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Effect of Blood Spots in Table Egg Albumen on Salmonella Growth²

USDA, Agricultural Research Service, Richard B. Russell Research Center, 950 College Station Road, Athens, GA 30605

² Corresponding author: douglas.smith{at}ars.usda.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Presence of blood spots in eggs has been correlated with a greater rate of Salmonella Enteritidis contamination. Therefore, this study was conducted to determine whether Salmonella inoculated into egg albumen with naturally occurring blood spots would survive or grow. In each of 3 trials, white shell table eggs with blood spots were collected from a commercial egg-processing plant after candling. In each trial, eggs were broken out, and approximately 4 mL of clear albumen (CLEAR) and 4 mL of bloody albumen (BLOOD) from each of 10 eggs were placed in sterile test tubes and inoculated with a nalidixic acid-resistant Salmonella Typhimurium. For inoculation, 0.1 mL of the Salmonella Typhimurium suspension (containing 7.1, 7.7, or 7.0 log cfu/mL in trials 1 to 3, respectively) was added to each tube. Tube contents were mixed and incubated at 25°C for 24 h. Immediately after inoculation (0 h) and again after 24 h, 0.1 mL from each tube was plated onto Brilliant Green-Sulfa agar with 200 ppm nalidixic acid and incubated at 37°C for 24 h. Results are reported as log colony-forming units per milliliter of albumen. No significant differences (P < 0.05) in mean Salmonella Typhimurium counts were found between CLEAR or BLOOD samples at 0 h (5.6 vs. 5.8, respectively), indicating that initial inoculation levels were consistent between treatments. After 24 h, CLEAR samples were slightly but significantly lower than BLOOD samples for Salmonella Typhimurium (6.5 vs. 6.8, respectively). Salmonella Typhimurium numbers increase somewhat in albumen with or without blood, but slightly greater numbers are produced in albumen with blood spots. In this experiment, blood in the albumen of table eggs contributed to the survival and growth of Salmonella Typhimurium inoculated into egg albumen.

Key Words: table egg • blood spot • Salmonella • albumen

	INTRODUCTION

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Presence of blood in table eggs is a longstanding quality issue addressed by the egg grading standards of the USDA (2000). Table egg processors and egg breaker plants use various forms of candling technology to detect and remove blood spot eggs before packaging or further processing.

The incidence of blood spots in table eggs varies but is generally reported by the commercial industry as less than 1% of eggs produced. Nutritional factors, such as a lack of vitamin A in the diet, have been implicated as increasing blood spot incidence (Bearse et al., 1960). Hen lines can be genetically selected for blood spot eggs (Becker and Bearse, 1973), with some selected flocks producing more than 50% of eggs containing blood spots (Merkley et al., 1973). Other factors may also affect the rate at which hens produce blood spot eggs, including the blood pressure of the hen or whether the birds are caged (Fry et al., 1968; Mench et al., 1986). Blood spots in eggs typically originate from the ovary of the hen during the ovulatory process or from the upper oviduct (Nalbandov and Card, 1944; Shirley, 1965).

Salmonella has been found in the ovaries of infected laying hens (Miyamoto et al., 1997; Gast et al., 2004), including Salmonella Typhimurium in commercial flocks (Barnhart et al., 1991). Therefore, laying hens, if infected with Salmonella, may produce eggs with blood spots that could contain this pathogen. In an indirect survey, table eggs with blood spots procured from several commercial flocks were nearly twice as likely to contain Salmonella Enteritidis as eggs without blood spots (Schlosser et al., 1999). The objective of this study was therefore to determine if blood spots present in albumen promote survival or growth of Salmonella in table eggs.

