Project description:Infectious pancreatic necrosis (IPN) is widespread disease with global distribution and highly contagious, which leads to severe economic problems both freshwater and saltwater, particularly in the Atlantic salmon (Salmo salar). In this work we analyzed and evaluated gene expression profiles in resistant and susceptible families associated to S. salar exposed to IPNV in order to understand the defense mechanisms mounted by resistant fish in combating infection. Experimentally, 29 families with known pedigree and variability were infected by immersion with 1x106 PFU/ml of IPNV, and a gamma frailty model analysis based on Kaplan-Meier mortality curves were obtained to identify and classify families as susceptible or resistant. Samples of head kidney tissue were obtained at day 1 and 5 post-infection from susceptible and resistant families and pooled to analyse their expression pattern with realt-ime PCR and one-color salmon-specific oligonucleotide microarray (21K). The microarray results showed clear differences in both families, where resistant fish presented an over-expression of genes associated with endocrine function, and a down-regulation of genes associated principally with tissue differentiation, metabolism protein degradation, cell signaling and immunological process. Moreover qPCR analysis using as target a number of transcripts associated with immunological processes showed the same trend observing a higher expression in susceptible than resistant families, indicating significant differences for CCL-19, CCRM-NM-3c, IL-12, IFN-M-NM-1, and IFN-M-NM-3. The one-color oligonucleotide microarrays included in this study was performed using pooled samples from each families at two different post-infection time condition. In the case of susceptible families were chosen at 1 day post-infection (dpi) the families 5352 (n = 2) and 5469 (n = 2) and at 5 dpi the families 5629 (n = 2) and 5637 (n = 2), whereas in resistant families were selected at 1 dpi the families 5459 (n =3) and 5761 (n =3), and at 5 dpi the families 5463 (n =3) and 5764 (n =3).

Project description:Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large genetic component underlying resistance to this disease has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. A global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus was undertaken. Full sibling salmon fry from two IPNV-resistant and two IPNV-susceptible families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Microarray interrogations were performed using a custom-designed, oligonucleotide microarray platform (Agilent) with 44 K probes per slide (Salar_2; Agilent Design ID:025520). The design is lodged with ArrayExpress ( under accession number A-MEXP-2065. Dual-label hybridisations were undertaken, with each experimental sample (Cy3 labelled) being competitively hybridised against a pooled reference control (Cy5 labelled) comprising equimolar amounts from each experimental RNA sample. The interrogations comprised 144 separate hybridisations; 2 genotypes (susceptible, resistant) Ã 2 families for each genotype Ã 2 challenge states (control, challenged) Ã 3 timepoints (1, 7, 20 dpi) Ã 4 biological replicates for resistant (2 from each of two tanks) and 8 biological replicates for susceptible (4 from each of two tanks). A preliminary analysis suggested evidence for a segregating QTL in both the susceptible families and therefore twice as many offspring were screened. It was later established that the evidence for QTL segregation in one of the families was inconclusive and therefore comparisons were made at the family level only. The analyses took the unbalanced design into account.

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