Comparison of Salmonellaenterica Serovar Typhimurium Colitis in Germfree Mice and Mice Pretreated with Streptomycin
ABSTRACT
Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of bacterial enterocolitis. Mice are generally protected from Salmonella serovar Typhimurium colonization and enterocolitis by their resident intestinal microflora. This phenomenon is called “colonization resistance” (CR). Two murine Salmonella serovar Typhimurium infection models are based on the neutralization of CR: (i) in specific-pathogen-free mice pretreated with streptomycin (StrSPF mice) antibiotics disrupt the intestinal microflora; and (ii) germfree (GF) mice are raised without any intestinal microflora, but their intestines show distinct physiologic and immunologic characteristics. It has been unclear whether the same pathogenetic mechanisms trigger Salmonella serovar Typhimurium colitis in GF and StrSPF mice. In this study, we compared the two colitis models. In both of the models Salmonella serovar Typhimurium efficiently colonized the large intestine and triggered cecum and colon inflammation starting 8 h postinfection. The type III secretion system encoded in Salmonella pathogenicity island 1 was essential in both disease models. Thus, Salmonella serovar Typhimurium colitis is triggered by similar pathogenetic mechanisms in StrSPF and GF mice. This is remarkable considering the distinct physiological properties of the GF mouse gut. One obvious difference was more pronounced damage and reduced regenerative response of the cecal epithelium in GF mice. Overall, StrSPF mice and GF mice provide similar but not identical models for Salmonella serovar Typhimurium colitis.
Salmonella enterica serovar Typhimurium infects a variety of mammalian hosts and can essentially cause two different types of disease. In humans and cattle Salmonella serovar Typhimurium infection leads to enteric disease associated with diarrhea. Bovine infection models have helped to identify Salmonella serovar Typhimurium virulence factors triggering enterocolitis (55). In susceptible mice, Salmonella serovar Typhimurium causes a typhoid-like disease characterized by rapid multiplication of bacteria in the liver and spleen but little intestinal pathology (12, 39). The intestinal tract of conventional specific-pathogen-free (SPF) mice is only poorly colonized by Salmonella serovar Typhimurium (∼104 CFU/g of contents) (2, 9) and other pathogenic bacteria upon oral infection (27, 42). This phenomenon, termed “colonization resistance” (CR) (49) or “microbial interference” (31), has been described as a complex mechanism in which host and the resident microflora cooperate to prevent the growth of other potentially pathogenic bacteria. The following different mechanisms for CR have been discussed: (i) the production of inhibitory substances, like bacteriocins, by indigenous microbes (36, 47), (ii) competition for nutrients and adhesion to mucin receptor sites (3, 13, 17, 25), (iii) a potential immunomodulatory effect of the commensal microflora on host defense mechanisms (e.g., defensin secretion stimulated by bacterial products like lipopolysaccharide, lipoteichoic acid, and muramyl-dipeptide) (1, 23, 35, 37), (iv) the inhibition of toxin production by indigenous microorganisms (7, 10, 11), and (v) the generation of a physiologically restrictive environment (pH, toxic metabolites) (4, 5, 29, 30).
Recently, we found that SPF mice pretreated with a single dose (20 mg/animal per os) of streptomycin (StrSPF mice) provide an animal model for Salmonella serovar Typhimurium colitis (2). The cecum and colon are inflamed, and only a little inflammation is observed in the ileum. Salmonella serovar Typhimurium colitis in StrSPF mice is associated with epithelial damage, polymorphonuclear granulocyte (PMN) infiltration, edema, and crypt abscesses, which are most evident at day 3 postinfection (p.i.) (2, 19). Similar inflammatory changes have been observed in several documented human cases (6) and in the bovine small and large intestines (48, 51). In contrast to the bovine or typical noncomplicated human infections, Salmonella serovar Typhimurium colitis in mice is associated with a parallel systemic infection, and susceptible natural resistance-associated macrophage protein 1-deficient mice (C57BL/6, BALB/c) die of this systemic disease on day 5 or 6 p.i. However, the systemic spread does not seem to cause or alter intestinal inflammation at 1 to 3 days postinfection (G. Paesold and W.-D. Hardt, unpublished results). Overall, StrSPF mice provide an interesting murine model for basic research on molecular aspects of acute Salmonella serovar Typhimurium colitis. Nevertheless, it should be kept in mind that direct transfer of results from any animal model to a human disease should be done with great caution.
In accordance with data from bovine models for Salmonella enterocolitis (50, 56), the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is a key virulence factor in StrSPF mice (2, 18). SPI-1 function enables bacteria to penetrate the intestinal epithelial barrier and reach the underlying tissues (e.g., lamina propria) (14, 19, 50). This process is associated with cytokine release and inflammation.
Germfree (GF) mice lack any intestinal microflora and are highly susceptible to colonization with various bacteria (52, 53), including Salmonella spp. (9, 31).
Due to the lack of the intestinal microflora, the intestine of GF mice has several distinct features, including altered mucus secretion (8), an underdeveloped gut-associated immune system, reduced immunoglobulin A (IgA) production (43), altered numbers of M cells (41), and reduced expression of antimicrobial peptides (21, 22). Reassociation of GF mice with a normal SPF flora can lead to acute self-limiting colitis (33), and colonization of GF mice with Salmonella serovar Typhimurium was found to cause diarrheal disease (26, 46). Due to the distinct physiological features of GF mice, it has not been clear whether Salmonella serovar Typhimurium colitis in GF mice differs from the disease in StrSPF mice.
Here, we compared StrSPF and GF mice as models for Salmonella serovar Typhimurium colitis. We studied colonization and the histopathologic features of cecal inflammation in StrSPF and GF mice. Wild-type and Salmonella serovar Typhimurium strains with a disrupted SPI-1 TTSS were included in the analyses to investigate which pathogenetic mechanisms trigger disease.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
For mouse infections, Salmonella serovar Typhimurium wild-type strain SL1344 (20) and its isogenic derivative SB161 (SL1344 ΔinvG [24]) were grown for 12 h at 37°C in Luria-Bertani broth containing 0.3 M NaCl, diluted 1:20 in fresh medium, and subcultured for 4 h with mild aeration. Bacteria were washed in ice-cold phosphate-buffered saline (PBS) and resuspended in cold PBS (5 × 107 CFU/50 μl).