	MATERIALS AND METHODS

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In each of 3 trials, eggs with blood spots were collected from a commercial facility after candling and rejection. Approximately 30 were sprayed with 70% ethanol, allowed to dry, and broken out in sterile Petri dishes to obtain 10 with distinct blood spots in the albumen. From each of these 10 eggs, approximately 4 mL of albumen containing the blood spot was collected by pipette and placed in a sterile 50-mL tube (BLOOD). An additional 4 mL of albumen from the same egg with no sign of blood in the clear albumen was collected in the same manner (CLEAR), resulting in 10 paired BLOOD and CLEAR samples per trial. Exact pipette measurement was difficult because eggs were broken out within 2 d of collection from the plant and the thick albumen was still very viscous. A suspension of Salmonella Typhimurium culture (0.1 mL, nalidixic acid-resistant) in PBS was added to each tube and shaken rapidly with a Mini-Vortexer (VWR Scientific Products, West Chester, PA) for 5 s. The albumen-Salmonella Typhimurium mixture (0.1 mL) was removed from each tube and prepared for serial dilutions and plating as described below to estimate initial contamination. The original Salmonella Typhimurium inoculum (0.1 mL) was also serially diluted and plated. All tubes were held 24 h at 25°C, then another 0.1 mL was removed and prepared for dilution and plating after vortexing for 5 s. Serial dilutions were prepared in PBS, then 0.1 mL was plated on the surface of Brilliant Green-Sulfa agar (Becton Dickinson, Sparks, MD) with added 200 ppm sodium salt of nalidixic acid (Sigma Chemical Co., St. Louis, MO). Plates were incubated at 37°C for 24 h, and colonies representative of Salmonella were counted.

The Salmonella Typhimurium culture used for the inoculum was prepared by plating a nalidixic acid-resistant strain of Salmonella Typhimurium onto Brilliant Green-Sulfa agar supplemented with 200 ppm nalidixic acid and incubating overnight at 37°C. Cells were harvested and suspended into PBS. Cellular concentration in the suspension was estimated using a spectrophotometer at 540 nm. Serial dilutions were performed to achieve the appropriate challenge level for each trial. The level of Salmonella Typhimurium inoculum added to albumen samples was 7.1, 7.7, and 7.0 log cfu/mL for trials 1, 2, and 3, respectively.

Numbers of bacteria were converted to log colony-forming units for statistical analysis. Differences between treatments of CLEAR and BLOOD were tested by ANOVA using SAS ANOVA procedures, and means were separated using the Tukey method (SAS Institute, 1999). No significant interactions were noted due to treatment or trial; therefore, tests were conducted on pooled data. Differences were calculated between 0- and 24-h samples for both CLEAR and BLOOD treatments; this overall difference between CLEAR and BLOOD treatments was then tested using a paired t-test (SAS Institute, 1999).

	RESULTS AND DISCUSSION

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Numbers of Salmonella recovered from either CLEAR or BLOOD albumen are shown in Table 1. There was not a significant difference (P < 0.05) between the initial (0 h) inoculated CLEAR or BLOOD albumen sample Salmonella Typhimurium average numbers (5.6 and 5.8 log cfu/mL, respectively). After 24 h, numbers of Salmonella Typhimurium increased in both CLEAR and BLOOD albumen samples. The increase was greater in the BLOOD samples, resulting in significantly more Salmonella Typhimurium recovered from these samples versus the CLEAR samples (6.8 vs. 6.5 log cfu/mL, respectively). The overall difference in numbers of Salmonella Typhimurium between 0 and 24 h was calculated for both CLEAR and BLOOD samples, then compared by treatment; BLOOD albumen numbers increased 1.0 log cfu, which was significantly greater than the 0.9 log cfu increase for CLEAR samples.

