- Email: [email protected]
In vitro invasion of laying hen ovarian follicles by Salmonella Enteritidis strains
Research Note In vitro invasion of laying hen ovarian follicles by Salmonella Enteritidis strains T. M. Dawoud,* P. Hererra,†‡§1 I. Hanning,†‡2 Y. M. Kwon,*†§ and S. C. Ricke*†‡§3 *Cell and Molecular Biology Program; †Center for Food Safety; ‡Department of Food Science; and §Department of Poultry Science, University of Arkansas, Fayetteville 72701 we used an in vitro invasion assay to determine ovarian follicle invasion by 5 SE strains. After inoculating the freshly collected ovarian follicles, all 5 SE strains were able to invade into the follicles after 2 h of incubation at 37°C. The mean percentage of SE invasion ranged from 0.016 to 0.034% and no significant difference was found among the SE strains. For Escherichia coli K-12 strain, which was used as a negative control, the mean percentage of invasion was 0.0003%. The in vitro follicle invasion by SE strains demonstrated in this study may reflect the ability of the strains to invade ovarian follicles in laying hens once SE cells reach ovaries through various routes.
Key words: Salmonella Enteritidis, laying hen, ovarian follicle, invasion, egg 2011 Poultry Science 90:1134–1137 doi:10.3382/ps.2010-01182
INTRODUCTION Human infections by foodborne bacterial pathogens are a critical public health problem. Among the foodborne bacterial pathogens are nontyphoid Salmonella serotypes, one of the major causes of human foodborne illnesses in the United States and worldwide (guardPetter, 2001). A recent study estimated that an average of 9.4 million incidents of foodborne illness from 31 major pathogens occur in the United States and that, of those, 11% are caused by nontyphoid Salmonella species as the second main causative pathogen. Furthermore, nontyphoid Salmonella species caused approximately 35% of the annual hospitalizations cases and 28% of deaths related to foodborne illness, leading other foodborne pathogens (Scallan et al., 2011). The transmission sources of Salmonella are wide ranging, but most incidences of this foodborne illness ©2011 Poultry Science Association Inc. Received October 12, 2010. Accepted January 16, 2011. 1 Current address: Food Safety and Inspection Service, 2736 Lake Shore Drive, Waco, TX 72708. 2 Current address: Department of Food Science and Technology, University of Tennessee, 2605 River Drive, Knoxville, TN 37996. 3 Corresponding author: [email protected]
have been traced to the consumption of poultry meat, eggs, and their food products (Rabsch et al., 2001; Andrews and Bäumler, 2005). The fact that these pathogens infect the host animals without observable sickness contributes to high numbers of human infections (Angulo and Swendlow, 1999; Poppe, 1999; Patrick et al., 2004). Among Salmonella enterica serotypes, the serotype Enteritidis is one of the most common serotypes implicated in human illness. This serotype has been accountable for 14% of all foodborne human cases of salmonellosis in the United States (Braden, 2006), making it the second most common Salmonella serotype after Salmonella Typhimurium. In the European Union, Salmonella Enteritidis (SE) is responsible for 62.5% of human salmonellosis (gantois et al., 2009). Salmonella Enteritidis has distinct ecological characteristics in association with chicken shell eggs, which raise unique concerns about public safety. Before the 1950s, there was no reported incidence of SE resulting from the consumption of raw or undercooked chicken eggs or their products in the United States (Bäumler et al., 2000; Rabsch et al., 2001; Andrews and Bäumler, 2005). From the late 1970s to the mid 1990s, the incidence of SE increased rapidly, reaching pandemic proportions, with most outbreaks associated with the consumption of raw or light-
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ABSTRACT Salmonella is the major foodborne bacterial pathogen worldwide. Among numerous serotypes, Salmonella Enteritidis (SE) is one of the most common Salmonella serotypes responsible for human infections in the United States. The main source of SE outbreaks is foods associated with raw or undercooked chicken eggs. Salmonella Enteritidis is the only serotype that routinely contaminates eggs. The transovarian transmission of SE and subsequent contamination of the eggs before egg shell formation is considered to be the main route of egg contamination by SE. To evaluate whether invasion of ovarian follicles is an important step during the production of eggs contaminated by SE,
RESEARCH NOTE
MATERIALS AND METHODS Bacterial Strains and Preparation of Bacterial Cultures Five different strains of SE and Escherichia coli (EC) K-12 strain were used in this study (Table 1). All frozen bacterial cultures were resuscitated by transfer into Luria-Bertani (LB) broth media (Difco, Sparks, MD) for 2 successive cycles of 24 h of incubation at 37°C. All SE strains and the EC strain were maintained and cultured in LB media. The bacterial cultures for ovarian follicle invasion assay were prepared by incubating the cultures until optical densities at 600 nm reached 0.2, which corresponds to a midlog phase.
