Veterinary Research Communications

, Volume 33, Issue 3, pp 273–280 | Cite as

Replication kinetics of Salmonella enteritidis in internal organs of ducklings after oral challenge: a quantitative time-course study using real-time PCR

  • S. X. Deng
  • A. C. ChengEmail author
  • M. S. Wang
  • X. R. Li
  • B. Yan
Original Article


This research was undertaken to understand the replication kinetics of Salmonella enteritidis (S. enteritidis) in the internal organs of ducklings after oral challenge over a 2 wk period. A serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) assay was used to detect genomic DNA of S. enteritidis in the blood and the internal organs at different time points respectively. The results showed that the spleen was positive at 12 h post inoculation (PI) and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas and kidney were positive at 20 h PI, and the final organ to show a positive results was the gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h–36 h PI, with the liver and spleen containing the highest concentration of S. enteritidis. The blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 2 wk for the spleen without causing apparent symptoms. To make the results meaningful, a side-by-side bacteriology method (IFA) was performed. The results of IFA were similar to the FQ-PCR assay. This research provided a significant data for understanding the life cycle of S. enteritidis in the internal organs, and may help to understand the pathogenesis of S.entertidis in the future.


Fluorescence-based quantitative polymerase chain reaction Interal organs Salmonella enteritidis Replication kinetics 



Real-time fluorescence-based quantitative polymerase chain reaction




Indirect immuno-fluorescent antibody staining


Medial lethal dose



This work was Supported by the National Key Technology R&D Program of China (No.2004BA901A03); National Scientific and Sechnical Support Program (No.2007Z06-017); The Cultvation Fund of the Key Scientific and Technical Innovation Project&Ministry of Education of China (No.706050); Program for New Century Excellent Talents in University (No.NCET-04-0906/NCET-06-0818); Sichuan Province Basic Research Program (No.04JY0290061), and Program for Key Disciplines Construction of Sichuan Province(No. SZD0418).