Animal experiments.
Animal experiments were performed in individually ventilated cages at the BZL (Universität Zürich) as described previously (2), using SPF female C57BL/6 mice (6 to 9 weeks old) from Harlan (Horst, Netherlands) or GF C57BL/6 mice raised in a germfree isolator at BZL Zürich.
In the StrSPF model, water and food were withdrawn 4 h prior to treatment with 20 mg of streptomycin intragastrically. After this, animals were supplied with water and food ad libitum. Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 h before the mice were infected with 5 × 107 CFU of serovar Typhimurium (50 μl of a suspension in PBS intragastrically by gavage). GF mice were raised in a germfree isolator and fed sterile chow. They were removed from the isolator and transferred into individually ventilated cages without water and food 2 to 4 h prior to infection. Under these conditions mice remained germfree for at least 10 to 20 h, as determined by plating of fecal pellets on appropriate culture media (data not shown). GF mice were infected with 5 × 107 CFU of serovar Typhimurium (50 μl of a suspension in PBS intragastrically). To both GF and streptomycin-pretreated mice, water was offered ad libitum immediately after infection, and food was supplied 2 h p.i. At different times after infection, the mice were sacrificed by cervical dislocation, and tissue samples from the cecum, spleen, and liver were removed for analysis. Animal experiments were approved by and were performed as required by Swiss national and institutional regulations.
Analysis of serovar Typhimurium loads in the intestine, mLN, spleen, and liver.
To analyze colonization, the spleen, liver, and mesenteric lymph nodes (mLN) were removed aseptically and homogenized in 4°C PBS containing 0.5% Tergitol and 0.5% bovine serum albumin as described previously (2). The bacterial loads were determined by plating on MacConkey agar plates containing streptomycin (50 μg/ml). The minimal detectable levels were 10 CFU/organ in the mLN, 20 CFU/organ in the spleen, and 100 CFU/organ in the liver. Cecal contents were collected at different times after infection, and the bacterial loads were determined by plating. The minimal detectable level was 10 CFU per sample.
Histological procedures.
Tissue samples were embedded in OCT (Sakura, Torrance, CA), snap-frozen in liquid nitrogen, and stored at −80°C. Cryosections (5 μm) were mounted on glass slides, air dried for 2 h at room temperature, and stained with hematoxylin and eosin (H&E). As indicated below, cecal tissues were fixed in 4% formalin and embedded in paraffin prior to sectioning and staining.
Cecal pathology was independently evaluated by two pathologists in a blinded manner using 5-μm-thick H&E-stained sections and the following histopathological scoring scheme, as previously described (2).
(i) Submucosal edema.
Submucosal edema (expressed as a percentage) was deduced from the extension of the submucosa and was scored by morphometric analysis according to the following formula: submucosal edema = (b − a)/c, where a is the area enclosed by the mucosa (mucosa and intestinal lumen), b is the area enclosed by the borderline between the submucosa and the tunica muscularis (submucosa, mucosa, and intestinal lumen), and c is the area enclosed by the outer edge of the tunica muscularis (tunica muscularis, submucosa, mucosa, and lumen; area of the whole cecal cross section). The scores for submucosal edema were as follows: 0, no pathological changes; 1, detectable edema (submucosal edema, <10%); 2, moderate edema (submucosal edema, 10 to 40%); 3, profound edema (submucosal edema, ≥40%).
(ii) PMN infiltration into the lamina propria.
PMN in the lamina propria were enumerated in 10 high-power fields (magnification, ×400; field diameter, 420 μm), and the average number of PMN per high-power field was calculated. The scores were determined as follows: 0, less than 5 PMN per high-power field; 1, 5 to 20 PMN per high-power field; 2, 21 to 60 PMN per high-power field; 3, 61 to 100 PMN per high-power field; 4, more than 100 PMN per high-power field.
(iii) Goblet cells.
The average number of goblet cells per high-power field (magnification, ×400) was calculated from 10 different regions of the cecal epithelium. The scores were determined as follows: 0, more than 28 goblet cells per high-power field (in the cecum of the normal SPF mice we observed an average of 6.4 crypts per high-power field, and the average crypt consisted of 35 to 42 epithelial cells, 25 to 35% of which were differentiated into goblet cells); 1, 11 to 28 goblet cells per high-power field;. 2, 1 to 10 goblet cells per high-power field; 3, less than 1 goblet cell per high-power field.
(iv) Epithelial integrity.
Epithelial integrity was scored as follows: 0, no pathological changes detectable in 10 high-power fields (magnification, ×400); 1, epithelial desquamation; 2, erosion of the epithelial surface (gaps of 1 to 10 epithelial cells per lesion); 3, epithelial ulceration (gaps of >10 epithelial cells per lesion) (at this stage, there was generally granulation tissue below the epithelium).
The combined pathological score for each tissue sample was determined by adding the averaged scores described above, and the scores indicated the following: 0, intestine intact without any signs of inflammation; 1 to 2, minimal signs of inflammation which were not signs of disease (this was frequently found in the cecum of SPF mice); 3 to 4, slight inflammation; 5 to 8 moderate inflammation; 9 to 13, profound inflammation.
Immunofluorescence experiments.
Cryosections (7 μm) were mounted on glass slides and air dried for 2 h at room temperature prior to immunostaining. Sections were fixed in 4% paraformaldehyde for 30 min, washed in PBS, permeabilized with Triton X-100 (0.1% in PBS, 10 min, room temperature), washed, and blocked in 10% (wt/vol) normal goat serum in PBS for 1 h. The sections were stained for 1 h with polyclonal rabbit anti-class I and II cytokeratin (1:100; Biomedical Technologies, Stoughton, Mass.), polyclonal rabbit anti-Ki-67 (1:100; Abcam, Cambridge, United Kingdom), or monoclonal rat anti-CD18 (1:100) and hamster anti-ICAM-1 (1:100; Becton Dickinson) in PBS containing 10% (wt/vol) goat serum. Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:100), Cy5-conjugated anti-rat IgG (1:100), and Cy3-conjugated anti-hamster IgG (1:100) polyclonal antibodies (Dianova, Germany) diluted in PBS containing 10% (wt/vol) goat serum served as secondary antibodies. DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole) (0.5 μg/ml; Sigma). F-actin was visualized by staining with tetramethylrhodamine (TRITC)-conjugated phalloidin (Molecular Probes). Stained slides were mounted with Vectashield (Vector Laboratories).