Collection and Preparation of Ovarian Follicles Ovarian follicles were collected from a flock of Single Comb White Leghorn hens maintained in the poultry science research facility at the University of Arkansas (Fayetteville) and other local farms. Birds used for follicle collection were between 60 and 80 wk of age and had not been subjected to a forced molting. After the hens were killed, ovarian follicles were removed from the ovaries of laying hens aseptically. All of the follicles obtained from each hen were rinsed with sterile 1× PBS and distributed evenly into 6 groups, with fol-
licles in different stages of maturation in each group. Then the follicles in each group were placed in 90 mL of sterile culture media, Hank’s balanced saline solution (HBSS) supplemented with sodium bicarbonate (EMD Chemicals Inc., Gibbstown, NJ) to a final concentration of 4.2 mM.
Ovarian Follicle Invasion Assay The ovarian follicle invasion assay previously devised by Howard et al. (2005) was used in our study with some modifications. After ovarian follicles were prepared as described above, bacterial cultures were added to 1× HBSS cell medium containing the collected ovarian follicles to a final concentration of 106 cfu/mL. Immediately after inoculation, an aliquot of the medium was collected and used to determine the total colony-forming units in the medium postinoculation of bacteria. Follicles were then incubated with bacteria for 2 h at 37°C. The follicles were subsequently removed from the inoculated medium, rinsed with sterile 1× PBS, and then placed in sterile 100 mL of 1× HBSS supplemented with gentamicin sulfate (Omnipur, EMD Chemicals Inc.) at a final concentration of 500 µg/mL. The follicles were incubated for an additional 4 h at 37°C. Treatment with gentamicin (500 µg/mL) was used to kill all free-living bacteria present in the culture media, selecting for those bacteria that were able to invade the ovarian follicles. Subsequent to 4 h of incubation with gentamicin, each group of follicles was removed from the culture media, rinsed, and stomached separately with 10 mL of sterile 1× PBS. These stomached samples were then used to determine the total colonyforming units recovered from the ovarian follicles. For enumeration of bacterial cells at t0 and tinv, each sample was serially diluted into 1× PBS and plated in triplicate sets onto brilliant green agar (Difco) plates supplemented with novobiocin (25 µg/mL) or LB agar (Difco) plates supplemented with novobiocin (25 µg/ mL) for enumeration of SE and EC strains, respectively. Inoculated plates were incubated for 24 h at 37°C and colony-forming units were counted. The average colony-forming units of t0 and tinv samples from triplicate sets were used for calculation of the percentage invasion using the following equation: % invasion = [cfu at tinv /cfu at t0] × 100, where cfu at t0 = the total colony-forming units in the media postinoculation of bacteria and cfu at tinv = the total colony-forming units recovered from the ovarian follicles contents. Four separate trials of ovarian follicle invasion assays were performed for each strain.
Statistical Analysis Each trial consisted of 6 groups, and each group was inoculated with a different bacterial strain (5 SE and 1 EC). The percentage invasion from 4 separate trials was analyzed using JMP 7 statistical software (SAS
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on June 6, 2015
ly cooked hens’ eggs (Ward et al., 2000; Velge et al., 2005). In fact, among more than 2,500 serotypes SE is the only serotype that routinely contaminates chicken eggs, suggesting that SE possesses unique genetic and phenotypic characteristics that enable routine egg contamination by this serotype. The modes of transmission are considered to be both vertical (transovarian) and horizontal (shell egg penetration) routes. The vertical route of transmission is a result of infection in the reproductive tract that causes the contamination of the eggs by SE before egg shell is formed (Gast and Beard, 1990; Okamura et al., 2001). In the horizontal route, SE penetrates the egg shell and multiplies within the egg after the shell is contaminated with SE from the external environment (Cox et al., 2000; Messens et al., 2006). Although experimental evidence supports transovarian transmission through the vertical route, it is still uncertain which steps in the process are associated with the unique ability of SE to contaminate eggs routinely (Gast et al., 2004). One possible mechanism is that SE cells, once transmitted to the reproductive tract, invade ovarian follicles at different stages of development before egg shell formation. In this study, we investigated the ability of SE strains to invade laying hens’ ovarian follicles by in vitro invasion assays in an effort to evaluate the importance of follicle invasion in the process leading to the production of SE-contaminated eggs.
