Erling Olaf Koppang

Constitutive high expression of interleukin-4/13A and GATA-3 in gill and skin of salmonid fishes suggests that these tissues form Th2-skewed immune environments.

Rainbow trout and Atlantic salmon interleukin-4/13A (IL-4/13A) genes were identified. They were found expressed at high level in thymus, gill, and skin, in concert with the transcription factor gene GATA-3. High expression levels of IL-4, IL-13, and GATA-3 were also detected in murine thymus, suggesting similar importance of the fish and mammalian homologues for early T cell development. In mammals, combined high expression of IL-4/13 and GATA-3 in tissues other than thymus is mostly indicative of Th2 responses. Th2-skewage may protect fish skin and gill from parasites and from damage by inflammatory Th1 and Th17 responses. The immune milieus of fish gill and skin are relevant to aquaculture, because these tissues are preferred sites for vaccine administration. The similarities between the immune milieus of fish gill and thymus may reflect an evolutionary relationship, since these tissues map close together lining the gill cavity. Expression patterns of IL-4/13A and interferon gamma (IFN-γ) in isolated trout gill cells and pronephrocytes were consistent with Th2 identity of IL-4/13A.

Antigen-sampling cells in the salmonid intestinal epithelium

Antigen uptake has been shown to occur in the teleost intestine, but so far, limited information is available on the distribution and nature of cells involved in the process, and M cells, known for their antigen-sampling abilities in mammals, have not been identified. Here, different intestinal segments from salmonid fish were exposed to gold-BSA to identify antigen-sampling cells. Sections from exposed intestine were examined by light and electron microscopy. Uptake of gold-BSA was restricted to very few dendritic-like cells and to a limited number of epithelial cells located in the mucosal folds in the second segment of the mid-intestine. Gold-positive epithelial cells displayed diverging and electron-dense microvilli with channels intruding into the cytoplasm. A lectin binding experiment demonstrated the presence of cells with mammalian M-cell characteristics in the identical regions. As the identified epithelial cells shared some morphological similarities with immature mammalian M cells, this phenotype may represent evolutionary early antigen-sampling enterocytes.

Expression of the Infectious Salmon Anemia Virus Receptor on Atlantic Salmon Endothelial Cells Correlates with the Cell Tropism of the Virus

ABSTRACT Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac2). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.

Intestinal morphology of the wild Atlantic salmon (Salmo salar).

The worldwide‐industrialized production of Atlantic salmon (Salmo salar) has increased dramatically during the last decades, followed by diseases related to the on‐going domestication process as a growing concern. Even though the gastrointestinal tract seems to be a target for different disorders in farmed fish, a description of the normal intestinal status in healthy, wild salmon is warranted. Here, we provide such information in addition to suggesting a referable anatomical standardization for the intestine. In this study, two groups of wild Atlantic salmon were investigated, consisting of post smolts on feed caught in the sea and of sexually mature, starved individuals sampled from a river. The two groups represent different stages in the anadromous salmon life cycle, which also are part of the production cycle of farmed salmon. Selected regions of gastrointestinal tract were subjected to morphological investigations including immunohistochemical, scanning electron microscopic, and morphometric analyses. A morphology‐based nomenclature was established, defining the cardiac part of the stomach and five different regions of the Atlantic salmon intestine, including pyloric caeca, first segment of the mid‐intestine with pyloric caeca, first segment of the mid‐intestine posterior to pyloric caeca, second segment of the mid‐intestine and posterior intestinal segment. In each of the above described regions, for both groups of fish, morphometrical measurements and regional histological investigations were performed with regards to magnitude and direction of mucosal folding as well as the composition of the intestinal wall. Additionally, immunohistochemistry showing cells positive for cytokeratins, α‐actin and proliferating cell nuclear antigen, in addition to alkaline phosphatase reactivity in the segments is presented. J. Morphol. 274:859–876, 2013.

CD4 homologues in Atlantic salmon.

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p56lck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals.