Images were recorded at a magnification of ×40 using a Perkin-Elmer Ultraview confocal imaging system and a Zeiss Axiovert 200 microscope. Red, green, and Cy5 fluorescence was recorded confocally, while DAPI fluorescence was imaged by epifluorescence microscopy. Images were combined using the Adobe Photoshop version 7.0.1 software, ensuring that all panels from each figure were processed in the same way.
Statistical analysis.
Statistical analysis of the individual pathological scores for submucosal edema, PMN infiltration, loss of goblet cells, and epithelial integrity and of the combined pathological score was performed using the exact Mann-Whitney U test and the SPSS version 11.0 software, as described previously (2). P values of <0.05 were considered statistically significant. Bacterial colonization was analyzed in a similar manner. To allow statistical analysis of the bacterial loads, the values for animals yielding “no CFU” were set to the minimal detectable value (for mLN, 10 CFU; for spleens, 20 CFU; for livers, 100 CFU; for intestinal contents, between 67 and 400 CFU [see above]). After this, the median values were calculated using Microsoft Excel XP, and statistical analysis was performed using the exact Mann-Whitney U test and the SPSS version 11.0 software. P values of <0.05 were considered statistically significant.
RESULTS
Time course of serovar Typhimurium colitis in GF and StrSPF mice.
StrSPF and GF mice are animal models for Salmonella serovar Typhimurium-induced colitis (2, 31). However, these two disease models have never been compared side by side. In a pilot experiment we compared the kinetics of colonization by Salmonella serovar Typhimurium and inflammation in StrSPF and GF mice. SPF mice harboring a normal undisturbed intestinal microflora served as a control. We infected seven SPF mice, six StrSPF mice (mice pretreated with streptomycin 24 h prior to infection), and five GF mice that were kept under germfree conditions until 2 h prior to infection with the Salmonella serovar Typhimurium wild-type strain (5 × 107 CFU intragastrically). At 4 h, 8 h, and 20 h p.i. the numbers of animals indicated below were sacrificed, and we analyzed colonization of the cecum, as well as pathological changes in the large intestine (as described in Materials and Methods). At most times we used two SPF, two GF, and two StrSPF mice. One mouse was used to examine the results at 8 h for GF mice, and three mice were used to test SPF mice at 20 h p.i. (Fig. 1).
In the SPF mice, the cecal colonization levels were low (105 to 106 CFU per g cecal contents) at 4 h and 8 h p.i. and decreased at 20 h p.i. (101, 103, and 106 CFU per g cecal contents) (Fig. 1A). For the StrSPF and GF mice only one of the two mice in each group showed cecal colonization at 4 h p.i. (105 to 106 CFU/g) (Fig. 1A). At 8 h and 20 h p.i., serovar Typhimurium had colonized the ceca of both StrSPF and GF mice at high densities (108 to 109 CFU/g). This is in line with earlier studies with StrSPF and GF mice (2, 9, 44, 46) and indicated that Salmonella serovar Typhimurium colonized StrSPF and GF mice with similar kinetics.
No inflammation was detected in ceca of infected SPF mice at any of the times investigated (the pathological scores ranged from 1 to 2) (Fig. 1B and 2A and D). In contrast, StrSPF and GF mice developed pronounced intestinal inflammation characterized by mucosal edema, PMN influx, and destruction of the cecal epithelium within 20 h (the pathological score for the cecum ranged from 6.5 to 9.5) (Fig. 1B and 2B, C, E, F, G, and H). Crypt elongation was observed in the StrSPF mice but not in the GF mice. This was investigated in more detail, as described below.
Thus, Salmonella serovar Typhimurium colonized the ceca of StrSPF and GF mice with similar efficiencies. Severe cecal and colonic inflammation developed in both of these groups of mice within 20 h but not in SPF mice. This is in line with the notion that the mere lack of bacterial microflora is sufficient to allow Salmonella serovar Typhimurium to colonize the murine large intestine and cause colitis.
SPI-1 is required for colitis in GF and StrSPF mice.
Salmonella serovar Typhimurium colitis was observed in GF and StrSPF mice. However, it was not clear whether the disease was attributable to similar mechanisms in the two cases. Was inflammation due to the reduced epithelial barrier function observed in GF animals? Might this have allowed penetration of bacterial products or living bacteria into the intestinal tissue? Or was it attributable to Salmonella serovar Typhimurium-specific virulence factors? The latter phenomenon has been observed in StrSPF mice, in which inflammation is strongly dependent on the SPI-1 TTSS (2, 18, 19). We hypothesized that Salmonella serovar Typhimurium strains with a disrupted SPI-1 TTSS should be attenuated in both mouse models if Salmonella serovar Typhimurium colitis is attributable to the same pathogenetic mechanism.
Six or seven GF mice (kept in a germfree isolator until 2 h prior to infection) or six or seven StrSPF mice were used for each experimental group. For intragastric infection we used either wild-type Salmonella serovar Typhimurium strain SL1344 (5 × 107 CFU; seven mice), an isogenic Salmonella serovar Typhimurium mutant (SB161 [= SL1344 ΔinvG]; 5 × 107 CFU; nonfunctional SPI-1 TTSS [24]; seven mice), or PBS (mock infection; six mice). At 24 h p.i., mice were sacrificed, and we analyzed cecal colonization and pathology, as well as bacterial loads in the mLN, spleen, and liver.
Both Salmonella serovar Typhimurium strains had efficiently colonized the cecal lumina and mesenteric lymph nodes but not the spleens of GF and StrSPF mice at 24 h p.i. (Fig. 3A, B, and C). Liver colonization was observed in several animals infected with SB161 (Fig. 3D). This is in line with previous data for StrSPF mice (2). Wild-type Salmonella serovar Typhimurium caused severe colitis in GF and StrSPF animals, while SB161 did not (Fig. 3E and Table 1) (P ≪ 0.05). Thus, Salmonella serovar Typhimurium colitis in GF and StrSPF mice required a functional SPI-1 TTSS.
