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Open in a separate window Figure 1 Contamination kinetics

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Oct 11, 2024

Open in a separate window Figure 1 Contamination kinetics. harbored intracellular bacteria or not. These data argue that resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also relevant to other virulence models to examine infections at a cellular and subcellular level in vivo. Murine salmonellosis has been used for decades as a model system for human typhoid fever because develop a systemic contamination in mice reminiscent of human typhoid (1C3). The ability of to successfully colonize and ultimately cause disease in susceptible mice is usually ascribed to numerous bacterial gene products essential for pathogenesis. Mutants Mevalonic acid of have been characterized in vitro, and genes important for the entry process and intracellular survival in tissue culture cells have been recognized (4, 5) and tested for virulence in the murine typhoid model. The involvement of these gene products in virulence is based on LD50 determinations and contamination kinetics of organs such as liver and spleen, but the actual role these factors play in disease has not been examined due to the lack of appropriate methods to study host pathogen interactions in vivo. Tissue culture experiments Mevalonic acid showed that is able to survive intracellularly Mevalonic acid in macrophages, suggesting that survival within host cells is an essential feature for virulence. This is important for disease, since mutants which fail to do so are avirulent in the mouse model (6). However, evidence to support macrophages as the host cell in vivo is still indirect, and conflicting data exist regarding the location of in the mouse, ranging from inside neutrophils (7), macrophages (8, 9), and hepatocytes (10, 11) to the extracellular space (12). In studies using light and electron microscopy, artificially high inoculums were used to visualize bacteria in the organs. Large doses of bacteria might trigger a septic shock response in the mouse which could further complicate the results. Here, we use confocal laser scanning microscopy (CLSM)1 (13) and computerized image analysis techniques with immunostained sections of liver as a tool to extend the knowledge gained from in vitro studies to the in vivo situation. Unlike standard microscopy, this method allowed us to infect mice with small infectious doses of (100 CFU), to examine early time points in the infection, and to focus on a single bacterium in a large tissue area. The relative ease of detection of bacteria is facilitated by the use of thick sections (30 m), which is usually 7C30 occasions the thickness used in standard immunohistochemistry (1C4 m) and 300C600 occasions that used in electron microscopy (50C 100 nm). By using this technology, we resolved the issue of targeting in vivo. By using antibodies directed against different host cell types, we recognized which cells infects and whether bacteria are located inside host cells. This study demonstrates that confocal microscopy is usually a suitable technology to study pathogenesis in vivo. Furthermore, this approach can be very easily applied to other contamination models of other pathogens in different tissues. Materials and Methods Bacterial Strains and Growth Condition. strain SL1344 (14) is usually a highly virulent strain of with an LD50 10 CFU when administered intraperitoneally or intravenously to susceptible BALB/c mice (reference 15 and Richter-Dahlfors, A., unpublished observation). SL1344 was produced in Luria-Bertani medium at 37C in standing overnight cultures. Rabbit polyclonal to ANKMY2 1 ml of the bacterial culture was centrifuged and resuspended in 1 ml chilly PBS. The OD600 was adjusted to 0.2 using chilly PBS. From this suspension, a dilution series was made in cold PBS to yield 103, 104, and 105 CFU/ml. The mice were immediately infected with 0.1 ml of the diluted cell suspensions. To determine the exact quantity of live bacteria in the inoculums used to challenge the mice, 0.1 ml of the dilutions was plated on Luria-Bertani plates, incubated overnight at 37C, and the colonies were counted. The size of the inoculum diverse between 0.65C1.15 102 CFU, 0.87C 1.15 103 CFU, and 0.87C1.15 104 CFU, respectively. Contamination Kinetics and Preparation of Thick Cryosections of S. typhimuriumCinfected Mouse.