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Table 1. Bacterial strains used in this study Strain designation
Strain
SE14 SE45 SE134 SE266 SE13076 EC-K12
Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Escherichia coli K-12
Reference or source LK5 PT 8 PT 13A PT 4 NCTC13349 BM4246 ATCC 13076
Edwards et al., 2000 National Veterinary Services Laboratories, Ames, IA National Collection of Type Cultures, Porton Down, Salisbury, UK Billy Hargis, University of Arkansas, Fayetteville, AR American Type Culture Collection, Manassas, VA The Coli Genetic Stock Center, Yale University, New Haven, CT
Institute, Cary, NC). The ANOVA test for significance was used, with the statistical significance set at P < 0.05.
RESULTS AND DISCUSSION
ACKNOWLEDGMENTS Figure 1. The mean percentage invasion of Salmonella Enteritidis (SE) strains and Escherichia coli (EC) K-12 strain into ovarian follicles.
This research was supported by the United States Department of Agriculture (Washington, DC; USDACSREES grant no. 2005-35201-15429).
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on June 6, 2015
After incubating the ovarian follicles with bacterial strains, all strains were able to penetrate into the ovarian follicles during the 2 h of incubation at 37°C. The mean percentage invasion of the ovarian follicles by SE and EC strains determined in this study is shown in Figure 1. The mean percentage invasion of the SE strains ranged from 0.016 to 0.034%, whereas it was 0.0003% for EC, which was used as the negative control. The percentage invasion was closely similar among the 5 SE strains. The percentage invasion of SE13076 (0.016%), which showed lowest invasion among all 5 SE strains, was 53-fold higher than that of EC K-12 strain (0.0003%). However, no significant (P > 0.05) difference was found in percentage invasion among all strains tested. The concentration of gentamicin sulfate used in this study (500 μg/mL) was determined previously to ensure that all bacteria in the media were killed and bacterial counts inside the follicles were not affected (Howard et al., 2005). As expected, when we plated culture media after 4 h of incubation with gentamicin sulfate at this final concentration, no viable bacteria were detected.
The goal of this study was to determine and compare the capability of different SE strains to invade laying hens’ ovarian follicles. Our study showed through the in vitro invasion assay that all tested SE strains were able to penetrate the ovarian follicles under the assay conditions used in this study. Also, the result indicated that all SE strains invaded the follicles consistently without significant variation among all strains. Developing eggs have been found to be contaminated with SE in the reproductive tract of laying hens after both natural and experimental infections (Humphrey et al., 1989; Bichler et al., 1996). Transovarian transmission of SE has been confirmed by others following experimental infection of SE-free laying hens. In the study by Gast et al. (2004), approximately 66.7% of ovary and oviduct samples from experimentally infected hens were contaminated with SE. In another study, SE was recovered from 71% of egg contents following experimental infection of laying hens (Braden, 2006). In the study by Howard et al. (2005), developing small white follicles were invaded more efficiently by SE strain PT 19A than were follicles at different stages of maturity. The attachment of SE to ovarian follicles was observed in follicular granulosa cells and the level of attachment varied among the ovarian follicles at different developmental stages (Thiagarajan et al., 1994). Mizumoto et al. (2005) also showed that SE was associated with follicular explants. Our study demonstrated that different SE strains have the ability to invade ovarian follicles with insignificant variation. Our results, combined with those from previous studies, suggest that SE strains can efficiently adhere to and penetrate hens’ ovarian follicles in a manner dependent on follicle maturity. The ovarian colonization by SE may lead to invasion of ovarian follicles, contributing to the production of contaminated eggs. However, the factors responsible for the higher prevalence of SE in eggborne infections compared with other Salmonella serotypes are still unclear. Future work should compare follicle invasion capacity between SE strains and that of other Salmonella serotypes.
