Multiplication and motility of Salmonella enterica serovars enteritidis, infantis, and montevideo in in vitro contamination models of eggs

The invasive ability of Salmonella enterica serovars Enteritidis, Infantis, and Montevideo in eggs was examined. Strains of these serovars originating from egg contents, laying chicken houses, and human patients were experimentally inoculated (0.1-ml dose containing 78 to 178 cells) onto the vitelline membrane of eggs collected from specific-pathogen-free chickens and incubated at 25 degrees C. The test strains were detected in 25 of 138 yolk contents by day 6, indicating the penetration of Salmonella organisms through the vitelline membrane. There were no significant differences in overall rates of penetration between serovars. The organisms were also detected in the albumen from 125 of 138 eggs tested by day 6. Growth to more than 10(6) CFU/ml was observed in 48 of the 125 albumen samples. An inoculum of 1000 Salmonella cells was added to 15 ml of albumen at the edge of a petri plate. A 10-mm-diameter cylindrical well, the bottom of which was sealed with a polycarbonate membrane with 3.0-microm pores, was filled with egg yolk and placed into the albumen at the center of the dish, which was maintained at 25 degrees C. Experiments were performed in triplicate with each strain. Salmonella organisms in all the albumen samples were detected by day 11. However, motility of the organisms toward the yolk was observed in only two dishes inoculated with the Salmonella Enteritidis strain from a human patient and in one dish inoculated with the Salmonella Infantis strain from liquid egg. The albumen samples obtained from the dishes inoculated with the Salmonella Enteritidis strain had high numbers of bacteria (>10(8) CFU/ml). The present study suggests that Salmonella organisms in egg albumen are unlikely to actively move toward the yolk, although depositionon or near the vitelline membrane can be advantageous for proliferation.     

Multiplication of Salmonella enteritidis in egg yolks after inoculation outside, on, and inside vitelline membranes and storage at different temperatures

Prompt refrigeration to restrict bacterial growth is important for reducing eggborne transmission of Salmonella enterica serovar Enteritidis (SE). The nutrient-rich yolk interior is a relatively infrequent location for initial SE deposition in eggs, but migration across the vitelline membrane can result in rapid bacterial multiplication during storage at warm temperatures. The objective of the present study was to measure the multiplication of SE in yolks after introduction at three different locations and subsequent storage at a range of temperatures. Using an in vitro egg contamination model, approximately 100 CFU of SE was inoculated either inside yolks, onto the exterior surface of vitelline membranes, or into the adjacent albumen. After storage of samples from each inoculation group at 10, 15, 20, and 25°C for 24 h, SE was enumerated in yolks. For all three inoculation locations, the final SE levels in yolks increased significantly with increasing storage temperatures. At all storage temperatures, significant differences in SE multiplication were observed between inoculation sites (yolk inoculation>vitelline membrane inoculation>albumen inoculation). At 25°C, final log concentrations of 7.759 CFU of SE per ml (yolk inoculation), 2.014 CFU/ml (vitelline membrane inoculation), and 0.757 CFU/ml (albumen inoculation) were attained in yolks after storage. These results demonstrate that, even when the initial site of SE deposition is outside the egg yolk, substantial multiplication supported by yolk nutrients can occur during the first day of storage and the risk of bacterial growth increases at higher ambient storage temperatures.     

Multiplication in egg yolk and survival in egg albumen of Salmonella enterica serotype Enteritidis strains of phage types 4, 8, 13a, and 14b

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.     

Effect of temperature on the growth response of Salmonella enteritidis inoculated onto the vitelline membranes of fresh eggs

The growth response of Salmonella Enteritidis (SE) on the vitelline membrane in vitro was studied with the use of a special tube devised specifically for the inoculation of SE onto the vitelline membrane and for the sampling of the yolk near the inoculation site. This latter ability allowed the detection of the movement of SE into the yolk. The growth of SE on the membrane was compared with that of SE inoculated into yolk and albumen in vitro and in ovo in fresh in-shell eggs. The incubation time was 2 days, and the incubation temperatures were 4, 8, 15, 27, and 37 degrees C. Comparison of the results obtained for in vitro growth showed that at 4, 8, and 15 degrees C, SE behaved as if it were in the albumen, with its numbers decreasing over time. At 27 and 37 degrees C, SE grew as if it were in yolk, with a maximum increase of 4.5 log CFU after 2 days at 37 degrees C. In no experiments involving growth on the vitelline membrane did SE appear in the yolk. Comparisons between in vitro and in ovo growth responses of SE in yolk and albumen indicate that SE growth on the membrane parallels that in the in-shell egg.     

