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1Department of Medicine, University of Rochester
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Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.
Wu, S., Lu, R., Zhang, Y., Sun, J. Chronic Salmonella Infected Mouse Model. J. Vis. Exp. (39), e1947, doi:10.3791/1947 (2010).
This protocol includes three portions: bacterial culture, mouse gavage, and Salmonella detection.
1. Salmonella growth condition.
2. Salmonella-infection model*.
*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.
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 PhoPc, PhoPc AvrA-, and PhoPc AvrA-/AvrA+, PhoPc 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|
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:
We have tested the Salmonella colonization in the mouse at different concentration: 1 x 103 colony-forming units to 1 x 108 colony-forming units (100 μl/mouse). At 1 x 106 to 10 x 108 colony-forming units, Salmonella was able to colonize the mice. Therefore, based on our previous publications1, 5 and unpublished data, the final concentration of Salmonella in this solution will not be measured before gavage.
In this experimental procedure, we used specific-pathogen-free female C57BL/6 mice (Taconic, Hudson, NY) that were 6-7 weeks old. Bacterial strains used include Salmonella typhimurium wild-type strain ATCC14028s (WT-SL), non-pathogenic Salmonella mutant strain PhoPc3, PhoPc AvrA-, and PhoPc AvrA-/AvrA+, PhoPc AvrA(table 2). Using this protocol, Salmonella typhimurium colonization can be detected in the mouse intestine over 6 months (Fig1). Salmonella infection and inflammation were measured by observing fecal matter and boy weight. When tissue samples and blood were collected, the length of intestine was measured as a feature of intestinal inflammation. The weights of spleens and livers were also examined. Mouse serum cytokine were tested by ELISA5. Further more, biochemical and molecular methods are further used to test the changes of inflammatory cytokine and pathology changes induced by Salmonella 5.
Typically, body weight loss and death occur within 4 weeks post infection (Table 1). Post Salmonella infection, there was significant body weight loss in the bacterial infected groups compared to the control group without bacterial treatment (*p< 0.05 or p<0.001 Table 1). The body weight was measured weekly. The survival proportions 4 weeks post infection were shown in Fig. 2. Overall, 92% of mice (n=50) infected with Salmonella strains PhoPc, 78% PhoPc AvrA-, or 70% PhoPc AvrA-/AvrA+ can survive with persistent Salmonella in intestine. After the acute infection, all survivors still carried Salmonella , which could be detected post infection 6 months using the method described in this proposal. Once mice survive 3 weeks post infection, mice gained body weight and less death occurred (Fig.2). Moreover, immunofluroscence staining data also show the invasion of Salmonella in intestinal mucosa 27 weeks post infection (Fig.3). These data showed that PhoPc, PhoPc AvrA-, or PhoPc AvrA-/AvrA+ can be used for the chronic infection model.
Mice with host resistance factor Nramp1+/+ are resistant to Salmonella infection 3-6. C57/BL6 mice with Nramp defective were use in the current studies. We found that 90% mice infected with pathogenic Salmonella strain 14028s cannot survive over 4 weeks (data not shown). To understand the long-term effects of wild-type S. Typhimurium infection, mice with Nampr1+/+ could be used.
This model is established in a BSL-2 laboratory. Our protocol is approved by the University of Rochester University Committee on Animal Resources (UCAR). Mice infected with Salmonella are likely to suffer discomfort and distress and become less active and move around slowly. Their fur may become ruffled and they may not feed or drink as normal. The animals will be observed closely and if any sign of discomfort such as unable to ambulate well enough to maintain hydration and caloric intake If a mouse showed indication that it had aspirated fluid or significant body weight loss (10% or more)7, and did not die immediately, the mouse was humanely euthanized. All laboratory personnel will be trained in observing the mice appropriately8, 9.
Many studies used the streptomycin-pretreatment have taken advantage of the colitis induced by Salmonella typhimurium for the studies on at the early phase of infection1,2,10. However, only few report on the chronic infection in mouse model in vivo11. Mice with Salmonella persistent existence in the GI 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. This model can also be used for E.coli pathogenesis and probiotics studies. Overall, this protocol will aid researchers using the mouse model to address fundamentally important biological and microbiological questions.
No conflicts of interest declared.
This work was supported by the NIDDK KO1 DK075386 grant and the American Cancer Society RSG-09-075-01-MBC to Jun Sun.
|Luria-Bertani broth||BD Biosciences||244610|
|Feeding needle||Popper & Sons, Inc.||7920|
|BBL CHROMagar Salmonella||BD Biosciences||214983|
|Disposable cell spreaders||Biologix Research Company||65-1010|
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