First stages of the murine intestinal infection with virulent and attenuated strains of Salmonella enteritidis.

GIACOMODONATO MN, GOREN NB, VACCARO MI, SORDELLI DO, GRASSO DH, ROPOLO AJ AND CERQUETTI MC

Salt Lake City, Utah, USA

Congreso; 102 nd General Meeting of American Society for Microbiology; 2002

American Society for Microbiology

Salmonella enteritidis is a gram-negative rod that causes disease in humans and animals. In the murine model, virulent strains of S. enteritidis express arrays of factors that allow the bacterium to induce inflammatory and secretory responses of the gut and it is able to survive and replicate in mononuclear phagocytes.  Wild type (wt) S. enteritidis causes the death of mice within a week following oral inoculation.  On the other hand, attenuated mutant E/1/3 of S. enteritidis is highly innocuous and yet it is able to colonize the host and induce a protective immune response.  We studied the early stages of intestinal infection in Balb/c mice inoculated with wt and attenuated strains of S. enteritidis.  Animals were sacrificed 6 hs after receiving 109 CFU of the bacterial suspension, intestines and Peyer´s patches (PP) were removed immediately.  Intestinal IFN-gamma was measured by ELISA.  Apoptosis was detected by TUNEL reactions and apoptotic cells were counted using a morphometric method.  Western blotting and RT-PCR were used to assess expression of iNOS protein and mRNA, and L-citrulline assays for studies on iNOS activity.  Immunostaining was used to localize the sites of expression of iNOS. Six hs after oral administration, wild type and attenuated strain E/1/3 of S. enteritidis increased intestinal IFN-gamma (1,262 + 453 pg/ml and 1,470 + 512 pg/ml, respectively) compared to untreated mice (361 + 116) (p< 0.05).  Similar results were obtained when IFN-gamma was investigated in isolated PP.  The percent of apoptotic lymphoid tissue in PP of animals immunized with mutant E/1/3 [median = 10; (range = 7-12)] or wt S. enteritidis [median = 9; (range = 5-13)] was significantly higher (p < 0.01) than that found in control mice [median = 1; (range = 0.5-2)].  Immunohistochemistry showed iNOS positive cells in PP.  Western blot and RT-PCR revealed increased expression of iNOS protein and iNOS mRNA in mice inoculated with wt and attenuated mutant E/1/3 compared with untreated animals.  In summary, our results show that highly attenuated mutant E/1/3 resembles the very first stages of the intestinal infection and demonstrate that this mutant is a good candidate for the construction of a live vaccine.