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Poult Sci 2006. 85:1678-1681
© 2006 Poultry Science Association
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PROCESSING, PRODUCTS, AND FOOD SAFETY: Research Note

In Vitro Study of Salmonella enteritidis and Salmonella typhimurium Definitive Type 104: Survival in Egg Albumen and Penetration through the Vitelline Membrane

J. Guan1, C. Grenier and B. W. Brooks

Ottawa Laboratory-Fallowfield, Canadian Food Inspection Agency, Nepean K2H 8P9, Canada

1 Corresponding author: guanj{at}inspection.gc.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Salmonella enteritidis and Salmonella typhimurium definitive type 104 (DT104) have been detected in the chicken oviduct, and their survival in egg albumen at the chicken body temperature of 42°C may be important in oviductal and transovarian contamination of intact shell eggs. Eight S. enteritidis and 24 S. typhimurium DT104 strains were tested for their in vitro survival in egg albumen. The concentration of the organisms declined more rapidly when incubated at 42°C than at 37°C and dropped to nondetectable levels within 96 h at the higher, but not at the lower, temperature. In another experiment, 3 S. enteritidis and 3 S. typhimurium DT104 strains were randomly selected, and dosages of 20 and 200 cells of each strain were inoculated onto the vitelline membranes of egg yolks, which were then submerged in the original albumen and incubated for 24 h at 42°C. Under these conditions, the organisms survived in albumen but did not penetrate the vitelline membrane. However, in a similar experiment, penetration did occur when the specimens were incubated at 30°C for 72 h. The results suggest that low numbers of S. enteritidis and S. typhimurium DT104 can be expected to survive in egg albumen during the 24-h period of egg formation in the oviduct but would be unlikely to invade the yolk before oviposition. However, depending on storage conditions following oviposition, S. enteritidis, as well as S. typhimurium DT104, could survive longer and may eventually invade the egg yolks.

Key Words: Salmonella • survival • penetration • egg albumen • vitelline membrane


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Salmonella enterica serovar Enteritidis is closely associated with poultry, and over the last 2 decades, it has become an important cause of foodborne human illnesses worldwide (Centers for Disease Control and Prevention, 2003). Many of these illnesses are related to consumption of raw or undercooked egg products that derive from contaminated intact shell eggs (Centers for Disease Control and Prevention, 2003). As a result, much work has been conducted to determine the causes for oviductal and transovarian egg contamination by Salmonella enteritidis. Previous work suggested that S. enteritidis migrates into eggs in the upper oviduct in association with albumen (Gast and Beard, 1990; Hoop and Pospischil, 1993). This evidence has been corroborated by the work of Keller et al. (1995, 1997), who found that the recovery rate of S. enteritidis is higher in the forming eggs taken from the oviduct than in the freshly laid eggs. Later, Lu et al. (2003) identified a gene of S. enteritidis that benefits S. enteritidis survival in albumen and may contribute to its oviductal and transovarian egg contamination. Thus, it appears that S. enteritidis survival in albumen during egg formation plays a role in contamination of intact shell eggs. Often, there are 10 or less cells of S. enteritidis per internally contaminated egg from naturally infected hens, and the cells are usually confined to the albumen (Humphrey et al., 1991; Poppe et al., 1992). However, Gast et al. (2005) and Murase et al. (2005) demonstrated that storage of contaminated eggs at 25 or 30°C allows small numbers of S. enteritidis to penetrate the vitelline membrane and rapidly multiply in the yolk contents, thereby increasing the risk to human health.

Along with S. enteritidis, the incidence of multiple antibiotic-resistant Salmonella enterica serovar Typhimurium definitive type 104 (DT104) has emerged as a world health problem (Helms et al., 2005). The spread of Salmonella typhimurium DT104 may be linked to the intensive use of antibiotics in human medicine and agriculture (Threlfall, 2002). Although S. typhimurium DT104 has been associated primarily with cattle and pigs, the number of poultry isolates has increased in recent years (Threlfall, 2002; Poppe et al., 2002). Studies have shown that commercial chicken layers become infected with S. typhimurium DT104 following challenge by oral and aerosol routes (Leach et al., 1999). Williams et al. (1998) demonstrated that S. typhimurium DT104 may contaminate the contents of intact shell eggs laid by chickens, but this requires oral inoculation with 107 cells, which exceeds what would likely occur under natural conditions. Thus, unlike S. enteritidis, little is known about the potential for oviductal and transovarian contamination of eggs by S. typhimurium DT104 and the threat that it may pose to human health.

