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Vinsamlegast notið þetta auðkenni þegar þið vitnið til verksins eða tengið í það: http://hdl.handle.net/1946/23037

Titill: 
  • Titill er á ensku Studies on immunostimulants and innate immune gene expression in Atlantic salmon: candidate tools for marker selection
Námsstig: 
  • Meistara
Útdráttur: 
  • Útdráttur er á ensku

    The aim of this project was to create tools to select robust fish with better innate immunity for breeding stock. The project was divided into three main stages. The first stage was to study basal expression of antimicrobial peptides (AMP) and inducible nitric oxide synthase (iNOS) of different Atlantic salmon (Salmo salar) families and investigate the effect of calcium supplemented β-Hydroxy-β-methyl butyrate (calcium-HMB) and β-Hydroxy-β-methyl butyrate (HMB) in the diet and water, as inductors of expression of these genes. The second stage was to test if increased expression of these genes has a defense role against bacterial and virus infection and the third stage was to initiate a search for single nucleotide polymorphism (SNP) markers in order to use them as selection markers for fish high-tolerance against pathogen infections.
    For the first stage, two different experiments were performed. In the first experiment, gill lamella tissue of 1330 fish (133 families and 10 fish per family) were collected in order to study basal expression of Cathelicidin-2 (CATH-2), an important component for the first line of immune defenses. In addition, 14 families were selected, treated with calcium-HMB and, then, gill tissues were collected and analyze in order to study the induction of CATH-2. Different basal expression of CATH-2 was observed and we could see induction of expression in two of the 14 families. Calcium-HMB treatment did not work as expected, possibly due to the presence of calcium; therefore, we decided to change the treatment. In the second experiment, gill and skin tissues of 12 families (12 individuals per family) of Atlantic salmon were collected in order to investigate induction of CATH-2, Hepcidin-1 (Hep-1) and inducible nitric oxide synthase (iNOS), by adding HMB in the diet and water. Up-regulation of CATH-2 in gill samples and CATH-2 and iNOS in skin samples was observed in all the families. Interestingly, Hep-1 expression in skin samples, rather than being enhanced, was in some cases down-regulated. These studies provide us with possible and powerful molecular tools to choose the most robust families.
    Once we had established conditions for inducing the expression of these AMPs and iNOS, we conducted a study in which a calculated number of 24-wells plates containing CHSE-214 cells, ASK cells and RTS-11 cells grown to 80% confluency, were pre-treated for 48h with the inducers HMB, PBA and vitaminD3. After this pre-treatment plates with CHSE214 were infected with infectious pancreatic necrosis virus (IPNV) and ASK cells with infections salmon anemia virus (ISAV) at a multiplicity of infection (MOI) of 0.1 (0.1 plaque forming units per cell) in a reference laboratory in Chile. In addition, a CHSE-214 cell plate and a RTS-11 cell plate were infected with Pisciricketssia salmonis in a different reference laboratory, also located in Chile. The findings of this study strongly suggest that pre-treating cells with concentrations of HMB known to induce expression of AMPs and iNOS, can lower IPNV and ISAV infectivity by 53% and 26% respectively. Unfortunately dilution of bacterial infection used for P. salmonis caused a violent infection and, at the moment of analysis, our internal housekeeping gene control was severely degraded preventing us from drawing any conclusions.
    The third stage of the project was approached by analyzing fin samples from the second experiment (144 samples) by ddRAD sequencing. Fin samples were collected from 12 families, 6 containing the major quantitative trait locus (QTL) for IPNV and 6 non-QTLs, all with different basal expression of CATH-2. Our previous experiments allow us to build a phenotype with high and low CATH-2-basal expression families. By performing ddRAD sequencing we hope to find SNPs, which can be used as biomarkers for selection of fish robustness. ddRAD sequencing was successfully performed but data analysis is still underway.

Samþykkt: 
  • 27.9.2015
URI: 
  • http://hdl.handle.net/1946/23037


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