Detailed histopathological analysis revealed that wild-type Salmonella serovar Typhimurium caused slightly more pronounced colitis in GF mice than in StrSPF mice (pathological scores, 9 to 12 and 5 to 8.5, respectively) (Fig. 3E and Table 2). While PMN infiltration and submucosal edema did not differ (P > 0.05), the loss of goblet cells (P = 0.001) and disruption of the intestinal epithelium (P = 0.002) were more pronounced in GF mice than in StrSPF mice (Table 2 and Fig. 3E and 4C, F, L, and O). In GF mice infected with wild-type Salmonella serovar Typhimurium cecal crypts were extremely shallow and the epithelium showed erosion and ulceration, while StrSPF mice displayed merely epithelial desquamation (Fig. 4, compare panels L and O with panels C and F).
It should be noted that the distinct mucosal architecture of GF mice may slightly affect the quantitative evaluation of inflammation (see Materials and Methods). The lamina propria and epithelium are less broad in GF mice. Thus, the pathological score for PMN infiltration (number of PMN per optical field; see Materials and Methods) might slightly underrepresent the density of PMN infiltrating the mucosa of GF animals. This should be kept in mind when pathological scores for StrSPF and GF mice are compared.
We analyzed the difference in epithelial damage between StrSPF and GF mice in more detail. Increased leukocyte infiltration might provide one possible explanation. We performed immunofluorescence analyses of CD18+ cells (e.g., PMN, macrophages, NK-cells, and CD11c+ dendritic cells) in cecal tissues. We also analyzed the expression of ICAM-1, an intercellular adhesion molecule predominantly expressed on vascular endothelium and high endothelial venules, as well as on a variety of activated lymphocytes. ICAM-1 expression is upregulated upon inflammation, mediating infiltration of CD18+ cells into target tissues. We found upregulation of ICAM-1 and concomitant infiltration of CD18+ cells in the cecal mucosa and also transmigration into the cecal lumina of StrSPF and GF mice infected with wild-type Salmonella serovar Typhimurium (Fig. 4I and R). Low expression of ICAM-1 and few CD18+ cells were detected in SB161-infected mice and mice mock infected with PBS (Fig. 4G, H, P, and Q). Thus, wild-type Salmonella serovar Typhimurium seems to trigger equivalent CD18+ infiltration and ICAM-1 upregulation in GF and StrSPF mice. Increased numbers of infiltrating CD18+ cells are not likely to account for the severe mucosal ulceration found in GF mice.
Salmonella serovar Typhimurium infection of GF and StrSPF mice for 2 days.
Overall, Salmonella serovar Typhimurium colitis is similar in StrSPF and GF mice, and in both cases the SPI-1 TTSS is required for the induction of colitis on day 1 p.i. To assess whether disease progression differed between StrSPF and GF mice, we also analyzed colitis at 48 h pi. We reasoned that subtle differences between StrSPF and GF mice might lead to more obvious differences after a prolonged infection period.
Nine or 10 GF mice (kept in a germfree isolator until 2 h prior to infection) or six or seven StrSPF mice were infected with either wild-type Salmonella serovar Typhimurium SL1344 (5 × 107 CFU) or an isogenic Salmonella serovar Typhimurium mutant (SB161 [= SL1344 ΔinvG]; 5 × 107 CFU) or were mock infected with PBS (Fig. 5). At 48 h p.i., mice were sacrificed, and we analyzed cecal colonization and pathology, as well as the bacterial loads in the mLN, spleen, and liver.
In GF mice, the cecum was colonized with high efficiency at days 1 and 2 postinfection by all strains analyzed (Fig. 3A and 5A). StrSPF mice were colonized with similar efficiency at day 1 p.i., but at day 2 p.i. wild-type Salmonella serovar Typhimurium was present at significantly lower numbers than SB161 (Fig. 5A). This was also observed in one other study (19) but not in our first study of the streptomycin-pretreated mouse model (2). The reason for this variability is currently unclear. We speculate that reduction of the Salmonella serovar Typhimurium density by the onset of an unidentified element of the inflammatory response might play a role. In any case, at day 3 p.i. the level of wild-type Salmonella serovar Typhimurium increased and reached 109 to 1010 CFU/g at day 4 p.i., which was approximately 10-fold higher than the concentration in mice infected with SB161 (19).
At macroscopic inspection, the ceca and colons of mice infected for 2 days with wild-type Salmonella serovar Typhimurium appeared to be small, white, and edematous and were filled with a purulent exudate. The ceca and colons of all SB161- and mock-infected mice did not show overt signs of disease. Histopathological examination of the cecal tissue of wild-type Salmonella serovar Typhimurium-infected GF and StrSPF mice revealed that the overall degrees of the inflammation differed slightly but significantly (P = 0.044) in GF and StrSPF mice at 48 h p.i. (Fig. 5E and 6C, F, I, and L). Individual scoring parameters showed pronounced differences (Fig. 5E and Table 3). As found at 24 h p.i., the epithelial damage and loss of goblet cells were more pronounced in GF mice (P < 0.05) (Table 3 and Fig. 6F and L), while PMN influx and submucosal edema did not differ significantly between GF and StrSPF animals. Overall, the level of inflammation at 48 h p.i. was low in mice inoculated with PBS or SB161 (Table 4 and Fig. 5E and 6A, B, D, E, G, H, J, and K). Similar scores are routinely observed in noninfected SPF animals and are not considered signs of colitis.
In some GF mice (Fig. 5E and 6M), the pathological scores of wild-type Salmonella serovar Typhimurium-infected tissues diverged significantly with respect to mucosal edema depending on the part of the cecum which was used for analysis (Fig. 6M). At the apical part of the cecum, where the cecal patch is located, no mucosal edema was present in some of the mice, in contrast to the medial part of the cecum (Fig. 6N). This phenomenon was not observed in cecal sections of StrSPF mice (unpublished observations). This indicated that there were local alterations in the severity of disease in the cecum of GF mice.