The objectives of this study were to assess the in vitro survival of S. enteritidis and S. typhimurium DT104 in egg albumen at the chicken body temperature of 42°C and to investigate the likelihood of the organisms invading the yolk before and after oviposition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Salmonella Inocula

All 8 S. enteritidis and 24 S. typhimurium DT104 strains used in this study were provided by C. Poppe (Health Canada, Guelph, Ontario). The 8 S. enteritidis strains were isolated from raw eggs, and the 24 S. typhimurium DT104 strains were isolated from the following various chicken samples: internal organs (6), intestines (6), carcass (1), fluff (1), feces (1), litter (4), and barn environment (6). All strains were cultured in 9 mL of Luria-Bertani (LB) broth (Fisher Scientific, Ottawa, Ontario, Canada) for 24 h, and the cultures were centrifuged at 10,000 x g for 5 min to pellet the cells. The cell pellets were washed once with 9 mL of 0.85% sterile saline and were suspended in saline to give an optical density of 0.2 at 600 nm, equivalent to 2.0 x 108 cells/mL. To prepare inocula and to determine the actual cell concentrations, the cell suspensions were serially diluted and plated onto LB agar. Colonies were counted after incubation of the plates for 24 h at 37°C.

In Vitro Survival of Salmonella in Separated Egg Samples

Eggs used in the study were from specific pathogen-free Single-Comb White Leghorn chickens raised at the Ottawa Laboratory-Fallowfield, Canadian Food Inspection Agency. Within 24 h after oviposition, eggs were disinfected by immersion in 70% ethanol, and under aseptic conditions, they were dried, cracked, and the contents were collected into a sterile container. Egg yolk and albumen were separated into 2 pools, and the albumen was passed through a strainer with a mesh size of 2 mm2. The albumen and the yolk were dispensed in 10-mL portions into 15-mL centrifuge tubes and were incubated at 37 or 42°C for 1 h for temperature equilibration before inoculation. Each of the 8 S. enteritidis and 24 S. typhimurium DT104 strains were inoculated into 10 mL of yolk or albumen samples to give a final concentration of approximately 1.0 x 103 cells/mL. The samples were incubated at 37 or 42°C for 5 d. Uninoculated samples from the albumen and yolk pools were included as negative controls. Every 24 h, 100 µL of suspension was removed from each sample and was serially diluted with sterile saline. The dilutions were spread-plated onto LB agar in duplicate, and colonies were counted after incubation of the plates at 37°C for 24 h. This in vitro experiment was repeated twice.

In Vitro Survival of Salmonella in Whole Egg Samples

Three S. enteritidis and 3 S. typhimurium DT104 strains were randomly selected to investigate the survival of low numbers of Salmonella in the albumen of whole egg samples and their ability to penetrate through the vitelline membrane. The in vitro egg-contamination model described by Gast et al. (2005) was adapted for this study. Briefly, each intact yolk was separated from albumen and transferred into a 50-mL centrifuge tube, following which 20 or 200 cells of each Salmonella strain were inoculated onto the vitelline membrane. Five minutes after the inoculation, albumen from the same egg was poured into the centrifuge tube to cover the yolk. Twenty whole egg samples were prepared for each treatment, and 20 uninoculated egg samples were included as negative controls. The samples were incubated at 42°C for 24 h to simulate the temperature and duration of egg formation in the chicken body before oviposition. After incubation, 5 mL of yolk contents was collected from each sample and tested for Salmonella, as described by Gast et al. (2005). The rest of the egg sample was mixed with 45 mL of tryptic soy broth (Becton, Dickinson and Co., Oakville, Ontario, Canada) supplemented with 35 mg/L of ferric sulfate (Fisher Scientific) in a stomacher bag and was agitated for 2 min using a stomacher 400 (Seward, London, Ontario, Canada). The mixture was incubated at 37°C for 24 h, and after the incubation, 10 µL of the mixture was streaked onto XLT4 (Becton, Dickinson and Co.) plates and incubated at 37°C for 24 h. Three typical Salmonella colonies were picked for further testing by PCR using invA primers (Malorny et al., 2003).

To investigate the potential for S. enteritidis and S. typhimurium DT104 to penetrate into the yolk contents of improperly stored eggs, 100 cells of each of the above 6 strains were inoculated onto the vitelline membrane of 40 whole egg samples, as described above. After 24 and 72 h incubation at 30°C, 20 samples for each strain were tested for Salmonella, as described above.