Laying season and egg shell cracks on the growth of Salmonella enteritidis in the egg albumen during storage

We studied the effects of laying seasons and egg shell cracks on the ability of egg albumen to support the growth of Salmonella Enteritidis (SE) in eggs. Hens eggs used were those laid in February, June, and October in a farm in Japan and stored at 10, 20, and 30 degrees C, and at 30 degrees C after storage at 10 degrees C, immediately after receipt or after cracking the shell. At several-day intervals during storage, the egg contents were poured into a dish, SE was inoculated into albumen, and then the growth of SE during 3 days incubation at 18 degrees C was measured. The results demonstrated that storage temperature and laying season affected the growth of SE in the egg albumen. The proportion of eggs upon which albumen allowed the growth of SE was higher in the eggs stored at 30 degrees C than those stored at 10 degrees C. The growth of SE in eggs was lowest in the following order of laying: February, October, and June. SE grew preferably in albumen of cracked eggs than intact eggs.     

Populations of Salmonella enteritidis in artificially inoculated chicken eggs as influenced by the temperatures under which eggs might be held from the day of lay until the day of processing

This study was undertaken to determine the levels of Salmonella Enteritidis in artificially inoculated eggs as affected by the temperatures under which eggs might be held from the day of lay until the day of processing. Unprocessed chicken eggs of different sizes (n=1920, with 480 being laid in each season) were inoculated in the albumen with a five-strain mixture of Salmonella at 102 CFU per egg. The eggs were stored at 4, 10, and 22 degrees C for 3 weeks and sampled twice a week to determine the populations of Salmonella and total aerobic bacteria. The season in which eggs were laid did not significantly impact the growth of the pathogen (P > 0.05). The mean populations of the inoculated Salmonella were not significantly different in eggs stored at 4 versus 10 degrees C (P > 0.05). Eggs stored at 22 degrees C had a mean Salmonella population that was 3.71 or 3.37 log higher than the Salmonella population of eggs stored at 4 or 10 degrees C (P > 0.05). The mean Salmonella population at 22 degrees C increased from the initial 2.12 log CFU/ml to 3.36 log CFU/ml after 2 weeks of storage and to 7.84 log CFU/ml after 3 weeks of storage. A sharp increase in the population of Salmonella occurred after 2 to 2.5 weeks of storage at 22 degree C. This study provided a scientific basis for the current egg handling and transporting temperature requirements and reinforced the importance of maintaining low temperatures in controlling and preventing the growth of Salmonella Enteritidis in eggs from the day of lay until the day of processing.     

Comparison of homogenization methods for recovering Salmonella Enteritidis from eggs

For Salmonella Enteritidis (SE) detection, shell eggs have been homogenized with stomachers, with electric blenders, and by hand massaging. However, to date, there have been no published reports addressing whether the method of homogenization affects the recovery of SE from raw eggs. Three inoculum levels (10, 126, and 256 SE cells per pool of 10 eggs) were used to conduct three experiments. The 10-egg pools were homogenized by one of four homogenization methods--mechanical stomaching, electric blending, hand massaging, and hand stirring-for 30 s. The homogenized eggs were then incubated at 37 degrees C, and SE colonies were enumerated after 24 and 48 h of incubation. After 24 h of incubation, no SE was recovered from egg samples from stomached or electrically blended pools inoculated with <10 cells, while levels of 106 CFU/ml were found for samples from whipped or hand-massaged pools inoculated with <10 cells. Similarly, after 24 h of incubation, the numbers of SE cells recovered from hand-massaged or hand-stirred egg pools inoculated with 126 cells were significantly larger than the numbers recovered from stomached or electrically blended egg pools inoculated with 126 cells. The number of SE cells recovered from samples homogenized with a blender was still significantly smaller than the numbers recovered from samples homogenized by the other three methods when the inoculum level was increased to 256 CFU per pool. However, the SE count for all samples approached 9 log10 CFU/ml after 48 h of incubation. It is concluded that the detection of small SE populations in shell egg samples could be improved with the use hand massaging and hand stirring for homogenization.     