RESEARCH NOTE
REFERENCES
Ricke. 2005. Ovarian laying hen follicular maturation and in vitro Salmonella internalization. Vet. Microbiol. 108:95–100. Humphrey, T. J., A. Baskerville, S. Mawer, B. Rowe, and S. Hopper. 1989. Salmonella enteritidis phage type 4 from the contents of intact eggs: A study involving naturally infected hens. Epidemiol. Infect. 103:415–423. Messens, W., K. Grijspeerdt, and L. Herman. 2006. Eggshell penetration of hen’s eggs by Salmonella enterica serotype Enteritidis upon various storage conditions. Br. Poult. Sci. 47:554–560. Mizumoto, N., K. Sasai, H. Tani, and E. Babe. 2005. Specific adhesion and invasion of Salmonella Enteritidis in the vagina of laying hens. Vet. Microbiol. 111:99–105. Okamura, M., Y. Kamijima, T. Miyamoto, H. Tani, K. Sasai, and E. Baba. 2001. Differences among six Salmonella serotypes in abilities to colonize reproductive organs and to contaminate eggs in laying hens. Avian Dis. 45:61–69. Patrick, M. E., P. M. Adcock, T. M. Gomez, S. E. Altekruse, B. H. Holland, R. V. Tauxe, and D. L. Swendlow. 2004. Salmonella enteritidis infections, United States, 1985–1999. Emerg. Infect. Dis. 10:1–7. Poppe, C. 1999. Epidemiology of Salmonella enterica serotype Enteritidis. Pages 3–18 in Salmonella enterica Serotype Enteritidis in Humans and Animals: Epidemiology, Pathogenesis, and Control. A. M. Saeed, R. K. Gast, M. E. Potter, and P. G. Wall, ed. Iowa State University Press, Ames. Rabsch, W., H. Tschäpe, and A. J. Bäumler. 2001. Non-typhoidal salmonellosis: Emerging problems. Microbes Infect. 3:237–247. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M. A. Widdowson, S. L. Roy, J. L. Jones, and P. M. Griffin. 2011. Foodborne illness acquired in the United States—Major pathogens. Emerg. Infect. Dis. 17:7–15. Thiagarajan, D., A. M. Saeed, and E. K. Asem. 1994. Mechanism of transovarian transmission of Salmonella enteritidis in laying hens. Poult. Sci. 73:89–98. Velge, P., A. Cloeckaert, and P. Barrow. 2005. Emergence of Salmonella epidemics: The problems related to Salmonella enterica serotype Enteritidis and multiple antibiotic resistance in other major serotypes. Vet. Res. 36:267–288. Ward, L. R., J. Threifall, H. R. Smith, S. J. O’Brien, H. Riemann, P. Kass, D. Cliver, A. J. Bäumler, B. M. Hargis, and R. M. Tsolis. 2000. Salmonella enteritidis epidemic. Science 287:1763.
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Andrews, H. L., and A. J. Bäumler. 2005. Salmonella species. Pages 327–339 in Foodborne Pathogens: Microbiology and Molecular Biology. P. M. Fratamico, A. K. Bhunia, and J. L. Smith, ed. Caister Academic Press, Norwich, UK. Angulo, F. J., and D. L. Swendlow. 1999. Epidemiology of human Salmonella enterica serotype Enteritidis infections in the United States. Pages 33–41 in Salmonella enterica Serotype Enteritidis in Humans and Animals: Epidemiology, Pathogenesis, and Control. A. M. Saeed, R. K. Gast, M. E. Potter, and P. G. Wall, ed. Iowa State University Press, Ames. Bäumler, A. J., B. M. Hargis, and R. M. Tsolis. 2000. Tracing the origins of Salmonella outbreaks. Science 287:50–52. Bichler, L. A., K. V. Nagaraja, and D. A. Halvorson. 1996. Salmonella enteritidis in eggs, cloacal swab specimens, and internal organs of experimentally infected White Leghorn chickens. Am. J. Vet. Res. 57:489–495. Braden, C. R. 2006. Salmonella enterica serotype Enteritidis and eggs: A national epidemic in the United States. Clin. Infect. Dis. 43:512–517. Cox, N. A., M. E. Berrang, and J. A. Cason. 2000. Salmonella penetration of egg shells and proliferation in broiler hatching eggs— A review. Poult. Sci. 79:1571–1574. Edwards, R. A., D. M. Schifferli, and S. R. Maloy. 2000. A role for Salmonella fimbriae in intraperitoneal infections. Proc. Natl. Acad. Sci. USA 97:1258–1262. Gantois, I., R. Ducatelle, F. Pasmans, F. Haesebrouck, R. Gast, T. J. Humphrey, and F. V. Immerseel. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiol. Rev. 33:718–738. Gast, R. K., and C. W. Beard. 1990. Production of Salmonella Enteritidis-contaminated eggs by experimentally infected hens. Avian Dis. 34:438–446. Gast, R. K., J. Guard-Bouldin, and P. S. Holt. 2004. Colonization of reproductive organs and internal contamination of eggs after experimental infection of laying hens with Salmonella heidelberg and Salmonella enteritidis. Avian Dis. 48:863–869. Guard-Petter, J. 2001. The chicken, the egg and Salmonella enteritidis. Environ. Microbiol. 3:421–430. Howard, Z. R., R. W. Moore, I. B. Zabala-Diaz, K. L. Landers, J. A. Byrd, L. F. Kubena, D. J. Nisbet, S. G. Birkhold, and S. C.