In GF and StrSPF mice we observed colonization of mLN by 24 h p.i. and colonization of mLN, livers, and spleens by 48 h p.i. (Fig. 3 and 5). At 48 h p.i. wild-type strain SL1344 and SB161 colonized the mLN of StrSPF mice slightly more efficiently than they colonized the mLN of GF mice (P < 0.05) (Fig. 5B). In the livers and spleens of GF mice, SB161 and wild-type strain SL1344 were present at slightly higher levels than they were in StrSPF mice. However, most of the differences were not statistically significant (Fig. 5C and D). Overall, the course of systemic Salmonella serovar Typhimurium infection was similar in GF and StrSPF animals at days 1 and 2 p.i.
Notably, GF mice infected with wild-type Salmonella serovar Typhimurium showed external signs of sickness (ruffled fur, hunched back) at day 2 p.i., while GF mice infected with SB161 or mock infected with PBS and all StrSPF mice appeared to be healthy. Considering the moderate level of systemic infection (102 to 104 CFU per organ) (Fig. 5C and D), this might indicate that GF mice are hyperreactive toward some virulence function of the SPI-1 TTSS.
In summary, GF and StrSPF mice exhibited only subtle differences in cecal colonization or pathology upon infection with both wild-type Salmonella serovar Typhimurium and SB161. The systemic infections were also quite similar, and the SPI-1 type III secretion system was required for colitis in both animal models. This suggested that Salmonella serovar Typhimurium colitis in StrSPF and GF mice is ruled by the same pathogenetic mechanisms.
GF mice fail to regenerate cecal epithelium upon infection with wild-type Salmonella serovar Typhimurium.
While several parameters of Salmonella serovar Typhimurium colitis were similar in StrSPF and GF mice, there was a clear difference in the integrity of the cecal epithelium. The epithelium of GF mice infected with wild-type Salmonella serovar Typhimurium was ulcerated and showed only a few signs of regeneration (Fig. 4O). In StrSPF mice colitis was accompanied by marked regeneration of epithelial cells, resulting in crypt and villus elongation (Fig. 4F). To analyze this in more detail, we performed immunofluorescence studies of serial cecal sections of PBS-, SB161-, and wild-type Salmonella serovar Typhimurium-infected StrSPF and GF mice from the experiment shown in Fig. 3. Epithelial cells were stained with a polyclonal rabbit anti-cytokeratin antibody, and regeneration was studied using an antibody directed against Ki-67, an antigen expressed in the nuclei of proliferating cells (16).
In StrSPF and GF mice infected with SB161 or mock infected with PBS, the epithelial cell layer overlying the lamina propria was intact (Fig. 7D, E, M, and N), and proliferating cells were detected in a region close to the bottom of the crypts (Fig. 7G, H, P, and Q), where the proliferative zone is located. The epithelial layer in wild-type Salmonella serovar Typhimurium-infected StrSPF mice was also intact (Fig. 7F). Here, crypt elongation was clearly visible. Strong proliferation of epithelial cells was also reflected by the distribution of Ki-67+ nuclei along large parts of the crypt and also in the tips (Fig. 7I). In contrast, intact crypts were only rarely observed in GF mice infected with wild-type Salmonella serovar Typhimurium, and the mucosa was almost entirely replaced by infiltrating cells and connective tissue. Cytokeratin-positive epithelial cells, including the rare Ki-67-positive proliferating cells (probably enterocytes), were present only as a thin disordered layer on top of the mucosa (Fig. 7O and R).
In conclusion, epithelial integrity and proliferation were comparable in noninflamed, SB161-treated, and PBS-treated StrSPF and GF mice. Wild-type Salmonella serovar Typhimurium-induced inflammation triggered marked epithelial proliferation in StrSPF mice. In GF mice massive epithelial damage but only a little regeneration was observed. This is likely to account for the morphological difference between the cecal mucosa of StrSPF and GF mice upon infection with wild-type Salmonella serovar Typhimurium.
DISCUSSION
Wild-type Salmonella serovar Typhimurium induces colitis in StrSPF and GF mice. Here, we compared the two animal models. Infection kinetics and most parameters of the intestinal inflammation were strikingly similar for GF and StrSPF mice. Most notably, in both GF and StrSPF mice, the SPI-1 TTSS was essential for induction of cecal inflammation.
GF and StrSPF mice both lack CR, but they differ in several aspects of gut physiology. StrSPF mice are associated with a “normal” microflora throughout their life until 24 h before infection. They have a fully developed gut-associated immune system, and the gut physiology matures in response to the colonization. In contrast, GF mice have never been in contact with a microflora and consequently have an “immature” gut physiology, including a reduced barrier function, altered mucus secretion, and no tolerance to bacterial products (21). Colonization of GF mice with a “normal” SPF flora was known to cause acute self-limited colitis (33). These observations suggested that GF mice might mount an inflammatory response to SB161, which lacks SPI-1-dependent protein secretion but produces ample amounts of microbe-associated molecular patterns (MAMP) (e.g., lipopolysaccharide, peptidoglycan, flagellin) and even colonizes the intestinal tissue, as indicated by the high number of SB161 cells present in the mLN. However, we found that this is not the case. Salmonella serovar Typhimurium requires the SPI-1 TTSS to “actively” induce colitis in GF mice. This is similar to the results obtained for StrSPF mice (2; this study) and suggests that the same pathogenetic mechanisms are responsible for Salmonella serovar Typhimurium colitis in StrSPF and GF mice. Physiological differences between GF and StrSPF mice (mucus, IgA production, M cells, developmental state of Peyer's patches) do not seem to be of great importance.
Exactly how the SPI-1 TTSS induces colitis is still a matter of debate. Effector proteins injected into intestinal cells via the SPI-1 TTSS could trigger signaling cascades which lead directly to the proinflammatory gene expression (14). Indeed, significantly elevated levels of various proinflammatory cytokines were detected in bovine ligated ileal loops infected with wild-type Salmonella serovar Typhimurium but not in loops infected with a mutant lacking the SPI-1 TTSS effector protein genes sipA, sopA, sopB, sopD, and sopE2 (2, 54). Alternatively, SPI-1-mediated cell invasion and penetration of the epithelial layer might simply increase Salmonella serovar Typhimurium loads in the mucosal tissue. This would lead to elevated extraluminal concentrations of bacterial products (MAMP). In this case, recognition of elevated MAMP levels by Toll-like receptors (45) would represent the primary trigger for inflammation. So far, it has not been clear which of these pathways is responsible for colitis triggered via the SPI-1 TTSS. A scenario including both mechanisms is also conceivable.