Effect of refrigeration on in vitro penetration of Salmonella enteritidis through the egg yolk membrane

Internally contaminated eggs have been implicated as leading sources of transmission of Salmonella Enteritidis (SE) to humans. Although SE is not often deposited inside the nutrient-rich yolks of naturally contaminated eggs, penetration through the vitelline membrane to reach the yolk contents could result in rapid bacterial multiplication. In previous studies, such penetration has been observed occasionally at warm temperatures during experiments with in vitro egg contamination models. The present study was conducted to determine whether refrigeration affects the frequency of in vitro SE penetration of the egg yolk membrane. After inoculation of small numbers of SE onto the outside of the vitelline membranes of intact yolks, immediate refrigeration of contaminated samples prevented the penetration of SE into the egg yolk contents during 24 h of storage. However, SE penetrated inside the yolk contents in 4% of contaminated egg samples refrigerated after 2 h of storage at 30 degrees C, 15% of samples refrigerated after 6 h of storage at 30 degrees C, and 40% of samples stored at 30 degrees C for 24 h (48 samples per treatment group). These results highlight the value of prompt refrigeration for restricting the opportunities for SE to multiply to high numbers inside the yolks of contaminated eggs.     

Optimization of iron supplementation for enhanced detection of Salmonella Enteritidis in eggs

Mixed raw egg contents were inoculated with approximately 10 CFU of Salmonella Enteritidis and supplemented with 0 to 7 mg of FeSO4 per g of egg contents. Egg contents were then incubated at 37 degrees C, and Salmonella Enteritidis colonies were enumerated for up to 106 h. Iron supplementation significantly enhanced the growth of Salmonella Enteritidis. Within the first 24 h of incubation, the optimum iron level for Salmonella Enteritidis growth in egg contents was between 0.2 and 2 mg of FeSO4 per g of egg contents. After 24 h of incubation at 37 degrees C. Salmonella Enteritidis counts in eggs supplemented with 0.5 mg of FeSO4 per g of egg contents consistently reached approximately 1 x 10(9) CFU/ml, whereas Salmonella Enteritidis counts in eggs without iron supplementation varied from less than 5 CFU/ml to 8.4 x 10(6) CFU/ml. A 3 by 3 factorial design was used to study the effect of type of preenrichment and level of iron supplementation on the growth of Salmonella Enteritidis in egg contents. No significant differences in Salmonella Enteritidis counts between preenrichment and nonpreenrichment treatments were observed when egg contents were supplemented with 0.5 mg of FeSO4 per g of egg contents. It was concluded that preenrichment was not necessary for isolation of Salmonella Enteritidis from eggs. The effect of iron supplementation on the sensitivity of detection by the direct plating method was investigated. The direct plating method detected a significantly higher percentage of Salmonella Enteritidis in raw egg contents supplemented with 0.5 mg of FeSO4 per g of egg contents (90%) than in raw egg contents without iron supplementation (63.3%).     

Outgrowth of Salmonellae and the physical property of albumen and vitelline membrane as influenced by egg storage conditions

This study was undertaken to determine the influence of storage time and temperature on the volume, weight, and pH of egg albumen, the physical strength of vitelline membrane, and the fate of Salmonella Enteritidis artificially inoculated into egg albumen. A fiber-optic probe was used for inoculation with Salmonella Enteritidis at 10(2), 10(4), or 10(6) cells per egg. Both fresh and inoculated eggs were stored at 4, 10, and 22 degrees C for 6 weeks. Five fresh uninoculated eggs from each storage group were collected each week, and the weight, volume, and pH of the egg albumen were measured. The forces, energies, and degrees of membrane deformation required to rupture the vitelline membranes also were determined from either albumen-free yolks or yolks surrounded by albumen. In separate experiments, five inoculated eggs were evaluated each week for populations of Salmonella Enteritidis. When the eggs were stored at 4 degrees C, the albumen retained significantly more volume and weight and had a relatively lower pH. The vitelline membranes from eggs stored at 4 and 10 degrees C required more force and energy for rupture. Salmonellae flourished at 22 degrees C, even in the albumen with the lowest initial population, 10(2) cells per egg. Storage at 4 and 10 degrees C inhibited the growth of salmonellae in the albumen of eggs with initial populations of 10(2), 10(4), or 10(6) cells per egg. In eggs with initial Salmonella populations of 10(6) cells per egg that were stored at 22 degrees C, the populations of reached as high as 10(10) cells per egg after 4 weeks of storage. Storage at 4 and perhaps 10 degrees C postponed the aging process of chicken eggs, preserved the antimicrobial agents of the albumen, and maintained the integrity of vitelline membrane. Low-temperature storage therefore had a significant impact on the safety and overall quality of the eggs.     

