Atlantic Salmon Cells Research Articles

Establishment of a genetically amenable fibroblast cell line from Atlantic salmon skin

The Atlantic salmon, Salmo Salar, is a societally important species of fish, both as a food source and as a component of aquatic biosphere. Its sustainable production is hampered by a wide range of infectious diseases, which is difficult to address due to the lack of in vitro tools to study the disease-host interaction. In this paper, we describe the establishment and characterization of a homogenous Atlantic salmon skin fibroblast (ASSF) cell line. This immortalized cell line grows well in standard media formulations and is capable of migration. It is transcriptionally stable over dozens of passages, and its transcriptome is distinct from other publicly available Atlantic salmon cell lines (SHK1 and ASK). Even though ASSF cells show limited cytopathic effects when challenged with Infectious Pancreatic Necrosis Virus (IPNV) molecular evidence reveals that they are infected and support IPNV production, especially compared to other cell lines like ASK or SHK1. The potential of the ASSF cell line as a tool for Atlantic salmon research is highlighted by its permissibility to genetic manipulation with various methods including CRISPR/cas9, transfection and transduction.

Exploring the Use of Alternative Promoters for Enhanced Transgene and sgRNA Expression in Atlantic Salmon Cells.

This study facilitates design of expression vectors and lentivirus tools for gene editing of Atlantic salmon. We have characterized widely used heterologous promoters and novel endogenous promoters in Atlantic salmon cells. We used qPCR to evaluate the activity of several U6 promoters for sgRNA expression, including human U6 (hU6), tilapia U6 (tU6), mouse U6 (mU6), zebrafish U6 (zU6), Atlantic salmon U6 (sU6), medaka U6 (medU6), and fugu U6 (fU6) promoters. We also evaluated several polymerase type II (pol II) promoters by luciferase assay. Our results showed that hU6 and tU6 promoters were the most active among all the tested U6 promoters, and heterologous promoters (CMV, hEF1α core) had higher activity compared to endogenous Atlantic salmon promoters sHSP8, sNUC3L, sEF1α. Among endogenous pol II promoters, sEF1α and sHSP8 displayed higher activity than sNUC3L, sHSP703, sHSP7C, sXRCC1L, and sETF. We observed that extending the promoter sequence to include the region up to the start codon (ATG) resulted in a significant increase in expression efficiency for sNUC3L and sEF1α. We also show that mutating the PRDM1 motif will significantly decrease the activity of the sEF1α promoter. The presence of the PRDM1 motif in sHSP8 promoter was also associated with relatively high expression compared to the promoters that naturally lacked this motif, such as sNUC3L. We speculate that this short sequence might be included in other promoters to further enhance the promoter activity, but further experiments are needed to confirm this. Our findings provide valuable insights into the activity of different promoters in Atlantic salmon cells and can be used to facilitate further transgenic studies and improve the efficiency of transgene expression in Atlantic salmon.

The use of salmonid epithelial cells to characterize the toxicity of Tenacibaculum maritimum soluble extracellular products.

This study assessed how the etiological agent of mouth rot in farmed Atlantic salmon, Tenacibaculum maritimum, induces toxicity in host salmonid barrier cells, and determined whether environmental changes are relevant for these effects. Tenacibaculum maritimum soluble extracellular products (ECPs) were collected and used to treat Atlantic salmon and rainbow trout intestinal barrier cell lines as a comparative model of bacterial-salmonid cell interactions. Cellular assays that examine cell membrane integrity, marker expression, and metabolic activity revealed that T. maritimum ECPs induced salmonid epithelial cell death through an apoptosis mechanism. Changes in salinity (25, 29, and 33 ppt) and temperature (12°C, 18°C, and 24°C) within the natural ranges observed in Pacific Northwest aquaculture facilities affected bacterial growth and cytotoxicity of T. maritimum ECPs. Our results suggest epithelial barriers as targets of T. maritimum-mediated toxicity in farmed mouth rot-infected Atlantic salmon. The induction of apoptosis by T. maritimum soluble ECPs may also help to explain the absence of overt inflammation typically reported for these fish.

Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing

BackgroundInfectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that represents a large problem for salmonid aquaculture worldwide. Current prevention and treatment methods are only partially effective. Genetic selection and genome engineering have the potential to develop ISAV resistant salmon stocks. Both strategies can benefit from an improved understanding of the genomic regulation of ISAV pathogenesis. Here, we used single-cell RNA sequencing of an Atlantic salmon cell line to provide the first high dimensional insight into the transcriptional landscape that underpins host-virus interaction during early ISAV infection.ResultsSalmon head kidney (SHK-1) cells were single-cell RNA sequenced at 24, 48 and 96 h post-ISAV challenge. At 24 h post infection, cells showed expression signatures consistent with viral entry, with genes such as PI3K, FAK or JNK being upregulated relative to uninfected cells. At 48 and 96 h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Uninfected bystander cells at 48 and 96 h also showed clear transcriptional differences, potentially suggesting paracrine signalling from infected cells. These bystander cells expressed pathways such as mRNA sensing, RNA degradation, ubiquitination or proteasome; and up-regulation of mitochondrial ribosome genes also seemed to play a role in the host response to the infection. Correlation between viral and host genes revealed novel genes potentially key for this fish-virus interaction.ConclusionsThis study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection and revealed host-virus interactions at the cellular level. Our results highlight various potential key genes in this host-virus interaction, which can be manipulated in future functional studies to increase the resistance of Atlantic salmon to ISAV.

A nutrition algorithm to optimize feed and medium composition using genome-scale metabolic models

The optimization of animal feeds and cell culture media are problems of interest to a wide range of industries and scientific disciplines. Both problems are dictated by the properties of an organism's metabolism. However, due to the tremendous complexity of metabolic systems, it can be difficult to predict how metabolism will respond to changes in nutrient availability. A common tool used to capture the complexity of metabolism in a computational framework is a genome-scale metabolic model (GEM). GEMs are useful for predicting the fluxes of reactions within an organism's metabolism. To optimize feed or media, in silico experiments can be performed with GEMs by systematically varying nutritional constraints and predicting metabolic activity. In this way, the influence of various nutritional changes on metabolic outcomes can be evaluated. However, this methodology does not guarantee an optimal solution. Here, we develop a nutrition algorithm that utilizes linear programming to search the entire flux solution space of possible dietary intervention strategies to identify the most efficient changes to nutrition for a desirable metabolic outcome. We illustrate the utility of the nutrition algorithm on GEMs of Atlantic salmon (Salmo salar) and Chinese hamster ovary (CHO) cell metabolism and find that the nutrition algorithm makes predictions that not only align with experimental findings but reveal new insights into promising feeding strategies. We show that the nutrition algorithm is highly versatile and customizable to meet the user's needs. For instance, we demonstrate that the nutrition algorithm can be used to predict feed/media compositions that maximize profit margins. While the nutrition algorithm can be used to define an optimal feed/medium ab initio, it can also identify minimal changes to be made to an existing feed/medium to drive the largest metabolic shift. Moreover, the nutrition algorithm can target multiple metabolic pathways simultaneously with only a marginal increase in computational expense. While the nutrition algorithm has its limitations, we believe that this tool can be leveraged in a broad range of biotechnological applications to enhance the feed/medium optimization process.

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.

Fluorescent Microplastic Uptake by Immune Cells of Atlantic Salmon (Salmo salar L.)