Clearly, the SPI-1 TTSS plays an important role in initiating intestinal inflammation in GF mice (this study) and StrSPF mice (2, 18). Does it also contribute to systemic infection, as observed in oral infections in the murine typhoid model (15)? In a recent study it was shown that the SPI-1 TTSS strongly and significantly enhances invasion of the large intestinal epithelium and lamina propria of StrSPF mice (19) (Fig. 1). However, generally this does not lead to dramatically enhanced systemic infection. Colonization of the liver and spleen by wild-type Salmonella serovar Typhimurium seems to be only slightly enhanced compared to colonization by SB161. A significant difference is detected only in some experiments (e.g., spleen colonization at day 2 p.i.) (Fig. 5C, StrSPF mice) (P < 0.05). This is in line with our first observations (2). Thus, in StrSPF (and GF) mice, invasion of the intestinal epithelium does take place but does not seem to represent the rate-limiting step in reaching the mesenteric lymph nodes. Rather, the data are consistent with bacterial transport via M cells or dendritic cells (34, 38). Future research will be aimed at characterizing this pathway in more detail.
Pathology was somewhat aggravated (severe epithelial ulceration) in GF mice infected with wild-type Salmonella serovar Typhimurium compared to StrSPF mice. We found that this difference is associated with sluggish epithelial regeneration in GF mice. In contrast, Salmonella serovar Typhimurium colitis in StrSPF mice is accompanied by enhanced epithelial cell regeneration, resulting in crypt elongation. In uninfected GF mice the transit time of epithelial cells from intestinal crypts to villus tips is known to be longer than that in mice associated with an SPF microflora (40). Possibly this is attributable to the lack of Toll-like receptor signaling in GF mice (37). The low intrinsic epithelial cell turnover in GF mice might therefore explain the failure to respond adequately (by fast epithelial regeneration) to the insult imposed by wild-type Salmonella serovar Typhimurium.
Streptomycin can have several toxic side effects (28, 32). Therefore, whether intoxication of intestinal epithelial cells might contribute to Salmonella serovar Typhimurium colitis in StrSPF mice (i.e., by loosening tight junctions or disruption of the brush border) remained a matter of discussion. Here, we found that GF mice, which do not require streptomycin pretreatment, develop Salmonella serovar Typhimurium-induced colitis. This argues against the hypothesis that streptomycin-mediated intoxication of enterocytes plays a role in the streptomycin-pretreated mouse model.
In summary, this study shows that GF and StrSPF mice provide similar but not identical murine models for Salmonella serovar Typhimurium-induced colitis. In both model systems, the lack of CR allowed Salmonella serovar Typhimurium colonization of the murine large intestine at high levels and induction of SPI-1 TTSS-dependent colitis. This opens the door to profit from the advantages offered by each animal model. While streptomycin pretreatment allows workers to use a wide variety of knockout and transgenic mice obtained from SPF-certified facilities, GF mice have a different advantage: they guarantee that Salmonella serovar Typhimurium is the only bacterium present in the intestine and that there is no contamination by remains of the indigenous microflora. This might be advantageous for a range of experiments, including “Affymetrix chip-type” gene expression analyses. We expect that both animal models will contribute to elucidation of the molecular pathways of Salmonella serovar Typhimurium colitis.
FIG. 1.
FIG. 2.
FIG.3.
FIG. 4.
FIG.5.
FIG. 6.
FIG.7.
TABLE 1.
| Comparison | P values | ||||||
|---|---|---|---|---|---|---|---|
| PBS vs SB161 | SB161 vs SL1344 | ||||||
| GF | StrSPF | GF | StrSPF | ||||
| Combined score | 0.169 | 0.016 | 0.001 | 0.001 | |||
| Edema | 1.000 | 0.008 | 0.001 | 0.004 | |||
| PMN infiltration | 1.104 | 0.066 | 0.001 | 0.001 | |||
| Goblet cells | 1.000 | 1.000 | 0.001 | 0.001 | |||
| Epithelium | 1.000 | 0.462 | 0.001 | 0.001 | |||
a
The data shown in Fig. 3E were analyzed using the Mann-Whitney U test (see Materials and Methods).
TABLE 2.
| Comparison | P values for GF mice vs StrSPF mice | ||||
|---|---|---|---|---|---|
| PBS | SB161 | SL1344 | |||
| Combined score | 1.000 | 0.022 | 0.001 | ||
| Edema | 1.000 | 0.008 | 0.077 | ||
| PMN infiltration | 1.000 | 0.559 | 0.918 | ||
| Goblet cells | 1.000 | 1.000 | 0.001 | ||
| Epithelium | 1.000 | 1.000 | 0.002 | ||
a
The data shown in Fig. 3E were analyzed using the Mann-Whitney U test (see Materials and Methods).
TABLE 3.
| Comparison | P values for GF mice vs StrSPF mice | ||||
|---|---|---|---|---|---|
| PBS | SB161 | SL1344 | |||
| Combined score | 0.023 | 0.757 | 0.044 | ||
| Edema | 0.089 | 0.293 | 0.130 | ||
| PMN infiltration | 0.093 | 0.818 | 0.098 | ||
| Goblet cells | 1.000 | 1.000 | 0.002 | ||
| Epithelium | 1.000 | 0.425 | 0.001 | ||
a
The data shown in Fig. 5E were analyzed using the Mann-Whitney U test (see Materials and Methods).
TABLE 4.
| Comparison | P values | ||||||
|---|---|---|---|---|---|---|---|
| PBS vs SB161 | SB161 vs SL1344 | ||||||
| GF | StrSPF | GF | StrSPF | ||||
| Combined score | <0.001 | 0.029 | <0.001 | 0.001 | |||
| Edema | 0.013 | 0.048 | 0.002 | 0.001 | |||
| PMN infiltration | <0.001 | 0.022 | <0.001 | 0.001 | |||
| Goblet cells | 1.000 | 1.000 | <0.001 | 0.001 | |||
| Epithelium | 0.033 | 0.070 | <0.001 | 0.007 | |||
a
The data shown in Fig. 5E were analyzed using the Mann-Whitney U test (see Materials and Methods).