Multiplication of Salmonella enteritidis on the yolk membrane and penetration to the yolk contents at 30 degrees C in an in vitro egg contamination model

Refrigeration to limit bacterial multiplication is a critical aspect of efforts to control the transmission of Salmonella enterica serovar Enteritidis (SE) to consumers of contaminated eggs. Although the nutrient-rich yolk interior is an uncommon location for SE contamination in freshly laid, naturally contaminated eggs, migration across the vitelline membrane could lead to rapid bacterial multiplication even when the initial site of deposition is outside the yolk. Multiplication on the yolk membrane (before, or in addition to, multiplication within the yolk contents) could be another source of increased risk to consumers. The present study used an in vitro egg contamination model to compare the abilities of four strains of SE to either multiply in association with the yolk membrane or migrate through that membrane to reach the yolk contents during 36 h of incubation at 30 degrees C. After inoculation onto the exterior surface of intact, whole yolks, all four SE strains penetrated the vitelline membrane to reach the yolk contents (at an overall frequency of 11.5%) after 12 h of incubation. The mean log concentration of SE was significantly higher in whole yolks (including yolk membranes) than in yolk contents at both 12 h (0.818 versus 0.167 CFU/ ml) and 36 h (2.767 versus 1.402 CFU/ml) of incubation. These results demonstrate that SE multiplication on the vitelline membrane may both precede and exceed multiplication resulting from penetration into the yolk contents during the first 36 h of unrefrigerated storage, reinforcing the importance of rapid refrigeration for protecting consumers from egg-transmitted illness.     

Incubation of egg contents pools at an elevated temperature (42 degrees C) does not improve the rapid detection of Salmonella enteritidis phage type 14b

Detecting internal Salmonella Enteritidis (SE) contamination in eggs is essential for protecting public health. Pooling together > or = 10 eggs for sampling allows many eggs to be screened for contamination, but such pools must be incubated (usually at 25 to 37 degrees C) to permit small numbers of SE to multiply before further testing. The present study determined whether incubating egg contents pools at an elevated temperature (42 degrees C) could increase the rate of multiplication of a phage type 14b strain of SE sufficiently to support the detection of contamination by a rapid lateral flow immunodiffusion method within a single day. Pools of 10 eggs were contaminated with approximately 10 CFU of SE, supplemented with concentrated broth enrichment medium, and incubated at either 37 or 42 degrees C. Incubation of contaminated egg pools at 42 degrees C resulted in significantly higher SE levels after 6, 8, 10, and 12 h. However, incubation at 42 degrees C could only generate a mean log SE concentration of 4.21 CFU/ml within a single working day (8 h), inadequate to support efficient detection by most rapid assays. Detection of SE contamination in egg pools by a rapid lateral flow immunodiffusion test was not achieved at a high frequency until 12 h of incubation at 42 degrees C.     

In vitro Survival and Growth of Salmonella Typhimurium inoculated on Yolk Membrane after long Term refrigerated Storage of Shell Eggs