The ubiquitous presence of microplastics and their marine ecotoxicity are major public concerns. Microplastics are ingested accidentally by the marine fauna or are taken up indirectly through the food chain. These particles can accumulate in cells and tissues and affect the normal biological functions of organisms, including their defense mechanisms. There is limited information available about the response of immune cells to microplastics; the degree of uptake by the cells, the response of different organs or the impact of environmental concentrations of microplastic are matters that remain unclear. Moreover, very little is known about the toxicity of different polymer types. This study aimed to shed light on the physical impact of small microplastics (1–5 μm) on cells from Atlantic salmon. Immune cells from intestine, blood, and head kidney were exposed to green fluorescent polyethylene microplastic (PE-MP), yellow fluorescent polystyrene microplastic (PS-MP) and both. High (50 mg/L), medium (5 mg/L), and low (0.05 mg/L) concentrations were tested for 1, 24, 48, and 72 h to study cell mortality and microplastic uptake. Quantitative data of microplastic uptake by fish immune cells were obtained for the first time by imaging flow cytometry. Salmon immune cells showed a relatively low ability to phagocytose microplastics. Less than 6% of the cells ingested the particles after 48 h of exposure to high concentrations. Cells also phagocytosed microplastics at low concentrations although at low rates (<0.1%). PE-MPs was phagocytosed by higher percentage of cells compared to PS-MPs and the former bioaccumulated in time while the latter decreased over time. However, each cell generally phagocytosed more PS-MPs particles than PE-MPs. Cells from different tissues showed different responses to the microplastic polymers. In conclusion, this study shows that immune cells of Atlantic salmon can phagocytose microplastics, and the impact is dependent on the microplastic type. PE-MPs, the most abundant polymer in the oceans and a widely used plastic in salmon aquaculture, was more easily taken up than PS-MPs. Furthermore, the study demonstrates how imaging flow cytometry can be applied in microplastics research.

Biofilm Produced In Vitro by Piscirickettsia salmonis Generates Differential Cytotoxicity Levels and Expression Patterns of Immune Genes in the Atlantic Salmon Cell Line SHK-1.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and sessile LF-89 generated the highest virulence levels, associated with differential expression of il-1β, il-8, nf-κb, and iκb-α genes in SHK-1 cells. These results suggest that there is no single virulence pattern or gene expression profile induced by the planktonic or sessile condition of P. salmonis, which are dependent on each strain and bacterial condition used.

The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV.

IgM+ and IgT+ B Cell Traffic to the Heart during SAV Infection in Atlantic Salmon.

B cells of teleost fish differentiate in the head kidney, and spleen, and either remain in the lymphatic organs or move to the blood and peripheral tissues. There is limited knowledge about piscine B cell traffic to sites of vaccination and infection and their functional roles at these sites. In this work, we examined the traffic of B cells in Atlantic salmon challenged with salmonid alphavirus (SAV). In situ hybridization (RNAScope) showed increased numbers of immunoglobin (Ig)M+ and IgT+ B cells in the heart in response to SAV challenge, with IgM+ B cells being most abundant. An increase in IgT+ B cells was also evident, indicating a role of IgT+ B cells in nonmucosal tissues and systemic viral infections. After infection, B cells were mainly found in the stratum spongiosum of the cardiac ventricle, colocalizing with virus-infected myocardial-like cells. From sequencing the variable region of IgM in the main target organ (heart) and comparing it with a major lymphatic organ (the spleen), co-occurrence in antibody repertoires indicated a transfer of B cells from the spleen to the heart, as well as earlier recruitment of B cells to the heart in vaccinated fish compared to those that were unvaccinated. Transcriptome analyses performed at 21 days post-challenge suggested higher expression of multiple mediators of inflammation and lymphocyte-specific genes in unvaccinated compared to vaccinated fish, in parallel with a massive suppression of genes involved in heart contraction, metabolism, and development of tissue. The adaptive responses to SAV in vaccinated salmon appeared to alleviate the disease. Altogether, these results suggest that migration of B cells from lymphatic organs to sites of infection is an important part of the adaptive immune response of Atlantic salmon to SAV.