Statistical Analysis

Significant differences (P < 0.5) among the Salmonella strains in their recovery from the yolk contents of the whole egg samples were determined by applying Fisher’s exact test (Daniel, 1999).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eight S. enteritidis strains and 24 S. typhimurium DT104 strains, in concentrations that ranged from 890 to 1,150 cells/mL, were used for studies on survival in separated albumen and yolk. No bacteria was isolated from the uninoculated albumen and yolk samples. Mean concentrations of the S. typhimurium DT104 strains remained similar to those of the S. enteritidis strains during the 120-h incubation in separated albumen at both 37 and 42°C (Figure 1Go). None of the 32 strains tested survived in the separated albumen after 96 h at 42°C, but all strains survived for 120 h at 37°C (Figure 1Go). All strains multiplied rapidly in yolk contents and reached approximately 9.0 log cells/mL after 24 h of incubation at 37 or 42°C (data not presented).


Figure 1
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  		Figure 1. Mean concentrations of 8 Salmonella enteritidis strains in separated egg albumen after incubation at 42°C ({blacksquare}) or 37°C ({square}) and 24 similarly prepared Salmonella typhimurium DT104 strains at 42°C (•) or 37°C ({circ}). Error bar represents 1 SD.

 
Three S. typhimurium DT104 and 3 S. enteritidis strains were randomly selected for studies on penetration through the vitelline membrane. After 24 h of incubation at 42°C, none of the 6 strains were recovered from the yolk contents in any of the whole egg samples (data not presented). However, all 6 strains were recovered from the albumen of 95 or 100% of the whole egg samples (Table 1Go). When the whole egg samples were incubated at 30°C, no Salmonella was recovered from the yolk contents of any samples after 24 h of incubation, but after 72 h, 5 of the 6 strains were recovered from the yolk contents (Table 2Go). The recovery rate varied from 0 to 15% for the S. typhimurium DT104 strains and 10 to 25% for the S. enteritidis strains, and the only significant difference (P < 0.5) was between S. enteritidis 01-1964 and S. typhimurium 01-1448. All randomly-picked colonies that were recovered from each sample were confirmed to be Salmonella by PCR.


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  		Table 1. Recovery of Salmonella from egg albumen incubated at 42°C1
 

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  		Table 2. Recovery of Salmonella from yolk contents incubated at 30°C1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Survival of S. enteritidis in albumen during egg formation before oviposition has been considered crucial for oviductal and transovarian egg contamination to occur (Keller et al., 1995, 1997; Lu et al., 2003). Thus, chicken body temperature (42°C) was used to investigate the in vitro survival of S. enteritidis and S. typhimurium DT104 in albumen. The poor survival of the organisms under such conditions may result from the combined stress of heat and the enzymatic activity of proteins such as ovotransferrin and lysozyme (Ibrahim et al., 2000, 2001). However, it was considered important that S. enteritidis and S. typhimurium DT104 could survive under such conditions for 24 h, because this is the average length of time for natural egg formation in the chicken oviduct (Johnson, 2000). To further simulate natural oviductal and transovarian egg contamination, low numbers of S. enteritidis and S. typhimurium DT104 were used in the in vitro study using whole egg samples. Again, S. enteritidis and S. typhimurium DT104 survived in albumen at 42°C for 24 h, but they did not penetrate through the vitelline membrane. It was recognized that the in vitro contamination model could only simulate the time and temperature and might not accurately represent conditions during in vivo egg formation, when the layers of albumen and other components are formed (Johnson, 2000). The pure culture of S. enteritidis and S. typhimurium DT104 strains used in this study may have been more sensitive to heat than host-adapted strains (Rychlik and Barrow, 2005), and yet the results indicated that these strains may survive in albumen during egg formation. When the whole egg samples were incubated at 30°C, S. enteritidis and S. typhimurium DT104 penetrated the vitelline membrane after 24 to 72 h. Possibly, penetration would have occurred earlier if a host-adapted preparation of the organisms had been used (Gast et al., 2005). Thus, although the present study indicated that low numbers of S. enteritidis and S. typhimurium DT104 could survive in the egg albumen during egg formation, the organisms would be unlikely to penetrate into the yolk contents before oviposition. However, penetration into the yolk contents could occur following oviposition if eggs were stored under conditions that favored multiplication of the organism.


   ACKNOWLEDGMENTS
 
Sincere appreciation is expressed to C. Poppe in Health Canada (Guelph, Ontario, Canada) for providing the Salmonella strains and to J. L. Spencer in the Canadian Food Inspection Agency (Ottawa, Ontario, Canada) for his helpful suggestions on the experiment design.