View this table: [in this window] [in a new window]   Table 1. Average numbers (±SEM) of a nalidixic acid-resistant strain of Salmonella Typhimurium inoculated into paired clear or bloody albumen samples (0 h) and incubated at 25°C for 24 h and the difference () between 0- and 24-h times (n = 30)

Prior research has shown contradictory growth profiles of Salmonella inoculated into egg albumen. Salmonella Typhimurium increased by 3 log when incubated in egg albumen at an incubation temperature of 25°C for 24 h (Schoeni et al., 1995). A higher incubation temperature of 37°C resulted in only a slight increase of Salmonella Typhimurium incubated for 24 h (Guan et al., 2006). Bradshaw et al. (1990) reported that Salmonella Enteritidis inoculated into albumen decreased by 50% after 4 h, then by 90% after 48 h when incubated at 37°C. Gast and Holt (2000) found Salmonella Enteritidis did not appreciably multiply in egg albumen at either a low or a higher initial dosage of bacteria, regardless of incubation temperature (10°, 17.5°, or 25°C) and day of incubation (1, 2, or 3). In this experiment, the conditions produced a Salmonella Typhimurium increase of approximately 1 log after 24 h of incubation at 25°C. The particular strain of Salmonella, freshness of the albumen used, and presence of blood could have contributed to differences in bacterial growth from previous reports.

Although the 24-h difference between CLEAR and BLOOD samples was significant, it was slight and probably of little practical significance, because the difference was much less than 1.0 log cfu/mL. Results indicate that blood could assist Salmonella growth in albumen, but the evidence is not exceptionally compelling. The experimental design utilized indirect inoculation of naturally occurring blood spots, which may not have maximized potential beneficial effects of blood in albumen for Salmonella growth. Also, the clear albumen from an egg containing a blood spot could have contained a small amount of blood not readily visible. Further studies using direct inoculation of naturally occurring or laboratory-created blood spots then placing them into albumen should provide direct evidence for determining any beneficial effects of blood spots in eggs on Salmonella survival and growth.

There is a risk that hens infected with Salmonella may transmit this pathogen into the egg. Early research showed that this passage was unlikely (Mundt and Tugwell, 1958; Baker et al., 1980). However, more recent research has shown that hens inoculated by various methods with different strains of Salmonella can pass this pathogen to internal egg contents (Miyamoto et al., 1997; Gast et al., 2004).

Once in the egg, blood presence in albumen could assist Salmonella growth by providing nutrients for growth or overwhelming the natural defenses of the egg. Ovotransferrin inhibits Salmonella growth in albumen by denying iron availability, an essential growth factor (Lock and Board, 1992). Blood in the albumen could provide an ample iron source for bacteria. Garibaldi and Bayne (1962) presented data that excess iron does block the antibacterial activity of ovotransferrin. The increase in albumen pH with increased storage time of eggs could also be ameliorated with an excess of blood. Blood acting as a Salmonella growth promoter was indirectly shown by Schlosser et al. (1999), in which more than 50,000 blood spot eggs were collected from 28 flocks by the Salmonella Enteritidis Pilot Project in Pennsylvania and compared with regular eggs from the same flocks. The blood spot egg Salmonella incidence was 2.56 per 10,000 eggs, versus an incidence of 1.43 per 10,000 eggs for non-blood spot eggs.

The risk of blood spots contributing to Salmonella-contaminated table eggs reaching the market is very minor, because table eggs are candled during processing to remove blood spot eggs. However, brown shell table eggs or eggs from small flocks sold directly to consumers may not be adequately candled. Also, broiler hatching eggs are not candled before incubation. These circumstances could provide Salmonella-contaminated eggs to specific markets or to hatcheries. Further studies will be needed to examine the possibility of vertical transmission of pathogens from breeder flocks to hatcheries via eggs containing blood spots and contaminated with Salmonella.

	FOOTNOTES

 
¹ Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

Received for publication December 31, 2007. Accepted for publication April 3, 2008.

	REFERENCES

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Mench, J. A., A. van Tienhoven, J. A. Marsh, C. C. McCormick, D. L. Cunningham, and R. C. Baker. 1986. Effects of cage and floor pen management on behavior, production, and physiological stress responses of laying hens. Poult. Sci. 65:1058–1069.[Web of Science][Medline]

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