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Statistical analyzes of the cytometric results

Poultry are the main source of human infection by Salmonella. As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was, therefore, to identify infected host���cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.

Antibodies used for cellular characterization of cell subpopulations identified by flow cytometry

Statistical analyzes of the cytometric results

Increasing resistance to acute Salmonellosis (that is, contamination level shortly after infection) is not sufficient to reduce the risk for consumers to be contaminated by Salmonella. Indeed, animals may remain contaminated at a low level for weeks or months. Increased resistance to the Salmonella carrier state, i.e., animals' ability to clear bacteria, is needed; it involves measuring bacterial contamination several weeks after inoculation with a low dose. To study such resistance traits, three convergent approaches were used. A quantitative trait loci (QTL) study was performed, taking advantage of inbred lines differing in resistance. Several QTLs controlling resistance at a younger age were identified and are currently being confirmed in a new cross before finer mapping, using advanced intercross lines. These inbred lines are also presently being compared using functional genomics. In parallel, a selection experiment for increased or decreased resistance at a younger and a later age was undertaken. Besides providing genetic models differing in their levels of resistance, it underlined the importance of the choice of selection criterion, whether marker assisted or not. Indeed, genes controlling resistance are strongly dependant on age; selecting for resistance at a younger age might result in increased susceptibility at an older age. Finally, the results of this experiment were used in a model of the intra-flock propagation of Salmonella. It showed that introducing a proportion of resistant animals within a flock of susceptible hens could dramatically change the evolution of contamination. Moreover, it demonstrated the magnitude of synergy between selection and vaccination, which should enhance the interest of increased resistance. The results show that selection for increased resistance to the Salmonella carrier state may be efficient, providing that the appropriate criteria of selection are used.

International audience; Intestinal explants were maintained for weeks in a growth medium containing collagenase for progressive digestion to derive finite cell lines from the ileum (64 lines) or from the colon (8 lines) of a boar. Two ileal cell lines retaining either a fibroblastic or an epithelioid morphology have been used to derive heteroploid cell lines (IPI-1 and IPI-2) immortalized by transfection with an SV40 plasmid (pSV3-neo). The IPI-1 cells were found of fibroblastic lineage. The IPI-2 cell line gave rise to morphologically heterogeneous colonies ranging from typical epithelial cells to colonies of more-elongated cells. A crisis occurred during subcultivation of IPI-2 leading to the isolation of the IPI-2I cell line with a 24 h doubling time and a 21% plating efficiency. Epithelial nature of IPI-2I cells was supported by ultrastructural analysis of the cell monolayers. Differentiated cells were found to express microvilli at the apical cellular membrane and desmosomes connecting adjacent cells. Stable epithelioid phenotypes were obtained only from the IPI-2I cell line by multiple subcloning. These cells were found to express characteristics of both epithelial and mesenchymal cells by positive immunostaining with monoclonal antibodies reacting either with keratin 18 filament of simple epithelia or with vimentin filament typical in vivo of mesoderm. The lack of villin expression and the absence of transepithelial resistance have to be related to a poor differentiated state of this cell line. All these immortalized cell lines were permissive to the replication of microorganisms pathogenic for pig (Salmonella chloleraesuis, Salmonella typhimurium and tissue culture-adapted strains of transmissible gastroenteritis virus). The collection of finite and continuous cell lines will help to develop in vitro methods for long-term propagation of freshly isolated epithelium or three-dimensional organ culture in pig. In addition, the IPI-2I cell line provides a new model to study the conversion from a transformed to a nontransformed phenotype as incorporation of 2% dimethyl sulfoxide in the growth medium to repress large tumor antigen expression led to the progressive disappearance of cytokeratin 18 positive cells with, over a week, the death of the surviving vimentin-positive cells.

The antibody allowing identification of epithelial cells required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the liver is shown for epithelial cells.

The anti-Bu antibody, that allows B lymphocytes identification, was FITC conjugated. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (FITC) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the spleen is shown for the B lymphocytes.

Antibodies used for cellular characterization of cell subpopulations identified by flow cytometry

The anti-CD3 antibody, that allows T lymphocytes identification, was conjugated with FITC. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (FITC) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the spleen is shown for the T lymphocytes.

OHEJP Project MoMIR-PPC

The antibody allowing identification of endothelial cells required a secondary Alexa FluorTM 488 conjugated anti-rabbit antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the aortic vessels is shown for endothelial cells.

The antibody allowing identification of thrombocytes required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris was eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results were expressed as a percentage. Some examples are presented. Staining in the gall bladder is shown for the thrombocytes.

The antibody allowing identification of endothelial cells required a secondary Alexa FluorTM 488 conjugated anti-rabbit antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the aortic vessels is shown for endothelial cells.

The antibody allowing identification of monocytes-macrophages required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were removed on the basis of morphological criteria, regions were defined on the basis of uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) was on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results were expressed as a percentage. Some examples are presented. Staining in the gall bladder is shown for the monocytes-macrophages.

The antibody allowing identification of monocytes-macrophages required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris was removed on the basis of morphological criteria, regions were defined on the basis of uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) was on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results were expressed as a percentage. Some examples are presented. Staining in the gall bladder is shown for the monocytes-macrophages.

The antibody allowing identification of monocytes-macrophages required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were removed on the basis of morphological criteria, regions were defined on the basis of uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) was on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results were expressed as a percentage. Some examples are presented. Staining in the gall bladder is shown for the monocytes-macrophages.

The anti-Bu antibody, that allows B lymphocytes identification, was FITC conjugated. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (FITC) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the spleen is shown for the B lymphocytes.

The anti-Bu antibody, that allows B lymphocytes identification, was FITC conjugated. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris was eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (FITC) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the spleen is shown for the B lymphocytes.

The anti-CD3 antibody, that allows T lymphocytes identification, was conjugated with FITC. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (FITC) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the spleen is shown for the T lymphocytes.

The antibody allowing identification of epithelial cells required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results are expressed as percentages. Some examples are presented. Staining in the liver is shown for epithelial cells.

The antibody allowing identification of thrombocytes required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris were eliminated on the basis of morphological criteria, regions were set according to uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) is on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results were expressed as a percentage. Some examples are presented. Staining in the gall bladder is shown for the thrombocytes.