Ivar Hordvik

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

BackgroundSalmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking.ResultsThe 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 (EF1AA and EF1AB) 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 EF1AB>EF1AA>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 EF1AB>EF1AA>S20>β-actin>18S rRNA>GAPDH.ConclusionOverall, this work suggests that the EF1AA and EF1AB genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.

Cloning of Two Distinct cDNAs Encoding Parvalbumin, the Major Allergen of Atlantic Salmon (Salmo salar)

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin β (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal s1 ). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high‐performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal s1 was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST‐ and ELISA‐inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA‐library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the β‐lineage of parvalbumins and include the entire coding region.

Cloning and sequence analysis of cDNAs encoding the MHC class II β chain in Atlantic salmon (Salmo salar)

Atlantic salmon (Salmo salar) cDNAs encoding the major histocompatibility complex (Mhc-Sasa) class II β chain were isolated from a leucocyte library by a polymerase chain reaction (PCR) approach. Three different cDNAs (c144, c22, and c157) encoding the entire mature β chain have been analyzed. Clone c144 differs from clone c157 in 12.6% of the nucleotides in the β1-encoding region. The corresponding differences between clones c144 and c22, and clones c22 and c157, are 10.3% and 5.2%, respectively. This variation is, at least in part, most likely attributable to allelism. The similarity indices between the highly conserved β2 domains from Atlantic salmon and corresponding sequences from humans (DQβ), chicken (BLβ), carp (TLAIIβ-1), and rainbow trout (O. M. No. 55) are 45%, 40%, 66%, and 97%, respectively. Variable residues in the β1 domains from Atlantic salmon correspond with polymorphic sites of β1 domains from higher vertebrates. The frequency of substitutions in the β1-encoding region exceeds that in the 3′-untranslated (UT) region with several folds, indicating extensive β1 polymorphism in Atlantic salmon.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers (C 144) X 70165, (C 157) X 70166, and (C 22) X 70167.

Identification and characterization of a novel intraepithelial lymphoid tissue in the gills of Atlantic salmon

In addition to being the respiratory organ in fish, the gills form a barrier against the external milieu. Innate and adaptive immune system components have been detected in the gills, but lymphoid cell accumulations similar to that seen in the mammalian mucosa have not been described. The present investigations revealed cell accumulations on the caudal edge of interbranchial septum at the base of the gill filaments in the Atlantic salmon. Cytokeratin immunohistochemical staining and identification of a basal membrane and desmosome cell junctions by electron microscopy showed that the cell accumulation was located intraepithelially. Major histocompatibility complex (MHC) class II+ cells were detected by immunohistochemistry, and laser capture micro‐dissection and subsequent RT‐PCR analysis revealed expression of T‐cell receptor transcripts in the investigated tissue, suggesting the presence of T cells. The intraepithelial tissue reported here may be a suitable location for immune surveillance of gill infections, as well as a target site for new vaccine approaches and investigations of epithelial immunity. This is the first description of a lymphocyte cell aggregation within a teleostian gill epithelium network, illustrating a phylogenetically early form of leukocyte accumulations in a respiratory organ.

Molecular cloning of major histocompatibility complex class I cDNAs from Atlantic salmon (Salmo salar)

The major histocompatibility complex (Mhc) has attracted much attention because of its immense polymorphism, its importance in transplantation, and its indisputable role in disease susceptibility in humans (Chen and Parham 1989; Hill et al. 1991) and in animals (Lie 1990). Previously, typical Mhc features reflected in allograft rejection and mixed leucocyte reactivity were the only indications that an Mhc also existed in teleost fish (Stet and Egberts 1991). The use of polymerase chain reaction (PCR) with degenerate oligonucleotides from conserved Mhc regions provided the first direct evidence for Mhc class I and class II genes in a fish, the teleost carp (Hashimoto et al. 1990). The primary aim of our study was to isolate and characterize expressed Mhc molecules in Atlantic salmon, and thereby provide data for further studies on evolutionary and disease aspects of the Mhc and its polymorphism. An Atlantic salmon-specific Mhc probe from leucocyte RNA was generated by PCR based on primers from conserved regions of known Mhc genes. The oligonucleotides and detailed strategies are described in an accompanying paper by Hordvik and co-workers (this issue). This salmon-specific probe was employed to screen a leucocyte )~gtl0 cDNA library based on a few individuals, from which Mhc-positive cDNAs were derived. The cDNAs analyzed in this report were established as subclones in pGEM-7z(+)R (Promega, Madison, WI) and sequencing was performed on double-stranded DNA with SP6, T7, and internal primers, using the procedure supplied by Multi-Pol TM DNA

Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue

In modern bony fishes, or teleost fish, the general lack of leucocyte markers has greatly hampered investigations of the anatomy of the immune system and its reactions involved in inflammatory responses. We have previously reported the cloning and sequencing of the salmon CD3 complex, molecules that are specifically expressed in T cells. Here, we generate and validate sera recognizing a peptide sequence of the CD3ε chain. Flow cytometry analysis revealed high numbers of CD3ε+ or T cells in the thymus, gill and intestine, whereas lower numbers were detected in the head kidney, spleen and peripheral blood leucocytes. Subsequent morphological analysis showed accumulations of T cells in the thymus and spleen and in the newly discovered gill‐located interbranchial lymphoid tissue. In the latter, the T cells are embedded in a meshwork of epithelial cells and in the spleen, they cluster in the white pulp surrounding ellipsoids. The anatomical organization of the salmonid thymic cortex and medulla seems to be composed of three layers consisting of a sub‐epithelial medulla‐like zone, an intermediate cortex‐like zone and finally another cortex‐like basal zone. Our study in the salmonid thymus reports a previously non‐described tissue organization. In the intestinal tract, abundant T cells were found embedded in the epithelium. In non‐lymphoid organs, the presence of T cells was limited. The results show that the interbranchial lymphoid tissue is quantitatively a very important site of T cell aggregation, strategically located to facilitate antigen encounter. The interbranchial lymphoid tissue has no resemblance to previously described lymphoid tissues.

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

BackgroundThe 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.ResultsLarge 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.ConclusionsAtlantic 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.

The impact of ancestral tetraploidy on antibody heterogeneity in salmonid fishes

Summary: The immunoglobulin heavy chain locus in teleost fish is structurally similar to that in mammals, comprising a series of variable gene segments upstream of two constant region genes coding for IgM and IgD. Atlantic salmon have been shown to possess two distinct heavy chain loci, related to the tetraploid ancestry of this fish family. The nature (and results) of the evolutionary processes following the tetraploidization event are the focus of this review. Salmonid fish did not return quickly to a diploid state, but are still in the process of re‐establishing disomic inheritance. Thus, a specific locus in one species may still be endowed with four alleles, while it may have been converted to a pair of isoloci in another species. Analyses of immunoglobulin heavy chain genes in Atlantic salmon (Salmo salar) have strongly indicated that the ancestral heavy chain locus was subjected to tetrasomy throughout the radiation of the genera Oncorhynchus and Salmo, and that disomic inheritance was established in the Salmo lineage in the comparatively recent past. The introduction of disomic inheritance at these loci has resulted in two subsets of IgM and IgD heavy chains in Atlantic salmon.

A zebrafish engrailed-like homeobox sequence expressed during embryogenesis

The zebrafish genome was found to contain two sequences which cross‐hybridize strongly with the engrailed gene of Drosophila. Several independent clones containing one of these cross‐hybridizing sequences were isolated from a zebrafish genomic library. Characterization of this region (ZF‐EN) by DNA sequencing showed that it shares about 70% sequence identity with the engrailed homeobox. More extensive homeobox homology (90%) was found relative to the murine En genes. The closest relationship exists between ZF‐EN and En‐2 where the C‐terminal domains (104 amino acids) encoded by these genes are almost identical. We also observed that ZF‐EN and En‐2 are very similar with respect to their transcript sizes and temporal expression patterns.

Molecular phylogeny of microsporidians with particular reference to species that infect the muscles of fish

Ribosomal DNA from eight species of microsporidians infecting fish have been sequenced. Seven of these species infect the skeletal muscle of fish (Pleistophora spp.) and one species infects migratory mesenchyma cells (Glugea anomala). These sequences, in addition to other available microsporidian rDNA sequences from a broad range of host taxa, have been used in phylogenetic analysis. This analysis revealed that muscle‐infecting microsporidians from fish are a polyphyletic group, indicating that characters supposed to be important in the classification of the genus Pleistophora have to be re‐evaluated. One character that probably has a polyphyletic origin is the amorphous coat, which has been extensively used in the definition of this genus. Furthermore, our results showed that the insect parasitizing Pleistophora spp. are not related to the true pleistophorans parasitic in skeletal muscle of fish. Phylogenetic analysis of small subunit rDNA sequences revealed disagreements between the molecular phylogeny and classifications based upon ultrastruclure. Many of the morphological characters claimed to be important in microsporidian classifications appeared to have arisen several times during evolution: for example, the diplokaryon and sporophorous vesicles.