Stein Tore Solem

Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide

Abstract The present paper describes the effect of lipopolysaccharides (LPS) from Aeromonas salmonicida and other Gram-negative bacteria on the respiratory burst, phagocytosis and bactericidal activity of head kidney macrophages from Atlantic salmon ( Salmo salar L.) in vitro . Macrophages were first cultured in the presence of various concentrations of LPS from A. salmonicida for 1, 2 and 5 days and then tested for respiratory burst activity (reduction of nitroblue tetrazolium) after exposure to phorbol myristate acetate (PMA). The most marked increase in respiratory burst activity of LPS-treated macrophages was observed after 5 days of incubation with 1, 10 and 100 μ g LPS ml −1 . The increase appeared to be dose-dependent with a maximal response at 10 μ g ml −1 . At this LPS-concentration and incubation time the respiratory burst activity was 3·9 times larger in the treated macrophages than in the control macrophages. LPS from three other Gram-negative bacterial salmon pathogens and two non-fish pathogens also enhanced the respiratory burst activity of salmon macrophages. Macrophages incubated with 10 and 50 μ g LPS ml −1 also showed a significant increase in PMA-stimulated H 2 O 2 -production after 5 days of incubation. LPS also stimulated the phagocytic activity of Atlantic salmon macrophages against opsonized and nonopsonized glucan particles, and glutaraldehyde-fixed sheep red blood cells. LPS-treated macrophages showed an increased ability to kill an avirulent A-layer lacking strain of A. salmonicida , but not a virulent A-layer positive strain.

Characterization of crustins from the hemocytes of the spider crab, Hyas araneus, and the red king crab, Paralithodes camtschaticus.

Crustins are distributed across the decapods and are believed to play a significant part in the humoral defense system of their host. In this study, two crustin isoforms from Hyas araneus hemocytes were purified and tested for antimicrobial activity against selected microorganisms. They show both antibacterial and antifungal activity, with highest activity against the Gram-positive bacteria Corynebacterium glutamicum. Sequencing of the transcripts showed them to have a mature peptide of 90 amino acids and differing in three positions in the mature peptide. They were named CruHa1 and CruHa2. Real-time RT-PCR revealed that they mainly are expressed in hemocytes. Screening a cDNA library detected a crustin sequence in Paralithodes camtschaticus hemocytes, coding for a mature peptide of 98 amino acids. It was named CruPc. Based on phylogenetic inference and primary structure, CruHa1 and CruHa2 were placed within the Type I group of crustins, while CruPc belongs to the Type II.

Analysis of two IgM isotypes in Atlantic salmon and brown trout.

Atlantic salmon (Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CmuA and CmuB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout (Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CmuA and CmuB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CmuB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cmu subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.

Diversity of the immunoglobulin heavy chain in the Atlantic salmon (Salmo salar L.) is contributed by genes from two parallel IgH isoloci.

Immunoglobulin heavy chain (IgH) variable (V) region cDNAs from the Atlantic salmon, Salmo salar L., have been isolated and analysed with respect to diversity and transcription of the two parallel IgH isoloci in this species. A total of nine V(H) families were defined according to the 80% identity criterion, of which seven were highly related (>80% identity) to the V(H) families defined in rainbow trout and arctic charr. The variability of the CDR1 and 2 was low, although mutational hot-spot consensus sequences were accumulated in these regions. The CDR3 showed largest variability, expressing at least eight different groups of D motifs diversified by fusion of the D motifs, possible N and P nucleotide insertions and exonuclease activity. Presumably functional transcripts expressing D motifs in all three reading frames were identified for two of the motifs. The cDNAs were mapped to either of the two parallel loci, and sequence analysis revealed that the repertoire of V(H) segments was contributed by transcription of genes from both of the IgH isoloci. Transcription of genes from both isoloci generated no obvious effects on variability in the CDR3 of the Atlantic salmon IgH chains, although one additional J(H)-segment with altered N-terminal was generated by the process of duplication and divergence. Thus, the issue of biological significance of the two IgH isoloci remains unclear.

Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus

Abstract Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser32 to Ala32 amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser32 amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzymes catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure–function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

Heterologous expression and purification of the infectious salmon anemia virus hemagglutinin esterase

This study presents the heterologous production and purification of a soluble and functional form of the hemagglutinin esterase (HE) of the infectious salmon anemia virus (ISAV) isolate 4 (Glesvaer/2/90). The HE possesses receptor binding and receptor destroying enzyme (RDE) activity and is probably involved in the infection process. The recombinant HE protein (recHE 4) was expressed in insect cells (Sf9) using the baculovirus expression vector system. Both the transmembrane region and the cytoplasmic tail were deleted, and a C-terminal His(6)-tag was attached to facilitate identification and purification of the recHE 4 protein. As determined by Western analysis the recHE 4 was secreted at 20 degrees C and not at 28 degrees C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HEs own secretion signal sequence is more important than the promoter with respect to the amount of secreted recHE 4 obtained under the conditions used. A one-step purification by nickel-affinity chromatography resulted in a highly purified recHE 4, identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Also, the recHE 4 is glycosylated and contains disulfide bridges within the molecule. Functional studies including the verification of the receptor destroying enzyme (RDE) activity as well as the binding to Atlantic salmon erythrocytes (hemagglutination) indicate that the recHE 4 has similar functions as its native counterpart. In conclusion, insect cells secrete a functional form of the ISAV 4 HE. This is suitable for further analyses on its function and immunogenicity.

Characterization of an antiserum against Atlantic cod (Gadus morhua L.) g-type lysozyme

In this study we describe the production and characterization of an antiserum against recombinant g-type lysozyme derived from Atlantic cod. This is also the first initial analyses of g-type lysozyme protein expression in tissues of Atlantic cod. Recombinant expression and purification of cod g-type lysozyme was used for immunization to rabbit and the rabbit sera were analysed for anti g-type lysozyme antibodies using enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. ELISA results showed that antibody titres were mounted between 12,800 and 25,600 as measured at an optical density corresponding to 50% of the maximal level. By Western blot analysis the rabbit immune serum detected a single ∼23 kDa band representing the size of the injected antigen, in both spleen and head kidney homogenates from the Atlantic cod. Immunohistochemisrty detected the native folded g-type lysozyme in tissues and revealed that g-type lysozyme positive cells were observed in haematopoietic tissue of the head kidney and in red pulp of spleen. In conclusion, the rabbit anti g-type lysozyme immune sera was developed and is effectively utilized for ELISA, Western analysis as well as for immunohistochemistry. This has allowed us to obtain new knowledge about this protein regarding localization and distribution in cod tissue.