  1. Abshire, K.Z. and Neidhardt, F.C., 1993. Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage. J Bacteriol, 175, 3734–3743PubMedGoogle Scholar
  2. Barrow, P.A., Hassan, J.O., Lovell, M.A. and Berchieri, A.,1990. Vaccination of chickens with aroA and other mutants of Salmonella typhimutium and S. enteritidis. Res Microbiol, 141,851–853 doi: 10.1016/0923-2508(90)90120-F PubMedCrossRefGoogle Scholar
  3. Bihl, F., Salez, L., Beaubier, M., Torres, D., Lariviere, L., Laroche, L., Benedetto, A., Martel, D., Lapointe, J. M., Ryffel, B. and Malo, D.,2003. Overexpression of Toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice. J Immunol, 170,6141–6150PubMedGoogle Scholar
  4. Cirillo, D.M., Valdivia, R.H., Monack, D.M. and Falkow, S.,1998. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol, 30,175–188 doi: 10.1046/j.1365-2958.1998.01048.x PubMedCrossRefGoogle Scholar
  5. Danese, P.N., Pratt, L.A. and Kolter, R., 2000.Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol, 182,3593–3596 doi: 10.1128/JB.182.12.3593-3596.2000 PubMedCrossRefGoogle Scholar
  6. Davey, M.E. and O’toole, G.A., 2000. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev, 64, 847–867 doi: 10.1128/MMBR.64.4.847-867.2000 PubMedCrossRefGoogle Scholar
  7. Deng, S.X., Cheng, A.C., Wang, M.S. and Cao, P.,2007. Study on the gastrointestinal tract distribution of Salmonella enteritidis in orally infected mice with a species-specific fluorescent quantitative polymerase chain reaction. World J Gastroenterol, 13, 6568–6574 doi: 10.3748/wjg.13.6568 PubMedCrossRefGoogle Scholar
  8. Deng, S.X., Cheng, A.C., Wang, M.S., Cao, P., Yan, B., Yin, N.C., Cao, S.Y. and Zhang, Z.H., 2008a. Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time PCR. World J Gastroenterol, 7,782–789 doi: 10.3748/wjg.14.782 CrossRefGoogle Scholar
  9. Deng, S.X., Cheng, A.C., Wang, M.S., and Cao P., 2008b. Serovar-specific real-time quantitative detection of Salmonella enteritidis in the gastrointestinal tract of ducks after oral challenge. Avian dis, 52,88–93 doi: 10.1637/8102-090107-Reg PubMedCrossRefGoogle Scholar
  10. Dunlap, N.E., Beniamin, W.H. Jr., McCall, R.D. Jr., Tilden, A.B. and Briles, D.E., 1991. A safe site for Salmonella typhimurium is within splenic cells during the early phase of infection in mice. Microb Pathog, 10,297–310 doi: 10.1016/0882-4010(91)90013-Z PubMedCrossRefGoogle Scholar
  11. Gast, R.K. and Beard, C.W.,1990. Production of Salmonella enteritidis-contaminated eggs by experimentally infected hens. Avian Dis, 34, 438–446 doi: 10.2307/1591433 PubMedCrossRefGoogle Scholar
  12. Gast, R.K. and Benson, S.T.,1995.The comparative virulence for chicks of Salmonella enteritidis phage type 4 isolates and isolates of phage types commonly found in poultry in the United States. Avian Dis, 39, 567–574 doi: 10.2307/1591810 PubMedCrossRefGoogle Scholar
  13. Guard-Petter, J.,2001. The chicken, the egg and Salmonella enteritidis. Environ Microbiol, 3,421–430 doi: 10.1046/j.1462-2920.2001.00213.x PubMedCrossRefGoogle Scholar
  14. Hensel, M., Shea, J.E., Waterman, S.R., Mundy, R., Nikolaus, T., Banks, G., Vazquez-Torres, A., Gleeson, C., Fang, F.C. and Holden, D.W.,1998. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol Microbiol, 30,163–174 doi: 10.1046/j.1365-2958.1998.01047.x PubMedCrossRefGoogle Scholar
  15. Hsu, H.S.,1989. Pathogenesis and immunity in murine salmonellosis. Microbiol Rev, 53,390–409PubMedGoogle Scholar
  16. Humphries, A.D., Townsend, S.M., Kingslev, R.A., Nicholson, T.L., Tsolis, R.M. and Baumler, A.J.,2001. Role of fimbriae as antigens and intestinal colonization factors of Salmonella serovars. FEMS Microbiol Lett, 201,121–125 doi: 10.1111/j.1574-6968.2001.tb10744.x PubMedCrossRefGoogle Scholar
  17. Lai, C. W., Chan, R. C., Cheng, A.F., Sung, J.Y. and Leung, J.W.,1992. Common bile duct stones: a cause of chronic salmonellosis. Am J Gastroenterol, 87,1198–1199PubMedGoogle Scholar
  18. Mastroeni, P., Chabalgoity, J.A., Dunstan, S.J., Maskell, D.J. and Dougan, G.,2001. Salmonella:immune responses and vaccines. Vet J, 161, 132–164 doi: 10.1053/tvjl.2000.0502 PubMedCrossRefGoogle Scholar