Acknowledgments
We are grateful to Mathias Heikenwalder and Cosima Pelludat for comments and critical reading of the manuscript.
This work was supported by grant 3100A0-100175/1 to W.D.H. from the Swiss National Foundation.
REFERENCES
1.
Ayabe, T., D. P. Satchell, C. L. Wilson, W. C. Parks, M. E. Selsted, and A. J. Ouellette. 2000. Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria. Nat. Immunol.1:113-118.
2.
Barthel, M., S. Hapfelmeier, L. Quintanilla-Martinez, M. Kremer, M. Rohde, M. Hogardt, K. Pfeffer, H. Russmann, and W. D. Hardt. 2003. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect. Immun.71:2839-2858.
3.
Bernet, M. F., D. Brassart, J. R. Neeser, and A. L. Servin. 1994. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut35:483-489.
4.
Bohnhoff, M., C. P. Miller, and W. R. Martin. 1964. Resistance of the mouse intestinal tract to experimental Salmonella infection. I. Factors which interfere with the initiation of infection by oral inoculation. J. Exp. Med.120:805-816.
5.
Bohnhoff, M., C. P. Miller, and W. R. Martin. 1964. Resistance of the mouse intestinal tract to experimental Salmonella infection. II. Factors responsible for its loss following streptomycin treatment. J. Exp. Med.120:817-828.
6.
Boyd, J. F. 1985. Pathology of the alimentary tract in Salmonella typhimurium food poisoning. Gut26:935-944.
7.
Brandao, R. L., I. M. Castro, E. A. Bambirra, S. C. Amaral, L. G. Fietto, M. J. Tropia, M. J. Neves, R. G. Dos Santos, N. C. Gomes, and J. R. Nicoli. 1998. Intracellular signal triggered by cholera toxin in Saccharomycesboulardii and Saccharomyces cerevisiae. Appl. Environ. Microbiol.64:564-568.
8.
Bry, L., P. G. Falk, T. Midtvedt, and J. I. Gordon. 1996. A model of host-microbial interactions in an open mammalian ecosystem. Science273:1380-1383.
9.
Collins, F. M., and P. B. Carter. 1978. Growth of salmonellae in orally infected germfree mice. Infect. Immun.21:41-47.
10.
Corthier, G., F. Dubos, and P. Raibaud. 1985. Modulation of cytotoxin production by Clostridium difficile in the intestinal tracts of gnotobiotic mice inoculated with various human intestinal bacteria. Appl. Environ. Microbiol.49:250-252.
11.
Czerucka, D., I. Roux, and P. Rampal. 1994. Saccharomyces boulardii inhibits secretagogue-mediated adenosine 3′,5′-cyclic monophosphate induction in intestinal cells. Gastroenterology106:65-72.
12.
Everest, P., J. Wain, M. Roberts, G. Rook, and G. Dougan. 2001. The molecular mechanisms of severe typhoid fever. Trends Microbiol.9:316-320.
13.
Freter, R., H. Brickner, M. Botney, D. Cleven, and A. Aranki. 1983. Mechanisms that control bacterial populations in continuous-flow culture models of mouse large intestinal flora. Infect. Immun.39:676-685.
14.
Galan, J. E. 2001. Salmonella interactions with host cells: type III secretion at work. Annu. Rev. Cell. Dev. Biol.17:53-86.
15.
Galan, J. E., and R. Curtiss III. 1989. Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc. Natl. Acad. Sci. USA86:6383-6387.
16.
Gerdes, J., U. Schwab, H. Lemke, and H. Stein. 1983. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer31:13-20.
17.
Gopal, P. K., J. Prasad, J. Smart, and H. S. Gill. 2001. In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli. Int. J. Food Microbiol.67:207-216.
18.
Hapfelmeier, S., K. Ehrbar, B. Stecher, M. Barthel, M. Kremer, and W. D. Hardt. 2004. Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun.72:795-809.
19.
Hapfelmeier, S., B. Stecher, M. Barthel, M. Kremer, A. Müller, M. Heikenwalder, T. Stallmach, M. Hensel, K. Pfeffer, S. Akira, and W. D. Hardt. 2005. The Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems allow Salmonella serovar Typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms. J. Immunol.174:1675-1685.
20.
Hoiseth, S. K., and B. A. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature291:238-239.
21.
Hooper, L. V. 2004. Bacterial contributions to mammalian gut development. Trends Microbiol.12:129-134.
22.
Hooper, L. V., T. S. Stappenbeck, C. V. Hong, and J. I. Gordon. 2003. Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nat. Immunol.4:269-273.
23.
Kaila, M., E. Isolauri, E. Soppi, E. Virtanen, S. Laine, and H. Arvilommi. 1992. Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr. Res.32:141-144.
24.
Kaniga, K., J. C. Bossio, and J. E. Galan. 1994. The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mol. Microbiol.13:555-568.
25.
Lee, Y. K., K. Y. Puong, A. C. Ouwehand, and S. Salminen. 2003. Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli. J. Med. Microbiol.52:925-930.
26.
Lima-Filho, J. V., L. Q. Vieira, R. M. Arantes, and J. R. Nicoli. 2004. Effect of the Escherichia coli EMO strain on experimental infection by Salmonella enterica serovar Typhimurium in gnotobiotic mice. Braz. J. Med. Biol. Res.37:1005-1013.
27.
Maier, B. R., A. B. Onderdonk, R. C. Baskett, and D. J. Hentges. 1972. Shigella, indigenous flora interactions in mice. Am. J. Clin. Nutr.25:1433-1440.
28.
McCracken, G. H., Jr. 1986. Aminoglycoside toxicity in infants and children. Am. J. Med.80:172-178.
29.
Meynell, G. G. 1963. Antibacterial mechanisms of the mouse gut. II. The role of Eh and volatile fatty acids in the normal gut. Br. J. Exp. Pathol.44:209-219.
30.