Received for publication January 15, 2006. Accepted for publication April 25, 2006.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Centers for Disease Control and Prevention. 2003. Outbreaks of Salmonella serotype Enteritidis infection associated with eating shell eggs—United States, 1999–2001. JAMA 289:540–541.[Free Full Text]

Daniel, W. W. 1999. The Fisher exact test. Pages 606–611 in Biostatistics: A Foundation for Analysis in the Health Sciences. 7th ed. W. W. Daniel, ed. John Wiley & Sons Inc., New York, NY.

Gast, R. K., and C. W. Beard. 1990. Isolation of Salmonella enteritidis from internal organs of experimentally infected hens. Avian Dis. 34:991–993.[Web of Science][Medline]

Gast, R. K., P. S. Holt, and T. Murase. 2005. Penetration of Salmonella enteritidis and Salmonella heidelberg into egg yolks in an in vitro contamination model. Poult. Sci. 84:621–625.[Abstract/Free Full Text]

Helms, M., S. Ethelberg, K. Molbak, and DT104 Study Group. 2005. International Salmonella Typhimurium DT104 infections, 1992–2001. Emerg. Infect. Dis. 11:859–867.[Web of Science][Medline]

Hoop, R. K., and A. Pospischil. 1993. Bacteriological, serological, histological and immunohistochemical findings in laying hens with naturally acquired Salmonella enteritidis phage type 4 infection. Vet. Rec. 133:391–393.[Abstract]

Humphrey, T. J., A. Whitehead, A. H. L. Gawler, A. Henley, and B. Rowe. 1991. Numbers of Salmonella enteritidis in the contents of naturally contaminated hens’ eggs. Epidemiol. Infect. 106:489–496.[Medline]

Ibrahim, H. R., Y. Sugimoto, and T. Aoki. 2000. Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism. Biochim. Biophys. Acta 1523:196–205.[Medline]

Ibrahim, H. R., U. Thomas, and A. Pellegrini. 2001. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeablilization action. J. Biol. Chem. 276:43767–43774.[Abstract/Free Full Text]

Johnson, A. L. 2000. Reproduction in the female. Pages 569–596 in Sturkie’s Avian Physiology. 5th ed. G. C. Whittow, ed. Acad. Press, San Diego, CA.

Keller, L. H., C. E. Benson, K. Krotec, and R. J. Eckroade. 1995. Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect. Immun. 63:2443–2449.[Abstract/Free Full Text]

Keller, L. H., D. M. Schifferli, C. E. Benson, S. Aslam, and R. J. Eckroade. 1997. Invasion of chicken reproductive tissues and forming eggs is not unique to Salmonella enteritidis. Avian Dis. 41:535–539.[Web of Science][Medline]

Leach, S. A., A. Williams, A. C. Davies, J. Wilson, P. D. Marsh, and T. J. Humphrey. 1999. Aerosol route enhances the contamination of intact eggs and muscle of experimentally infected laying hens by Salmonella typhimurium DT104. FEMS Microbiol. Lett. 171:203–207.[Web of Science][Medline]

Lu, S., P. B. Killoran, and L. W. Riley. 2003. Association of Salmonella enterica serovar Enteritidis yafD with resistance to chicken egg albumen. Infect. Immun. 71:6734–6741.[Abstract/Free Full Text]

Malorny, B., J. Hoorfar, C. Bunge, and R. Helmuth. 2003. Multicenter validation of the analytical accuracy of Salmonella PCR: Towards an international standard. Appl. Environ. Microbiol. 69:290–296.[Abstract/Free Full Text]

Murase, T., P. S. Holt, and R. K. Gast. 2005. Growth of Salmonella enterica serovar Enteritidis in albumen and yolk contents of eggs inoculated with this organism onto the vitelline membrane. J. Food Prot. 68:718–721.[Web of Science][Medline]

Poppe, C., R. P. Johnson, C. M. Forsberg, and R. J. Irwin. 1992. Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment. Can. J. Vet. Res. 56:226–232.[Web of Science][Medline]

Poppe, C., K. Ziebell, L. Martin, and K. Allen. 2002. Diversity in antimicrobial resistance and other characteristics among Salmonella typhimurium DT104 isolates. Microbiol. Drug Resist. 8:107–122.

Rychlik, I., and P. A. Barrow. 2005. Salmonella stress management and its relevance to behaviour during intestinal colonisation and infection. FEMS Microbiol. Rev. 29:1021–1040.[Web of Science][Medline]

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