Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Salmon Macrophage early response to Aeromonas salmonicida

ABSTRACT: Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Enriched macrophages isolated from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0 h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0 h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response. Keywords: time course Enriched macrophages from 24 responder fish that showed positive respiratory burst in response to phorbol myristate acetate were plated in individual wells of 96-well flat-bottom polystyrene tissue culture plates. A. salmonicida were added to the macrophages, and incubated for 0.5, 1.0 or 2.0 h. Control wells received 10 ul of HBSS. Three replicate infections were performed for each type of bacteria. Hybridizations were carried out in duplicate, reversing the fluors for each sample on the second chip.

ORGANISM(S): Salmo salar

SUBMITTER: Katherine Ewart

PROVIDER: E-GEOD-8526 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The early response of Atlantic salmon (Salmo salar) macrophages exposed in vitro to Aeromonas salmonicida cultured in broth and in fish.

Vanya Ewart K K Williams Jason J Richards Robert C RC Gallant Jeffrey W JW Melville Krista K Douglas Susan E SE

Developmental and comparative immunology 20070820 4

Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the early macrophage respon ...[more]

PMID: 17825909

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Project description:An EST database from immune tissues was used to design the first high density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60mers) were successfully designed for 2716 out of 3482 unique sequences of the database. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida challenged fish at 3 days post-infection. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide and dye bias) factors using one-colour (1C) and two-colour (2C) -labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random effects variance revealed that technical variation was mostly negligible and biological variation represented the main factor, even using pooled samples. One-colour approach performed at least as well as 2C. A relevant proportion of genes turn out to be differentially labelled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defence response were detected at three days post-challenging. An unbalanced hierarchical design was planned to evaluate the sources of variation in microarray analysis. This design enabled us to consider most sources of noise in microarray analysis and evaluate their weight in the variability of the signal using an equilibrate number of replicates at each level and a not too large number of microarrays. According to this hierarchy, the following within slide sources of variation were assessed from top to bottom: biological (B), RNA extraction (E), labeling (L) and hybridization (H). Additionally, we studied variation between slides (S), the bias produced by labeling with different dyes (Cy3 and Cy5) and compared the performances of one-colour (1C) and two-colour (2C) -labeling approaches. A total of 24 microarrays grouped in three 8x15k slides were used for this design. One pool of five individuals was used as control and other two other pools of five individuals each (B1 and B2) were used as biological replicates. Three replicates were used for evaluating the variation associated with RNA extraction (E1, E2, E3), labeling (L1, L2, L3) and hybridization (H1, H2, H3) within slide. Some of these replicates were in turn hybridized in different slides to assess interslide variation. Three 2C hybridizations with dye swapping (6 microarrays) were performed in the same slide for comparison of 1C and 2C approaches and to analyze dye bias. Normalization within each microarray was carried out using the loess method. Normalization using all microarray data was done by performing the Aquantile method implemented in the limma package. To estimate the variation associated with replicates at the different steps of microarray analysis or to different slides we applied the Pearson correlation coefficient using log2 absolute treatment and treatment/control ratio values. Also an ANOVA of random effects was used to split the total variance into its components. For Dye bias evaluation and 1C vs. 2C comparisons we f estimated the proportion of common differentially expressed (DE) genes after Bonferroni correction at P <0.05 and P<0.01 at fold changes (FC) >1.5, 2 and 3. The degree of concordance between the different dyes and between 1C and 2C approaches was estimated as the proportion of common DE genes at each P and FC.

Dataset Information

Salmon Macrophage early response to Aeromonas salmonicida

Publications

The early response of Atlantic salmon (Salmo salar) macrophages exposed in vitro to Aeromonas salmonicida cultured in broth and in fish.

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