Salmonella enterica serovar Typhimurium has been isolated from commercial egg production facilities in the United States. Given its importance as a causative organism for food-borne salmonellosis, identifying approximate timelines for bacterial invasion of the egg is needed. The objective of this study was to examine net growth of S. Typhimurium in egg components over time. In trial 1 eggs were collected over a 24 hour period from a flock of single comb white leghorn hens while in trial 2 eggs were picked up from a commercial laying source once a week over the course of eight weeks and stored. Eggs were held at refrigeration temperature and each week, subsets of eggs were cracked, separated into yolk and albumen components, and inoculated with 10⁸ CFU/ml of novobiocin and nalidixic acid (NO/NA) resistant S. Typhimurium onto the vitelline membrane of the egg. Yolks were then covered with albumen. Eggs were incubated for twenty-four hours at 25°C. After incubation eggs were again separated into albumen, yolk, and vitelline membrane samples. In trial 1, S. Typhimurium net growth occurred in albumen by the second week and continued from 4 to 8 weeks while in trial 2 net growth only occurred at week 5 and 7. S. Typhimurium net growth on vitelline membranes occurred by 2 weeks and continued from 4 to 8 weeks in trial 1 while no net growth occurred in trial 2 over the 8 week period. Yolk samples showed no net increases in S. Typhimurium populations over the 8 week period. An erratum to this article is available at .     

In vitro Survival and Growth of Salmonella Typhimurium inoculated on Yolk Membrane after long Term refrigerated Storage of Shell Eggs

Salmonella enterica serovar Typhimurium has been isolated from commercial egg production facilities in the United States. Given its importance as a causative organism for food-borne salmonellosis, identifying approximate timelines for bacterial invasion of the egg is needed. The objective of this study was to examine net growth of S. Typhimurium in egg components over time. In trial 1 eggs were collected over a 24 hour period from a flock of single comb white leghorn hens while in trial 2 eggs were picked up from a commercial laying source once a week over the course of eight weeks and stored. Eggs were held at refrigeration temperature and each week, subsets of eggs were cracked, separated into yolk and albumen components, and inoculated with 10⁸ CFU/ml of novobiocin and nalidixic acid (NO/NA) resistant S. Typhimurium onto the vitelline membrane of the egg. Yolks were then covered with albumen. Eggs were incubated for twenty-four hours at 25°C. After incubation eggs were again separated into albumen, yolk, and vitelline membrane samples. In trial 1, S. Typhimurium net growth occurred in albumen by the second week and continued from 4 to 8 weeks while in trial 2 net growth only occurred at week 5 and 7. S. Typhimurium net growth on vitelline membranes occurred by 2 weeks and continued from 4 to 8 weeks in trial 1 while no net growth occurred in trial 2 over the 8 week period. Yolk samples showed no net increases in S. Typhimurium populations over the 8 week period.     

Determination of 14C residue in eggs of laying hens administered orally with [14C] sulfaquinoxaline

Ten layer hens were dosed for 5 consecutive days with 6.2 mg kg^-1 [¹⁴C] sulfaquinoxaline (SQX). Eggs were collected from the hens during the 5-day dosing period and during a 10-day post-dose withdrawal period. Egg yolk and albumen were separated and assayed for total radioactive residues (TRR) using a combustion oxidizer and liquid scintillation counting techniques. Significant amounts of radioactivity were detected on the second day of dosing (greater than 24 h after the initial dose) in both egg yolk and albumen. First eggs were collected about 8 h after dosing; the second-day eggs were collected during 8-h period after the second dose. Radioactive residues reached a maximum on the fifth day of dosing in albumen, whereas on the second day of withdrawal in egg yolk, the peak TRR levels in albumen were about threefold higher than in yolk. Thereafter, the TRR levels declined rapidly in albumen and were detectable up to withdrawal day 6, whereas the TRR levels in egg yolk declined more slowly and were detectable up to withdrawal day 10. High-performance liquid chromatography analysis indicated that the parent drug sulfaquinoxaline was the major component in both the egg albumen and yolk. Additionally, this work suggests that egg yolk is the appropriate matrix for monitoring SQX residues     

Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media.

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degrees C for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degrees C, or 250 cells per egg when eggs were stored at 30 degrees C, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs.     

Use of capillary tubes and plate heat exchanger to validate U.S. Department of Agriculture pasteurization protocols for elimination of Listeria monocytogenes in liquid egg products

D-values for a five-strain cocktail of Listeria monocytogenes in five different liquid egg products (whole egg, egg yolk, egg white, egg yolk + 5% sucrose + 5% NaCl, and egg yolk + 10% NaCl) were determined using 100-microl capillary tubes. The egg products were inoculated with approximately 1 x 10(10) organisms/ml and heated in capillary tubes to temperatures ranging from 53 to 69 degrees C for various time intervals. Using a pilot scale plate heat exchanger, the U.S. Department of Agriculture (USDA) protocols for pasteurization were also evaluated using egg products inoculated with approximately 1 x 10(7) L. monocytogenes/ml. Results of experiments with capillary tubes suggested that all processes would result in less than the 9D process recommended by USDA. Moreover, although pasteurization with a plate heat exchanger provided greater lethality than did capillary tubes, all products still received less than a 5.4D process. Hence, these results suggest that the current USDA protocol may not be adequate to assure a large margin of safety.     

