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

GIACOMODONATO MN; GOREN N; VACCARO MI; SORDELLI DO; GRASSO D; ROPOLO A; CERQUETTI MC

Salt Lake City, Utah, USA.

Congreso; 102nd General Meeting of the 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 factors that allow the bacterium to induce inflammatory and secretory response and 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 to induce a protective immune response. We studied the early stages of intestinal infection in 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 INF-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 both, wt and attenuated strain E/1/3 of S. enteritidis increased intestinal INF-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 INF-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 demonstrated that this mutant is a good candidate for the construction of a live vaccine.