Kinetics of transcriptional response against poly (I:C) and infectious salmon anemia virus (ISAV) in Atlantic salmon kidney (ASK) cell line

Vaccine adjuvants induce host innate immune responses improving long-lasting adaptive immunity against vaccine antigens. In vitro models can be used to compare these responses between adjuvants and the infection targeted by the vaccine. We utilized transcriptomic profiling of an Atlantic salmon cell line to compare innate immune responses against ISAV and an experimental viral vaccine adjuvant: poly (I:C). Induction of interferon and interferon induced genes were observed after both treatments, but often with different amplitude and kinetics. Using KEGG ortholog database and available software from Bioconductor we could specify a complete bioinformatic pipeline for analysis of transcriptomic data from Atlantic salmon, a feature not previously available. We have identified important differences in the transcriptional profile of Atlantic salmon cells exposed to viral infection and a viral vaccine adjuvant candidate, poly (I:C). This report increases our knowledge of viral host-pathogen interaction in salmon and to which extent these can be mimicked by adjuvant compounds.

Tilapia Lake Virus Does Not Hemagglutinate Avian and Piscine Erythrocytes and NH4Cl Does Not Inhibit Viral Replication In Vitro.

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH4Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH4Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH4Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

Erythroid Progenitor Cells in Atlantic Salmon (Salmo salar) May Be Persistently and Productively Infected with Piscine Orthoreovirus (PRV).

Piscine orthoreovirus (PRV-1) can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus targets erythrocytes in the acute peak phase, followed by cardiomyocytes, before the infection subsides into persistence. The persistent phase is characterized by high level of viral RNA, but low level of viral protein. The origin and nature of persistent PRV-1 are not clear. Here, we analyzed for viral persistence and activity in various tissues and cell types in experimentally infected Atlantic salmon. Plasma contained PRV-1 genomic dsRNA throughout an 18-week long infection trial, indicating that viral particles are continuously produced and released. The highest level of PRV-1 RNA in the persistent phase was found in kidney. The level of PRV-1 ssRNA transcripts in kidney was significantly higher than that of blood cells in the persistent phase. In-situ hybridization assays confirmed that PRV-1 RNA was present in erythroid progenitor cells, erythrocytes, macrophages, melano-macrophages and in some additional un-characterized cells in kidney. These results show that PRV-1 establishes a productive, persistent infection in Atlantic salmon and that erythrocyte progenitor cells are PRV target cells.

The long-chain monounsaturated cetoleic acid improves the efficiency of the n-3 fatty acid metabolic pathway in Atlantic salmon and human HepG2 cells.

The present study aimed to determine if the long-chain MUFA cetoleic acid (22 : 1n-11) can improve the capacity to synthesise the health-promoting n-3 fatty acids EPA and DHA in human and fish models. Human hepatocytes (HepG2) and salmon primary hepatocytes were first enriched with cetoleic acid, and thereafter their capacities to convert radio-labelled 18 : 3n-3 (α-linolenic acid, ALA) to EPA and DHA were measured. Increased endogenous levels of cetoleic acid led to increased production of radio-labelled EPA + DHA in HepG2 by 40 % and EPA in salmon hepatocytes by 12 %. In order to verify if dietary intake of a fish oil rich in cetoleic acid would have the same beneficial effects on the n-3 fatty acid metabolic pathway in vivo as found in vitro, Atlantic salmon were fed four diets supplemented with either sardine oil low in cetoleic acid or herring oil high in cetoleic acid at two inclusion levels (Low or High). The diets were balanced for EPA + DHA content within the Low and within the High groups. The salmon were fed these diets from 110 to 242 g. The level of EPA + DHA in liver and whole-body retention of docosapentaenoic acid and EPA + DHA relative to what was eaten, increased with increased dietary cetoleic acid levels. Thus, it is concluded that cetoleic acid stimulated the synthesis of EPA and DHA from ALA in human HepG2 and of EPA in salmon hepatocytes in vitro and increased whole-body retention of EPA + DHA in salmon by 15 % points after dietary intake of cetoleic acid.