  19. Mittrucker, H.W., Kaufmann, S.H.,2000.Immune response to infection with Salmonella typhimurium in mice. J Leukoc Biol, 67, 457–463PubMedGoogle Scholar
  20. Mittrucker, H.W., Kohler, A. and Kaufmann, S.H., 2002. Characterization of the murine T-lymphocyte response to Salmonella enterica serovar typhimurium infection. Infect Immun, 70,199–203 doi: 10.1128/IAI.70.1.199-203.2002 PubMedCrossRefGoogle Scholar
  21. Nnalue, N.A., Shnyra, A., Hultenby, K. and Lindberg, A.A., 1992. Salmonella choleraesuis and Salmonella typhimurium associated with liver cells after intravenous inoculation of rats are localized mainly in Kupffer cells and multiply intracellularly. Infect Immun, 60,2758–2768PubMedGoogle Scholar
  22. Ohl, M.E. and Miller, S.I.,2001. Salmonella: a model for bacterial pathogenesis. Annu Rev Med, 52,259–274 doi: 10.1146/ PubMedCrossRefGoogle Scholar
  23. Okamura, M., Miyamoto, T., Tani, H., Sasai, K. and Baha, E.,2001. Difference in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella enteritidis and other Salmonella serovars. Avian Dis, 45,962–971 doi: 10.2307/1592875 PubMedCrossRefGoogle Scholar
  24. Okamura, M., Lillehoj, H.S., Raybourne, R.B., Babu, U.S., Heckert, R.A., Tani, H., Sasai, K., Baba, E. and Lillehoj, E.P.,2005. Differential responses of macrophages to Salmonella enterica serovars enteritidis and typhimurium.Vet immunol immunpathol, 107,327–335 doi: 10.1016/j.vetimm.2005.05.009 CrossRefGoogle Scholar
  25. Prouty, A.M., Schwesinger, W.H. and Gun, J.S.,2002. Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp. Infect Immun, 70,2640–2649 doi: 10.1128/IAI.70.5.2640-2649.2002 PubMedCrossRefGoogle Scholar
  26. Qi, X.F., Yang X.Y., Cheng, A.C., Wang, M.S., Zhu, D.K., and Jia, Y.Y., 2008. Quantitative analysis of virulent duck enteritis virus loads in experimentally infected ducklings. Avian Dis, 52,338–344 doi: 10.1637/8120-100207-ResNote.1 PubMedCrossRefGoogle Scholar
  27. Rescigno, M. and Borrow, P.,2001. The host-pathogen interaction: new themes from dendritic cell biology. Cell, 106,267–270 doi: 10.1016/S0092-8674(01)00454-8 PubMedCrossRefGoogle Scholar
  28. Sierro, F., Dubois, B., Coste, A., Kaiserlian, D., Kraehenbuhl, J.P. and Sirard, J.C., 2001.Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells. Proc Natl Acad Sci USA, 98, 13722–13727 doi: 10.1073/pnas.241308598 PubMedCrossRefGoogle Scholar
  29. Takata, T., Liang, J., Nakano, H. and Yoshimura, Y.,2003. Invasion of Salmonella enteritidis in the tissues of reproductive organs in laying Japanese quail: an immunocytochemical study. Poult Sci, 82:1170–1173PubMedGoogle Scholar
  30. Van Velkinburgh, J.C. and Gunn, J.S.,1999. PhoP-PhoQ-regulated loci are required for enhanced bile resistance in Salmonella spp. Infect Immun, 67,1614–1622PubMedGoogle Scholar
  31. Vasquez-Torres, A., Xu, Y., Jones-Carson, J., Holden, D.W., Lucia, S.M., Dinauer, M.C., Mastroeni, P. and Fang, F.C.,2000. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science, 287,1655–1658 doi: 10.1126/science.287.5458.1655 CrossRefGoogle Scholar
  32. Yan, B., Cheng, A.C., Wang, M.S., Deng, S.X., Zhang, Z.H., Yin, N.C., Cao, P. and Cao, S.Y.,2008. Application of indirect immunofluorescent staining method for detection Salmonella enteritidis in paraffin slices and antigen location in infected duck tissues. World J Gastroenterol, 7, 776–781 doi: 10.3748/wjg.14.776 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • S. X. Deng
    • 1
    • 4
  • A. C. Cheng
    • 1
    • 2
    Email author
  • M. S. Wang
    • 1
    • 2
    • 3
  • X. R. Li
    • 1
  • B. Yan
    • 1
  1. 1.Avian Diseases Research CenterCollege of Veterinary Medicine of Sichuan Agricultural UniversityYaanChina
  2. 2.Key Laboratory of Animal Diseases and Human Health of Sichuan ProvinceYaanChina
  3. 3.College of Life Science and Technology of Southwest University for NationalitiesChengduChina
  4. 4.Agricultural office of Dalingshan townDongguanChina

Personalised recommendations