Use of capillary tubes and plate heat exchanger to validate U.S. Department of Agriculture pasteurization protocols for elimination of Salmonella enteritidis from liquid egg products

D values for a five-strain cocktail of Salmonella Enteritidis in five different liquid egg products (whole egg, egg yolk, egg white, egg yolk + 5% sucrose + 5% NaCl, and egg yolk + 10% NaCl) were determined using 100-microl capillary tubes. The egg products were inoculated with approximately 1 X 10(10) organisms/ml and heated in capillary tubes to temperatures ranging from 51 to 68 degrees C for various time intervals. Using a pilot scale plate heat exchanger, the U.S. Department of Agriculture (USDA) protocols for pasteurization were also evaluated using egg products inoculated with approximately 1 x 10(7) Salmonella Enteritidis/ml. Results of experiments with capillary tubes suggested that almost all processes would result in less than the 9D process recommended by the USDA. However, when the egg products were pasteurized using the plate heat exchanger, a greater than 9D process was achieved for Salmonella Enteritidis in all products except egg yolk containing 5% sucrose + 5% NaCl, which received approximately a 4D process.     

Pathogen prevalence and microbial levels associated with restricted shell eggs

Restricted shell eggs that do not meet quality standards for retail but maintain acceptable quality for inclusion in further processed eggs are often diverted to further processing. A study was conducted to characterize the microbiological populations present on and in these eggs. On a single day, restricted eggs were collected from three shell egg processing plants a total of three times (replicates). Six shells or egg contents were combined to create a pool. Ten pools of shells and contents were formed for each plant per replicate. Shells and membranes were macerated in 60 ml of diluent. Contents were stomacher blended to form a homogeneous mixture. Total aerobic microorganisms and Enterobacteriaceae were enumerated. The prevalence of Salmonella, Campylobacter, and Listeria was determined by cultural methods. Average aerobic counts were 4.3 log CFU/ml for the shells and 2.0 log CFU/ml for the contents. There were plant x replicate differences for both (P < 0.05 and P < 0.01, respectively). The average Enterobacteriaceae level associated with the shell was 2.4 log CFU/ml and less than 0.1 log CFU/ml for the egg contents, with 36.7% of the samples being positive. One shell sample (0.5% of total samples) was Campylobacter positive. Two shell samples (1.1% of total samples) were Salmonella positive. Twenty-one percent of samples were positive for Listeria (33 shells and 5 contents). Although current pasteurization guidelines are based on Salmonella lethality, the results of this study reiterate the need to revisit the guidelines to determine the effectiveness for other pathogenic species.     

Growth of Salmonella serovars in hens’ egg albumen as affected by storage prior to inoculation

Different Salmonella enterica serovars, including Enteritidis, were tested for growth at 20°C in separated albumen upon inoculation with 39 cfu ml⁻¹. The albumen was fresh or stored for up to 3 weeks prior to inoculation (p.i.) either in the shell egg or separated from the yolk. The serovar Enteritidis did not behave differently than the other serovars indicating that the association between human S. Enteritidis infections and eggs is not due to its growth behaviour in albumen. A pronounced growth occurred more frequently and up to a one-log unit higher level in fresh albumen than in albumen stored p.i. This was at least partly explained by a pH effect. Since growth in the separated albumen was similar when the albumen had been stored p.i. in the absence or presence of yolk, we have no indication that nutrients or factors negating the inhibitory properties of the albumen leak out from the yolk during storage. Growth of Salmonella inoculated at a level of 8 cfu in the albumen of fresh and stored whole shell eggs was studied to simulate a more natural situation. In this case, growth also occurred more frequently when inoculated in the albumen of fresh eggs compared to eggs stored p.i. It can be concluded from our study that cooling practices are recommended shortly after lay to prevent Salmonella from growing in eggs.