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Key words: Salmonella Enteritidis, laying hen, ovarian follicle, invasion, egg 2011 Poultry Science 90:1134–1137 doi:10.3382/ps.2010-01182
INTRODUCTION Human infections by foodborne bacterial pathogens are a critical public health problem. Among the foodborne bacterial pathogens are nontyphoid Salmonella serotypes, one of the major causes of human foodborne illnesses in the United States and worldwide (guardPetter, 2001). A recent study estimated that an average of 9.4 million incidents of foodborne illness from 31 major pathogens occur in the United States and that, of those, 11% are caused by nontyphoid Salmonella species as the second main causative pathogen. Furthermore, nontyphoid Salmonella species caused approximately 35% of the annual hospitalizations cases and 28% of deaths related to foodborne illness, leading other foodborne pathogens (Scallan et al., 2011). The transmission sources of Salmonella are wide ranging, but most incidences of this foodborne illness ©2011 Poultry Science Association Inc. Received October 12, 2010. Accepted January 16, 2011. 1 Current address: Food Safety and Inspection Service, 2736 Lake Shore Drive, Waco, TX 72708. 2 Current address: Department of Food Science and Technology, University of Tennessee, 2605 River Drive, Knoxville, TN 37996. 3 Corresponding author: [email protected]
have been traced to the consumption of poultry meat, eggs, and their food products (Rabsch et al., 2001; Andrews and Bäumler, 2005). The fact that these pathogens infect the host animals without observable sickness contributes to high numbers of human infections (Angulo and Swendlow, 1999; Poppe, 1999; Patrick et al., 2004). Among Salmonella enterica serotypes, the serotype Enteritidis is one of the most common serotypes implicated in human illness. This serotype has been accountable for 14% of all foodborne human cases of salmonellosis in the United States (Braden, 2006), making it the second most common Salmonella serotype after Salmonella Typhimurium. In the European Union, Salmonella Enteritidis (SE) is responsible for 62.5% of human salmonellosis (gantois et al., 2009). Salmonella Enteritidis has distinct ecological characteristics in association with chicken shell eggs, which raise unique concerns about public safety. Before the 1950s, there was no reported incidence of SE resulting from the consumption of raw or undercooked chicken eggs or their products in the United States (Bäumler et al., 2000; Rabsch et al., 2001; Andrews and Bäumler, 2005). From the late 1970s to the mid 1990s, the incidence of SE increased rapidly, reaching pandemic proportions, with most outbreaks associated with the consumption of raw or light-
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Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on June 6, 2015
ABSTRACT Salmonella is the major foodborne bacterial pathogen worldwide. Among numerous serotypes, Salmonella Enteritidis (SE) is one of the most common Salmonella serotypes responsible for human infections in the United States. The main source of SE outbreaks is foods associated with raw or undercooked chicken eggs. Salmonella Enteritidis is the only serotype that routinely contaminates eggs. The transovarian transmission of SE and subsequent contamination of the eggs before egg shell formation is considered to be the main route of egg contamination by SE. To evaluate whether invasion of ovarian follicles is an important step during the production of eggs contaminated by SE,
RESEARCH NOTE
MATERIALS AND METHODS Bacterial Strains and Preparation of Bacterial Cultures Five different strains of SE and Escherichia coli (EC) K-12 strain were used in this study (Table 1). All frozen bacterial cultures were resuscitated by transfer into Luria-Bertani (LB) broth media (Difco, Sparks, MD) for 2 successive cycles of 24 h of incubation at 37°C. All SE strains and the EC strain were maintained and cultured in LB media. The bacterial cultures for ovarian follicle invasion assay were prepared by incubating the cultures until optical densities at 600 nm reached 0.2, which corresponds to a midlog phase.