Individual monitoring of immune response in Atlantic salmon Salmo salar following experimental infection with piscine myocarditis virus (PMCV), agent of cardiomyopathy syndrome (CMS)

Piscine myocarditis virus (PCMV) is a double-stranded RNA virus structurally similar to the Totiviridae family. PCMV is the causative agent of cardiomyopathy syndrome (CMS), a severe cardiac disease that affects farmed Atlantic salmon (Salmo salar). A recent study characterized the host immune response in infected salmon through a transcriptome immune profiling, which confirmed a high regulation of immune and anti-viral genes throughout infection with PCMV. Previously we developed a novel model based on repeated non-lethal blood sampling, enabling the individual monitoring of salmonids during an infection. In the present work, we used this model to describe the host immune response in the blood cells of Atlantic salmon after intramuscular infection with PCMV-containing tissue homogenate over a 77-day period. At the final stage heart samples were also collected to verify the PCMV load, the pathological impact of infection and to compare the transcript profiles to blood. The expression level of a range of key immune genes was determined in the blood and heart samples by real-time PCR. Results indicated selected immune genes (mx, cd8α and γip) were up-regulated in the heart tissue of infected animals at the terminal time point, in comparison to the non-infected fish. When analyzing the blood samples over the course of infection, a significant n up-regulation of mx gene was also observed. The time and number of peaks in the kinetics of expression was different between individuals. The PCMV load and CMS pathology was verified by real-time PCR and histopathology, respectively. No pathogen and no pathology could be detected during the course of the experiment except at the terminal stage (viral load by qPCR and pathology by histology). This study emphasizes the value of non-lethal monitoring for evaluating the health status of fish at early stages of infection and in the absence of clinical signs.

Comparison of gene expression in post-smolt Atlantic salmon challenged by LF-89-like and EM-90-like Piscirickettsia salmonis isolates reveals differences in the immune response associated with pathogenicity.

Piscirickettsiosis is the main bacterial disease affecting the Chilean salmon farming industry and is responsible for high economic losses. The aim of this study was to describe and comparatively quantify the immune response of post-smolt Atlantic salmon infected by cohabitation with fish bearing LF-89-like and EM-90-like Piscirickettsia salmonis. The expression of 17 genes related to the immune response was studied in head kidney from cohabitant fish by RT-qPCR. Our results at the transcriptomic level suggest that P.salmonis is able to manipulate the kinetics of cytokine production in a way that might constitute a virulence mechanism that promotes intracellular bacterial replication in cells of Atlantic salmon. This strategy involves the creation of an ideal environment for the microorganism based on induction of the inflammatory and IFN-mediated response, modulation of Th1 polarization, reduced antigen processing and presentation, modulation of the evasion of the immune response mediated by CD8+ T cells and promotion of the CD4+ T-cell response during the late stage of infection as a mechanism to escape host defences. This response was significantly exacerbated in fish infected by PS-EM-90 compared with fish infected by PS-LF-89, a finding that is probably associated with the higher pathogenicity of PS-EM-90.

Resolution of Kudoa thyrsites infection is associated with infiltration of MHIIβ+ cells in Atlantic salmon, Salmo salar

Kudoa thyrsites is a myxozoan parasite of the skeletal muscle in a wide range of fish hosts with a global distribution. In British Columbia, Canada, infections in farmed Atlantic salmon (Salmo salar) incur significant economic losses due to post-mortem myoliquefaction. Despite obvious commercial importance, little is known about the life-cycle or host-parasite relationship of K. thyrsites. Atlantic salmon can recover from experimental infections and the recovery process is characterized by a gradual loss of plasmodium structure and replacement with fibrous connective tissue. The cellular mechanisms responsible for this process are not known although macrophage-like phagocytes containing mature spores have been observed in chronically infected fish. To address the possibility of protective adaptive immunity in recovered fish, Atlantic salmon were exposed to infective seawater for 500 or 1000 degreedays (DD). The fish were maintained in UV-sterilized seawater and muscle samples were examined histologically at 2000, 3500 and 4312 DD. Previously exposed fish and unexposed controls were exposed to seawater-containing K. thyrsites spores, from 4312 to 4812 DD and histological examinations conducted at 6312 DD. Prevalence and severity of K. thyrsites declined significantly between 2000 and 4312 DD and there was no statistical difference between the exposure groups. Following re-exposure, the prevalence and severity of infection were significantly lower in previously exposed salmon compared with controls. Significant infiltration of MHIIβ+ cells was detected in the musculature of infected salmon compared to uninfected salmon. The association of these cells with infected myocytes proceeded in 4 stages: initial contact and envelopment of the myocyte, infiltration of the myocyte, envelopment of the plasmodium and complete degradation of the plasmodium and dissemination of spores by positive cells. While not yet identified, the MHIIβ+ cells appear to play an important role in the host response to K. thyrsites. This study provides evidence for acquired resistance to reinfection of Atlantic salmon with K. thyrsites.

