Marc Young1, Jens-Eike Taeubert1, Juergen Geist2, Michael Pfaffl3, Ralph Kuehn1
1Unit of Molecular Zoology, Chair of Zoology, Department of Animal Science, Technische Universität München;
2Aquatic Systems Biology Unit, Department of Ecology and Ecosystem Management, Technische Universität München;
3Physiology Weihenstephan, Department of Animal Sciences, Centre of Life and Food Sciences, Technical University of Munich
The brown trout syndrome (BTS) is an annually reoccurring disease that causes species-specific die-off of Salmo trutta in BTS-impacted pre-alpine rivers of Europe. Based on experimental evidence to date it is hypothesized that BTS is caused by a yet unidentified pathogen. To validate this working hypothesis, as well as to discern the type of pathogen likely to be responsible for BTS, gene expression analyses were conducted on Salmo trutta suffering from BTS in order to identify and investigate pathogen-specific immune responses during the progression of the disease. Salmo trutta obtained from a single source were held in flow-through tanks supplied with river water at two separate geographic locations (BTS-impacted river section / treatment group; Non-impacted river section / control group) along the same river for a duration of 98 days, with 3 individuals being sampled from each tank (treatment and control) in 7 day intervals (total 14 time points). In a first step, cDNA microarrays were employed in order to screen for regulated pathogen-specific immune responses. Spot identification, intensity quantification and quality control were carried out with the GenePix® Pro 6 software. Analysis of the microarray data were conducted using the open source R software package Linear Model for Microarray Data. Microarray data were background-subtracted using the Kooperberg model-based background correction function, normalized within arrays with the Loess method followed by normalization between arrays using the scale method. Linear models were fitted to the expression data and moderated t-statistics were computed using the empirical eBayes method. Features with a p-value ≤ 0.001 and log 2 -fold change ≥ 1 were deemed significant. The microarray analysis revealed that Salmo trutta suffering from BTS exhibit increased hepatic expression of important anti-viral genes. Subsequently in a second step relative gene expression for selected set of anti-viral genes were measured by RT-qPCR in order to construct more concise temporal gene expression patterns for anti-viral response genes in the liver of BTS-afflicted Salmo trutta over the course of the disease. The online Primer3 tool was used to design primers for selected anti-viral target genes as well as for three candidate reference genes. The web based comprehensive tool RefFinder identified Ubiquitin to be the most stable candidate reference gene. The ∆∆Ct method without efficiency correction as described by Pfaffl was used to determine the relative levels of mRNA expression for the anti-viral target genes normalized to the expression values of the reference gene Ubiquitin between treatment and control group for each time point. Both the microarray and the RT-qPCR analysis reveal that Salmo trutta suffering from BTS up-regulate different anti-viral genes throughout the progression of the BTS. Overall the results from the gene expression analyses suggest that BTS is caused by an unknown pathogenic virus.
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