Collection and Preparation of Ovarian Follicles Ovarian follicles were collected from a flock of Single Comb White Leghorn hens maintained in the poultry science research facility at the University of Arkansas (Fayetteville) and other local farms. Birds used for follicle collection were between 60 and 80 wk of age and had not been subjected to a forced molting. After the hens were killed, ovarian follicles were removed from the ovaries of laying hens aseptically. All of the follicles obtained from each hen were rinsed with sterile 1× PBS and distributed evenly into 6 groups, with fol-
licles in different stages of maturation in each group. Then the follicles in each group were placed in 90 mL of sterile culture media, Hank’s balanced saline solution (HBSS) supplemented with sodium bicarbonate (EMD Chemicals Inc., Gibbstown, NJ) to a final concentration of 4.2 mM.
Ovarian Follicle Invasion Assay The ovarian follicle invasion assay previously devised by Howard et al. (2005) was used in our study with some modifications. After ovarian follicles were prepared as described above, bacterial cultures were added to 1× HBSS cell medium containing the collected ovarian follicles to a final concentration of 106 cfu/mL. Immediately after inoculation, an aliquot of the medium was collected and used to determine the total colony-forming units in the medium postinoculation of bacteria. Follicles were then incubated with bacteria for 2 h at 37°C. The follicles were subsequently removed from the inoculated medium, rinsed with sterile 1× PBS, and then placed in sterile 100 mL of 1× HBSS supplemented with gentamicin sulfate (Omnipur, EMD Chemicals Inc.) at a final concentration of 500 µg/mL. The follicles were incubated for an additional 4 h at 37°C. Treatment with gentamicin (500 µg/mL) was used to kill all free-living bacteria present in the culture media, selecting for those bacteria that were able to invade the ovarian follicles. Subsequent to 4 h of incubation with gentamicin, each group of follicles was removed from the culture media, rinsed, and stomached separately with 10 mL of sterile 1× PBS. These stomached samples were then used to determine the total colonyforming units recovered from the ovarian follicles. For enumeration of bacterial cells at t0 and tinv, each sample was serially diluted into 1× PBS and plated in triplicate sets onto brilliant green agar (Difco) plates supplemented with novobiocin (25 µg/mL) or LB agar (Difco) plates supplemented with novobiocin (25 µg/ mL) for enumeration of SE and EC strains, respectively. Inoculated plates were incubated for 24 h at 37°C and colony-forming units were counted. The average colony-forming units of t0 and tinv samples from triplicate sets were used for calculation of the percentage invasion using the following equation: % invasion = [cfu at tinv /cfu at t0] × 100, where cfu at t0 = the total colony-forming units in the media postinoculation of bacteria and cfu at tinv = the total colony-forming units recovered from the ovarian follicles contents. Four separate trials of ovarian follicle invasion assays were performed for each strain.
Statistical Analysis Each trial consisted of 6 groups, and each group was inoculated with a different bacterial strain (5 SE and 1 EC). The percentage invasion from 4 separate trials was analyzed using JMP 7 statistical software (SAS
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on June 6, 2015
ly cooked hens’ eggs (Ward et al., 2000; Velge et al., 2005). In fact, among more than 2,500 serotypes SE is the only serotype that routinely contaminates chicken eggs, suggesting that SE possesses unique genetic and phenotypic characteristics that enable routine egg contamination by this serotype. The modes of transmission are considered to be both vertical (transovarian) and horizontal (shell egg penetration) routes. The vertical route of transmission is a result of infection in the reproductive tract that causes the contamination of the eggs by SE before egg shell is formed (Gast and Beard, 1990; Okamura et al., 2001). In the horizontal route, SE penetrates the egg shell and multiplies within the egg after the shell is contaminated with SE from the external environment (Cox et al., 2000; Messens et al., 2006). Although experimental evidence supports transovarian transmission through the vertical route, it is still uncertain which steps in the process are associated with the unique ability of SE to contaminate eggs routinely (Gast et al., 2004). One possible mechanism is that SE cells, once transmitted to the reproductive tract, invade ovarian follicles at different stages of development before egg shell formation. In this study, we investigated the ability of SE strains to invade laying hens’ ovarian follicles by in vitro invasion assays in an effort to evaluate the importance of follicle invasion in the process leading to the production of SE-contaminated eggs.