Evaluating the potential role of vacuolization in enhancing or interfering with fish virus detection and replication

Vacuolization is an often observed characteristic of virus infection on cell lines during cell based virus detection and isolation protocols and is commonly considered as a cytopathic effect (CPE) resulting from virus infection. However, caution should be exercised when vacuolization is used to indicate CPE as in addition to being induced by viruses, vacuolization in cells can occur spontaneously, as a response to cellular damage, during natural sequestration of materials and fluids, and fusion of many smaller vesicles formed by cellular processes such as autophagy and material transport. In vitro, additional vacuolization inducing factors during virus isolation may include use of antibiotic and antifungal, tissue preserving agents (RNA Later), temperature, and medium levels. Therefore, the goal of this research is to examine the capacity of these factors to induce cellular vacuolization during fish virus isolation procedures. Four cell lines used were EPC, CHSE-214 and two Atlantic salmon cell lines Asimf20 (Atlantic Salmon Intestinal Myofibroblast) and ASP309 (Atlantic Salmon Pituitary). Amphotericin B at concentrations between 2.5 to 10 μg/mL induced vacuoles in all cell lines except EPC. RNA Later at dilutions of 0.01 to 0.001 (v/w) induced vacuoles in all cell lines. Vacuoles were more likely to appear in cell lines when incubated at lower temperature (4C) and lower infection volumes. Additionally, the effects of vacuolization inducing agents on fish virus stability and replication was examined with two fish viruses, viral hemorrhagic septicemia virus (VHSV) and Chum salmon reovirus (CSV). RNA Later reduced the titre of enveloped VHSV by one log while having no observable effect on non-enveloped CSV. Due to the multifactorial induction of vacuoles in cells, care must be taken during virus isolation procedures to control or reduce alternative factors that may be causing vacuoles formation in cells to prevent misinterpretation of results.

Food deprivation causes rapid changes in the abundance and glucidic composition of the cutaneous mucous cells of Atlantic salmon Salmo salar L.

Cutaneous mucus is the first physical and chemical barrier of fish. This slime layer is secreted by mucous cells located in the epidermis and is mainly composed of glycoproteins that have their origin in the diet. Therefore, food deprivation can potentially change the abundance and glucidic nature of skin mucous cells, thus changing the mucus properties. To test this hypothesis, we conducted an experiment with Atlantic salmon, Salmo salar L. Changes in the number and glucidic nature of epidermal mucus cells were analysed using standard techniques. The outcome of this study shows that food deprivation caused a rapid decrease in the density of epidermal mucous cells in Atlantic salmon. Lectin histochemistry revealed a change in the presence and stainability of some sugar residues in the mucous cells of unfed fish compared with fed fish. Given that the primary reason for mucus secretion in fish is for protection against infections, we speculate that the changes in the mucus properties caused by nutritional stress may affect their disease resistance. This fact is particularly important for fish that spend a period of time deprived of food, either as a part of their natural life cycle, or as part of farming practices.

Increase in CD8+CD3+ spleen cells in virus infected Atlantic salmon

Increase in CD8+CD3+ spleen cells in virus infected Atlantic salmon