Granulomatous Uveitis Associated with Vaccination in the Atlantic Salmon

This study addressed histologic and immunopathologic changes in ocular tissues and investigated the distribution of major histocompatibility class II (MHC class II)-positive cells in Atlantic salmon (Salmo salar) suffering from severe postvaccination disease. Twenty-nine fish with generalized inflammation, probably a result of vaccination, were investigated. One individual that had escaped vaccination was included in the study. Material was investigated by cultivation methods for fungi and bacteria. Histology using conventional staining procedures and immunohistochemistry with antisera against MHC class II β chain were performed. No growth was observed from the cultivation investigations. Histology revealed occlusion of the lumen in the larger choroid vessels and in the choriocapillaris, inflammatory infiltrations and loss of structure in the choroid rete, and, in some cases, aggregations of multinucleated giant cells (MGC) and Splendore-Hoeppli material. Immunohistochemistry demonstrated massive MHC class II+ cellular infiltrations in the uveal tract. Such infiltrations were also seen in the ventral ciliary cleft, a condition that is associated with glaucoma. Immunoreactive cells included dendritelike cells, epithelioid cells, and MGCs. The endothelia of smaller vessels were frequently MHC class II+, and immunoreactive infiltrations were seen in the optic nerve in several individuals. No pathologic changes were detected in the unvaccinated individual. In conclusion, generalized inflammatory reactions in fish may lead to severe ocular inflammation, occlusion of uveal vessels, and perivascular changes with MHC class II+ upregulation in cells in the uveal tract and optic nerve.

Antiviral functions of CD8+ cytotoxic T cells in teleost fish

Cytotoxic T-cells (CTLs) play a pivotal role in eliminating viruses in mammalian adaptive immune system. Many recent studies on T-cell immunity of fish have suggested that teleost CTLs are also important for antiviral immunity. Cellular functional studies using clonal ginbuan crucian carp and rainbow trout have provided in vivo and in vitro evidence that in many respects, virus-specific CTLs of fish have functions similar to those of mammalian CTLs. In addition, mRNA expression profiles of CTL-related molecules, such as CD8, TCR and MHC class I, have shown that in a wide range of fish species, CTLs are involved in antiviral adaptive immunity. These findings are a basis to formulate possible vaccination strategies to trigger effective antiviral CTL responses in teleost fish. This review describes recent advances in our understanding of antiviral CTL functions in teleost fish and discusses vaccination strategies for efficiently inducing CTL activities.

Inflammation in Fish As Seen from A Morphological Point of View with Special Reference to the Vascular Compartment

Modern bony fishes or teleosts are increasingly being used as model organisms for human diseases. Ambitious mapping programmes have revealed parts of or entire genomes of several species. This information suggests that there are several similarities between the mammalian and teleost immune systems, but also important differences. These differences are especially evident in the anatomical and functional constructions. However, compared to mammals, morphological studies of the immune system and in particular the inflammatory responses in fish are scarce, much due to a general lack of good cell markers. This review seeks to give an overview of the current knowledge of the teleost immune system related to inflammation and morphological research. The emphasis is placed on coronary changes which may be observed in salmonids over the size of 10 cm. Here, the immunopathological picture has some resemblance to that observed in similar changes in humans.

Infectious salmon anaemia virus infection of Atlantic salmon gill epithelial cells

Infectious salmon anaemia virus (ISAV), a member of the Orthomyxoviridae family, infects and causes disease in farmed Atlantic salmon (Salmo salar L.). Previous studies have shown Atlantic salmon endothelial cells to be the primary targets of ISAV infection. However, it is not known if cells other than endothelial cells play a role in ISAV tropism. To further assess cell tropism, we examined ISAV infection of Atlantic salmon gill epithelial cells in vivo and in vitro. We demonstrated the susceptibility of epithelial cells to ISAV infection. On comparison of primary gill epithelial cell cultures with ISAV permissive fish cell cultures, we found the virus yield in primary gill epithelial cells to be comparable with that of salmon head kidney (SHK)-1 cells, but lower than TO or Atlantic salmon kidney (ASK)-II cells. Light and transmission electron microscopy (TEM) revealed that the primary gill cells possessed characteristics consistent with epithelial cells. Virus histochemistry showed that gill epithelial cells expressed 4-O-acetylated sialic acid which is recognized as the ISAV receptor. To the best of our knowledge, this is the first demonstration of ISAV infection in Atlantic salmon primary gill epithelial cells. This study thus broadens our understanding of cell tropism and transmission of ISAV in Atlantic salmon.

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.