Chronic Salmonella Infected Mouse Model

Podsumowanie

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

Streszczenie

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Protokół

This protocol includes three portions: bacterial culture, mouse gavage, and Salmonella detection.

1. Salmonella growth condition.

1. Prepare the Salmonella Luria-Bertani broth (LB) plate, incubate at 37° C overnight.
2. Pick a clone from the LB plate and put into 7 ml LB in a 12 ml tube, and shake at 37°C for about 5 hours.
3. Inoculate 50 ml LB with 0.05 ml of a stationary phase culture and incubate at 37°C without shaking for about 18 hours.
4. Spin overnight bacterial culture at room temperature with 6000 rpm for 10 minutes, suspend the bacteria in HBSS using ratio 100:3 (LB: HBSS). For example:
   Every 50 ml LB culture will be suspended in 1.5 ml HBSS.
5. For the animal gavage, further dilute the bacterial culture at 1:10 ratio. For example:
   Every 1.5 ml LB culture will be suspended in 15 ml HBSS.

2. Salmonella-infection model*.

1. Prepare the streptomycin solution. Each mouse will be given 7.5 mg of streptomycin in 100 μl HBSS. For example, prepare total 90 mg of streptomycin in 1.2 ml HBSS for 10 mice. Always have some extra volume when preparing the streptomycin solution.
2. Withdraw water and food 4 hours before oral gavage treatment.
3. Gavage mice with streptomycin with 7.5 mg of streptomycin (100 μl of HBSS for control mice). Grab the skin over the mouse shoulder firmly with the thumb and middle finger, stretch the head and neck with the index finger to make the esophagus straight. Direct the ball-tip of the feeding needle along the roof of the mouth and toward the right side of the back of the pharynx, then gently pass down into the esophagus and inject the 100 μl solution. No resistance should be felt.
4. At 20 hours after streptomycin treatment, withdrawn water and food again before the mice are infected with bacteria.
5. Gavage each mouse with 100 μl suspension in HBSS or treated with sterile HBSS (control) by oral gavage. The gavage procedure is the same as 2.3) gavage mice with streptomycin.

*Animal experiments were performed by using specific-pathogen-free female C57BL/6 mice (Taconic, Hudson, NY) that were 6-7 weeks old as previously described¹. The protocol was approved by the University of Rochester University Committee on Animal Resources (UCAR).

3. Detection of Salmonella in intestine.

1. Collect mouse fecal (about 100 mg).
2. Transfer fecal sample to a 1.5 micro centrifuge tube with 1ml PBS and vortex vigorously.
3. Centrifuge for 10 min at 800 rpm. Transfer the supernatant into a clean microfuge tube.
4. Centrifugation at 6000 rpm for 5min. Supernatant is discarded and 200 μL PBS is added to the pellets and vortexed.
5. Streak the 200 μl PBS with a disposable cell spreader on a BBL CHROMagar plate to detect Salmonella. Salmonella species appear mauve (rose to purple, see Fig.1). If 200 μl yields too many colonies on the plate, use 100 μl or 50 μl for the streak.

4. Representative Results.

When the protocol is done correctly, Salmonella typhimurium colonization can be detected in the mouse intestine over 6 months. Salmonella could be detected by fecal culture over 6 months (Fig.1). As a typical out come of this model, body weigh loss and death occur within 4 weeks post infection. Dependent on the Salmonella strains used for infection, some mice may no survive over 6 months.

Figure 1. Intestinal Salmonella in the Salmonella species appear mauve (rose to purple) in color, due to metabolic differences in the presence of selected chromogens. Other bacteria are either inhibited or produce blue-green or colorless colonies.

Figure 2. Survival proportions of mice infected with Salmonella mutant strain PhoP^c, PhoP^c AvrA-, and PhoP^c AvrA-/AvrA+, PhoP^c AvrA for 4 weeks (28 days).

Table 1. Body weight of mice infected with Salmonella for 4 weeks.

	0	1 week	2 weeks	3 weeks	4 week
control	16.78 ± 1.05	16.91 ± 1.28	18.26 ± 1.23	19.31 ± 1.26	20.26 ± 1.15
PhoPc	16.89 ± 1.03	17.14 ± 1.19	17.43 ± 1.63*	18.68 ± 1.78	20.05 ± 1.11
PhoPcAvrA-	16.91 ± 1.12	16.96 ± 1.39	17.06 ± 2.14**	18.71 ± 2.18	20.15 ± 1.56
PhoPc AvrA-/AvrA+	16.94 ± 0.96	17.17 ± 1.02	17.63 ± 1.42*	18.44 ± 2.03	20.09 ± 1.17

*compared to control group p<0.05
** compared to control group p<0.01

Table 2. Salmonella strains used in this study.

Name	Description	Reference or source
Salmonella 14028s	Wild-type S. typhimurium	ATCC
PhoPc	Non-pathogenic complex regulator mutant	Miller et al., 1990
PhoPc AvrA-	AvrA- mutation	Collier-Hyams et al., 2002
PhoPcAvrA-/AvrA+	PhoPcAvrA- with complemented plasmid encoding AvrA	Collier-Hyams et al., 2002

Dyskusje

To use this system, it is necessary for the researcher to learn how to gavage the animals. We detail a methodology for prepare bacterial culture and gavage the mice. We also show how to monitor the Salmonella persistence in the gastrointestinal (GI) tract. The critical steps in this protocol including:

1. Streptomycin-pretreatment: Streptomycin-pretreatment could get rid of some commensal gut flora and make the mice susceptible to the Salmonella infection².
2. Salmonell...

Materiały

Name	Company	Catalog Number	Comments
HBSS	Sigma-Aldrich	ABCD1234
Luria-Bertani broth	BD Biosciences	244610
Feeding needle	Popper & Sons, Inc.	7920
Streptomycin	MP Biomedicals	100556
BBL CHROMagar Salmonella	BD Biosciences	214983
Disposable cell spreaders	Biologix Research Company	65-1010