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Table 1. Bacterial strains used in this study Strain designation
Strain
SE14 SE45 SE134 SE266 SE13076 EC-K12
Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Escherichia coli K-12
Reference or source LK5 PT 8 PT 13A PT 4 NCTC13349 BM4246 ATCC 13076
Edwards et al., 2000 National Veterinary Services Laboratories, Ames, IA National Collection of Type Cultures, Porton Down, Salisbury, UK Billy Hargis, University of Arkansas, Fayetteville, AR American Type Culture Collection, Manassas, VA The Coli Genetic Stock Center, Yale University, New Haven, CT
Institute, Cary, NC). The ANOVA test for significance was used, with the statistical significance set at P < 0.05.
RESULTS AND DISCUSSION
ACKNOWLEDGMENTS Figure 1. The mean percentage invasion of Salmonella Enteritidis (SE) strains and Escherichia coli (EC) K-12 strain into ovarian follicles.
This research was supported by the United States Department of Agriculture (Washington, DC; USDACSREES grant no. 2005-35201-15429).
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on June 6, 2015
After incubating the ovarian follicles with bacterial strains, all strains were able to penetrate into the ovarian follicles during the 2 h of incubation at 37°C. The mean percentage invasion of the ovarian follicles by SE and EC strains determined in this study is shown in Figure 1. The mean percentage invasion of the SE strains ranged from 0.016 to 0.034%, whereas it was 0.0003% for EC, which was used as the negative control. The percentage invasion was closely similar among the 5 SE strains. The percentage invasion of SE13076 (0.016%), which showed lowest invasion among all 5 SE strains, was 53-fold higher than that of EC K-12 strain (0.0003%). However, no significant (P > 0.05) difference was found in percentage invasion among all strains tested. The concentration of gentamicin sulfate used in this study (500 μg/mL) was determined previously to ensure that all bacteria in the media were killed and bacterial counts inside the follicles were not affected (Howard et al., 2005). As expected, when we plated culture media after 4 h of incubation with gentamicin sulfate at this final concentration, no viable bacteria were detected.
The goal of this study was to determine and compare the capability of different SE strains to invade laying hens’ ovarian follicles. Our study showed through the in vitro invasion assay that all tested SE strains were able to penetrate the ovarian follicles under the assay conditions used in this study. Also, the result indicated that all SE strains invaded the follicles consistently without significant variation among all strains. Developing eggs have been found to be contaminated with SE in the reproductive tract of laying hens after both natural and experimental infections (Humphrey et al., 1989; Bichler et al., 1996). Transovarian transmission of SE has been confirmed by others following experimental infection of SE-free laying hens. In the study by Gast et al. (2004), approximately 66.7% of ovary and oviduct samples from experimentally infected hens were contaminated with SE. In another study, SE was recovered from 71% of egg contents following experimental infection of laying hens (Braden, 2006). In the study by Howard et al. (2005), developing small white follicles were invaded more efficiently by SE strain PT 19A than were follicles at different stages of maturity. The attachment of SE to ovarian follicles was observed in follicular granulosa cells and the level of attachment varied among the ovarian follicles at different developmental stages (Thiagarajan et al., 1994). Mizumoto et al. (2005) also showed that SE was associated with follicular explants. Our study demonstrated that different SE strains have the ability to invade ovarian follicles with insignificant variation. Our results, combined with those from previous studies, suggest that SE strains can efficiently adhere to and penetrate hens’ ovarian follicles in a manner dependent on follicle maturity. The ovarian colonization by SE may lead to invasion of ovarian follicles, contributing to the production of contaminated eggs. However, the factors responsible for the higher prevalence of SE in eggborne infections compared with other Salmonella serotypes are still unclear. Future work should compare follicle invasion capacity between SE strains and that of other Salmonella serotypes.
RESEARCH NOTE
REFERENCES
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