Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon

Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed bi-phasic responses coinciding with the copepod-chalimus transition

The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development. Results Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated. Conclusions Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses

Atlantic Salmon Reovirus Infection Causes a CD8 T Cell Myocarditis in Atlantic Salmon (Salmo salar L.)

Heart and skeletal inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) is a disease characterized by a chronic myocarditis involving the epicardium and the compact and spongious part of the heart ventricle. Chronic myositis of the red skeletal muscle is also a typical finding of HSMI. Piscine reovirus (PRV) has been detected by real-time PCR from farmed and wild salmon with and without typical changes of HSMI and thus the causal relationship between presence of virus and the disease has not been fully determined [1]. In this study we show that the Atlantic salmon reovirus (ASRV), identical to PRV, can be passaged in GF-1 cells and experimental challenge of naïve Atlantic salmon with cell culture passaged reovirus results in cardiac and skeletal muscle pathology typical of HSMI with onset of pathology from 6 weeks, peaking by 9 weeks post challenge. ASRV replicates in heart tissue and the peak level of virus replication coincides with peak of heart lesions. We further demonstrate mRNA transcript assessment and in situ characterization that challenged fish develop a CD8+ T cell myocarditis

Fibroblasts Express Immune Relevant Genes and Are Important Sentinel Cells during Tissue Damage in Rainbow Trout (Oncorhynchus mykiss)

Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1β, IL-8, IL-10, TLR-3 and TLR-9. IL-1β and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1β, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1β. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1β, IL-8 and TGF-β already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells

Områdeberedskap på norsk sokkel - også for flyttbare innretninger?

Master's thesis in Risk Management and Societal SafetyDenne oppgaven er gjennomført som avsluttende del på mastergradsstudium i risikostyring og sikkerhetsledelse ved Universitetet i Stavanger. Veileder for oppgaven har vært førsteamanuensis Bjørn Ivar Kruke ved Universitetet i Stavanger. Bakgrunnen for oppgaven er Petroleumstilsynet som viser til en signifikant økning i storulykkespotensiale for flyttbare innretninger (Petroleumstilsynet, 2012). I 2000 ferdigstilte Norsk olje og gass sine retningslinjer for etablering av områdeberedskap på norsk sokkel (Norsk olje og gass, 2012). Formålet med retningslinjene er å uttrykke en norm for beredskap ved bruk av primært marine og luftbårne ressurser. I dag finnes det 4 ulike samarbeidsavtaler for områdeberedskap (Sørfeltene, Troll/Oseberg, Tampen og Halten Nordland) Oppgaven tar utgangspunkt i om alt er lagt til rette for å maksimere potensiale i områdeberedskapen? Det kan se ut som at flyttbare innretninger og områdeberedskap har noen utfordringer for å realisere det fulle potensialet. Dette leder frem til problemstillingen: Hvordan blir flyttbare innretninger integrert i områdeberedskap? Problemstillingen blir etterfulgt av tre forskningsspørsmål. Teorien lagt til grunn i oppgaven er mellom andre Quarantelli (2006) som skiller begrepene "disasters" fra "catastrophy". Videre om hva som legges til grunn for god beredskapsplanlegging og ledelse. Teoriene til Weick, Sutcliffe og Obstfeld (1999) om høypålitelighetsorganisasjoner (HRO) viser hvordan en kan oppnå og identifisere pålitelighet i en organisasjon. Forskningsprosjektet er gjennomført som et kvalitativt studie. Metoder for datainnsamling har vært intervju, dokumentanalyse og deltagende observasjon. Et av hovedfunnene fra oppgaven er at områdeberedskap ikke er bygget opp for å ivareta flyttbare innretninger. Det kreves at den enkelte flyttbare innretningen skal tilpasse seg den eksisterende beredskapen. Videre kan ikke forholdene beskrives som å ligge til rette for en smidig inngang til områdeberedskap. Løsningen kan være et større fokus på brodokumenter. Dette for å oppnå en bedre erfaringsoverføring og læring. I de tilfeller der den flyttbare innretningen står alene innenfor områdeberedskap, må områdeberedskap i større grad involveres i planleggingsprosesser for beredskapssamarbeidet. Andre funn er at områdeberedskap ikke påvirker håndteringen av en hendelse på en flyttbar innretning. Derimot vil områdeberedskap har stor betydning for håndteringen av konsekvensene, uavhengig av hvilken definerte fare- og ulykkessituasjon som legges til grunn. Videre at den flyttbare innretningen må gis muligheten til å stille gjensidige krav for å inngå i samarbeidet om beredskapen. Den bør få like vilkår til å utnytte potensiale i områdeberedskap. Først og fremst bør den flyttbare innretningen få ta del i et felles styring- og loggføringssystem, slik som faste innretninger har. Dette vil forbedre den horisontale koordineringen og den vertikale informasjonen. Det bør også stilles større krav til læring av feil etter øvelser og samtrening. Brodokumentet må bli et levende dokument på like linje som innretningens egen beredskapsplan. I studien blir det konkludert med at utfordringen er når storulykken inntreffer. Det kan synes som at der en storulykke på en fast innretning vil være en alvorlig krise, vil tilsvarende storulykke være en katastrofe på en flyttbar innretning. Dette som en følge av mangelfull integrering av flyttbare innretninger i områdeberedskap

Primary structure, developmentally regulated expression and potential duplication of the zebrafish homeobox gene ZF-21.

We report the molecular cloning and characterization of a cDNA derived from a zebrafish gene (ZF-21) related to the mouse homeobox containing gene Hox2.1. Interesting information about the differential conservation of various domains was gained from comparisons between the putative protein sequences from ZF-21 (275 amino acids) and Hox2.1 (279 aa). A separate DNA binding domain including the ZF-21 homeodomain and 36 additional flanking residues is completely identical to the C-terminal part of Hox2.1. As a consequence, these two mouse and zebrafish proteins must have identical DNA binding properties. A lower level of sequence identity between the N-terminal coding regions of ZF-21 and Hox2.1 reduces the total protein homology to 81%. However, short stretches of perfect homology in these N-terminals suggests that the essential biochemical functions are the same. As expected for true homologues, the ZF-21 and Hox2.1 genes also share extensive similarities with respect to non-coding sequences and temporal expression during embryogenesis. The finding of a potential ZF-21 duplication is discussed in relation to functional and evolutionary aspects of vertebrate homeobox genes

Expression studies of lipid metabolism genes in Atlantic salmon \u96 effects of replacing dietary fish oil with rapeseed oil

NRC publication: Ye