiglobulin test as early as 9 days after inoculation of hens with S. enteritidis. Yolk antibody titers reached peak levels at 3 to 5 wk postinoculation and remained at detectable levels for at least 7 wk postinoculation in eggs from both orally inoculated and horizontally contact-exposed hens. Eggs laid by hens from commercial flocks implicated in epidemiological investigations of human S. enteritidis outbreaks were also tested. Serogroup D-specific yolk antibodies were detected in 5 to 22% of eggs from hens in houses identified as infected by bacteriological culturing of internal organs of hens for S. enteritidis.

A move from conventional cages to either an enriched cage or a noncage system may affect the safety or quality, or both, of the eggs laid by hens raised in this new environment. The safety of the eggs may be altered either microbiologically through contamination of internal contents with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. Quality may be affected through changes in the integrity of the shell, yolk, or albumen along with changes in function, composition, or nutrition. Season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account. An understanding of these different effects is prudent before any large-scale move to an alternative housing system is undertaken. [ABSTRACT FROM AUTHOR]