Lars Austbø

Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.

Immune responses in Atlantic salmon (Salmo salar) following protective vaccination against Infectious salmon anemia (ISA) and subsequent ISA virus infection

Infectious salmon anemia (ISA) is an orthomyxoviral disease that has had devastating effects on farmed Atlantic salmon. ISA is still a disease resulting in continued loss of revenues and therefore development of effective vaccines is of great importance. Commercial vaccines against ISA are available, but the efficacy is poorly described. There is little information about vaccine-induced immune factors preventing ISA virus (ISAV) infection today. In this study we assessed the protective effects and immunogenicity of vaccines containing three different quantities of the inactivated ISAV antigen. Our findings indicated that immunization induced effective protection in Atlantic salmon with a relative percent survival (RPS) as high as 86. The level of protection was correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produced detectable ISAV-specific and neutralizing antibodies. While ISAV infection was detectable in non-vaccinated control fish challenged by cohabitation, no infection was detected in fish immunized with high antigen amounts. After challenge, transcriptional analysis of selected immune-related genes demonstrated activation of innate immune responses in ISAV-infected control fish, but not in vaccine protected fish. This study furthers the knowledge about vaccine efficacy and vaccine-induced immunity to ISAV challenge in Atlantic salmon.

Healthy goats naturally devoid of prion protein

Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.

Transcriptional response of immune genes in gills and the interbranchial lymphoid tissue of Atlantic salmon challenged with infectious salmon anaemia virus.

Previously, it has been assumed that fish lack organized mucosa-associated lymphoid structures. Recently, an interbranchial lymphoid tissue (ILT) was described in salmonid gills at a site with substantial exposure to antigen. In this study, immune responses were examined in gills, mid-kidney and the laser-dissected ILT of Atlantic salmon (Salmo salar L.) infected with infectious salmon anaemia virus (ISAV). A strong innate response was observed in gills and mid-kidney and even in the laser-dissected ILT, despite the fact that no virus could be traced in this tissue. A small delayed increase in IgT transcripts, exclusively in the ILT, could indicate that this tissue has a role as a secondary lymphoid organ with clonal expansion of IgT expressing B-cells. Compared to the other examined tissues, gills displayed the earliest replication of the virus, further supporting this tissue as the main entry route for infection with ISAV.

Pigment-producing granulomatous myopathy in Atlantic salmon: a novel inflammatory response.

Melanin comprises a complex group of pigmented polymers whose primary function is ascribed to dermal solar protection, but may also have an interesting role in innate immunity. In ectothermic vertebrates, melanogenesis is reported in leukocyte populations, but it is not known if this occurs in connection with inflammatory reactions. Melanin accumulations in ectopic locations, in particular muscle, represent a serious quality problem in salmon production. Here, we investigated such changes for the expression of dopachrome tautomerase and tyrosinase as well as some important immune genes and pathogens. Furthermore, the nature of the pathological changes was addressed by morphological methods. Gene transcripts encoding key enzymes in melanogenesis, suggesting a de novo melanin synthesis in pigmented muscle, were found. MHC class II transcripts were up-regulated and there was no indication of bacterial or viral infection. The histological examination revealed granulomatous inflammation with distribution of MHC class II positive cells and T cells, analogous to the pattern found in mammals. Importantly, in contrast to mammals pigmented cells were contributing in the inflammation. We demonstrate that melanin production occurs in granulomatous inflammation in salmon, revealing a close and hitherto unreported link between the pigmentary and immune systems.

Transcriptional Characterization of the T Cell Population within the Salmonid Interbranchial Lymphoid Tissue

Previously, our group has shown that the interbranchial lymphoid tissue (ILT) is a distinct structure largely consisting of T cells embedded in a meshwork of epithelial cells, with no direct resemblance to previously described lymphoid tissues. In this study, we aim to focus on the T cell population and the possibility of the ILT being a thymus analog. By characterizing structural responsiveness to Ag challenge, the presence of recombination activating genes, and different T cell–related transcripts, we attempt to further approach the immunological function of the ILT in salmonid gills. In addition to eight healthy individuals, a group of eight infectious salmon anemia virus–challenged fish were included to observe T cell responses related to infection. The results showed reduced size of ILT in the infected group, no expression of RAG-1 and -2, and a high degree of T cell diversity within the ILT. Taking into account that the ILT can be regarded as a strategically located T cell reservoir and possibly an evolutionary forerunner of mammalian MALTs right at the border to the external environment, the alteration in transcription observed may likely represent a shift in the T cell population to optimize local gill defense mechanisms.

Immune parameters in the intestine of wild and reared unvaccinated and vaccinated Atlantic salmon (Salmo salar L.).

Forming a barrier to the outside world, the gut mucosa faces the challenge of absorbing nutrients and fluids while initiating immune reactions towards potential pathogens. As a continuation to our previous publication focusing on the regional intestinal morphology in wild caught post smolt and spawning Atlantic salmon, we here investigate selected immune parameters and compare wild, reared unvaccinated and vaccinated post smolts. We observed highest transcript levels for most immune-related genes in vaccinated post smolts followed by reared unvaccinated and finally wild post smolts, indicating that farming conditions like commercial feed and vaccination might contribute to a more alerted immune system in the gut. In all groups, higher levels of immune transcripts were observed in the second segment of mid-intestine and in the posterior segment. In the life stages and conditions investigated here, we found no indication of a previously suggested population of intestinal T cells expressing MHC class II nor RAG1 expression.

Identification of differentially expressed genes in ileal Peyer's Patch of scrapie-infected sheep using RNA arbitrarily primed PCR

BackgoundIn scrapie and prion diseases, the knowledge concerning genes involved in host response during the early infection period in the lymphoid tissues, still remains limited. In the present study, we have examined differential gene expression in ileal Peyers patches and in laser microdissected follicles of sheep infected with scrapie.MethodsIleal Peyers patches and laser microdissected follicles were of scrapie and control lambs with susceptible genotypes for classical scrapie. Potential regulated genes were found using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and fingerprinting. The differentially expressed genes were confirmed using real-time RT-PCR.ResultsThe expression of three genes (MAPRE3, LOC729073 and DNAJC3), were found to be significantly altered in scrapie infected lambs (P < 0.05).ConclusionThe three genes have not previously been associated with prion diseases and are interesting as they may reflect biological processes involved in the molecular pathogenesis of prion diseases.

Uptake of yeast cells in the Atlantic salmon (Salmo salar L.) intestine.

The intestinal mucosa is an important port of entry for many pathogens. Information of antigen uptake mechanisms is essential to understand and to possibly prevent infections. In teleosts, several studies have aimed at investigating particulate uptake in the gastrointestinal system that seems to vary dependent on fish species and antigen. In the present study, particulate uptake in the Atlantic salmon intestine by anal intubation of yeast cells has been investigated. In the anal intubated fish, yeast were found in the epithelium close to nuclei of macrophage-like cells and inside large mononuclear cells in the intestinal lumen, indicating uptake and possible transport of large antigen particles over the epithelium by macrophage-like cells.

Identification of Differentially Expressed Genes Associated with Osteochondrosis in Standardbred Horses Using RNA Arbitrarily Primed PCR

The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified two genes not previously correlated with OC as differentially expressed. The two genes, which were identical to TLK2 and an equine EST, are good targets for future research on OC.