Meynell, G. G., and T. V. Subbaiah. 1963. Antibacterial mechanisms of the mouse gut. I. Kinetics of infection by Salmonella typhimurium in normal and streptomycin-treated mice studied with abortive transductants. Br. J. Exp. Pathol.44:197-208.
31.
Nardi, R. M., M. E. Silva, E. C. Vieira, E. A. Bambirra, and J. R. Nicoli. 1989. Intragastric infection of germfree and conventional mice with Salmonella typhimurium. Braz. J. Med. Biol. Res.22:1389-1392.
32.
Norris, C. H. 1988. Drugs affecting the inner ear. A review of their clinical efficacy, mechanisms of action, toxicity, and place in therapy. Drugs36:754-772.
33.
Ogawa, H., K. Fukushima, I. Sasaki, and S. Matsuno. 2000. Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria. Am. J. Physiol. Gastrointest. Liver Physiol.279:G492-G499.
34.
Penheiter, K. L., N. Mathur, D. Giles, T. Fahlen, and B. D. Jones. 1997. Non-invasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer's patches. Mol. Microbiol.24:697-709.
35.
Perdigon, G., C. Maldonado Galdeano, J. C. Valdez, and M. Medici. 2002. Interaction of lactic acid bacteria with the gut immune system. Eur. J. Clin. Nutr.56(Suppl. 4):S21-S26.
36.
Portrait, V., S. Gendron-Gaillard, G. Cottenceau, and A. M. Pons. 1999. Inhibition of pathogenic Salmonella enteritidis growth mediated by Escherichia coli microcin J25 producing strains. Can. J. Microbiol.45:988-994.
37.
Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, and R. Medzhitov. 2004. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell118:229-241.
38.
Rescigno, M., M. Urbano, B. Valzasina, M. Francolini, G. Rotta, R. Bonasio, F. Granucci, J. P. Kraehenbuhl, and P. Ricciardi-Castagnoli. 2001. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol.2:361-367.
39.
Santos, R. L., S. Zhang, R. M. Tsolis, R. A. Kingsley, L. G. Adams, and A. J. Baumler. 2001. Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes Infect.3:1335-1344.
40.
Savage, D. C., J. E. Siegel, J. E. Snellen, and D. D. Whitt. 1981. Transit time of epithelial cells in the small intestines of germfree mice and ex-germfree mice associated with indigenous microorganisms. Appl. Environ. Microbiol.42:996-1001.
41.
Savidge, T. C., M. W. Smith, P. S. James, and P. Aldred. 1991. Salmonella-induced M-cell formation in germ-free mouse Peyer's patch tissue. Am. J. Pathol.139:177-184.
42.
Shedlofsky, S., and R. Freter. 1974. Synergism between ecologic and immunologic control mechanisms of intestinal flora. J. Infect. Dis.129:296-303.
43.
Shroff, K. E., K. Meslin, and J. J. Cebra. 1995. Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. Infect. Immun.63:3904-3913.
44.
Stecher, B., S. Hapfelmeier, C. Muller, M. Kremer, T. Stallmach, and W. D. Hardt. 2004. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun.72:4138-4150.
45.
Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol.21:335-376.
46.
Tannock, G. W., and D. C. Savage. 1976. Indigenous microorganisms prevent reduction in cecal size induced by Salmonella typhimurium in vaccinated gnotobiotic mice. Infect. Immun.13:172-179.
47.
Toure, R., E. Kheadr, C. Lacroix, O. Moroni, and I. Fliss. 2003. Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes. J. Appl. Microbiol.95:1058-1069.
48.
Tsolis, R. M., R. A. Kingsley, S. M. Townsend, T. A. Ficht, L. G. Adams, and A. J. Baumler. 1999. Of mice, calves, and men. Comparison of the mouse typhoid model with other Salmonella infections. Adv. Exp. Med. Biol.473:261-274.
49.
van der Waaij, D., J. M. Berghuis-de Vries, and L.-V. Lekkerkerk. 1971. Colonization resistance of the digestive tract in conventional and antibiotic-treated mice. J Hyg.69:405-411.
50.
Wallis, T. S., and E. E. Galyov. 2000. Molecular basis of Salmonella-induced enteritis. Mol. Microbiol.36:997-1005.
51.
Watson, P. R., E. E. Galyov, S. M. Paulin, P. W. Jones, and T. S. Wallis. 1998. Mutation of invH, but not stn, reduces Salmonella-induced enteritis in cattle. Infect. Immun.66:1432-1438.
52.
Wells, C. L., M. A. Maddaus, R. P. Jechorek, and R. L. Simmons. 1988. Role of intestinal anaerobic bacteria in colonization resistance. Eur. J. Clin. Microbiol. Infect. Dis.7:107-113.
53.
Zachar, Z., and D. C. Savage. 1979. Microbial interference and colonization of the murine gastrointestinal tract by Listeria monocytogenes. Infect. Immun.23:168-174.
54.
Zhang, S., L. G. Adams, J. Nunes, S. Khare, R. M. Tsolis, and A. J. Baumler. 2003. Secreted effector proteins of Salmonella enterica serotype Typhimurium elicit host-specific chemokine profiles in animal models of typhoid fever and enterocolitis. Infect. Immun.71:4795-4803.
55.
Zhang, S., R. A. Kingsley, R. L. Santos, H. Andrews-Polymenis, M. Raffatellu, J. Figueiredo, J. Nunes, R. M. Tsolis, L. G. Adams, and A. J. Baumler. 2003. Molecular pathogenesis of Salmonella enterica serotype Typhimurium-induced diarrhea. Infect. Immun.71:1-12.
56.
Zhang, S., R. L. Santos, R. M. Tsolis, S. Stender, W. D. Hardt, A. J. Baumler, and L. G. Adams. 2002. The Salmonella enterica serotype Typhimurium effector proteins SipA, SopA, SopB, SopD, and SopE2 act in concert to induce diarrhea in calves. Infect. Immun.70:3843-3855.
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Received: 31 August 2004
Revision received: 21 October 2004
Accepted: 2 February 2005
Published online: 1 June 2005
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Comparison of Salmonellaenterica Serovar Typhimurium Colitis in Germfree Mice and Mice Pretreated with Streptomycin. Infect Immun
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