Atlantic Salmon Erythrocytes Research Articles

Salmon Erythrocytes Sequester Active Virus Particles in Infectious Salmon Anaemia.

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes.

Aeromonas salmonicida subsp. salmonicida (hereafter A.salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A.salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A.salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A.salmonicida have an intraerythrocytic phase. Here, we evaluate whether A.salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A.salmonicida did not kill primary S.salar erythrocytes, even in the presence of high bacterial loads, but A.salmonicida invaded the S.salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A.salmonicida showed bacteraemia 5days post-infection and the presence of intraerythrocytic A.salmonicida. Our results reveal a novel intraerythrocytic phase during A.salmonicida infection.

Antiviral Responses and Biological Concequences of Piscine orthoreovirus Infection in Salmonid Erythrocytes.

Salmonid red blood cells are the main target cells for Piscine orthoreovirus (PRV). Three genotypes of PRV (PRV-1,2,3) infect Atlantic salmon (Salmo salar), Chinook salmon (Onchorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), rainbow trout (Onchorhynchus mykiss) and brown trout (Salmo trutta), and can cause diseases like heart and skeletal muscle inflammation (HSMI), jaundice syndrome, erythrocyte inclusion body syndrome (EIBS) and proliferative darkening syndrome (PDS). Purified PRV administrated to fish has proven the causality for HSMI and EIBS. During the early peak phase of infection, salmonid erythrocytes are the main virus-replicating cells. In this initial phase, cytoplasmic inclusions called “virus factories” can be observed in the erythrocytes, and are the primary sites for the formation of new virus particles. The PRV-infected erythrocytes in Atlantic salmon mount a strong long-lasting innate antiviral response lasting for many weeks after the onset of infection. The antiviral response of Atlantic salmon erythrocytes involves upregulation of potential inhibitors of translation. In accordance with this, PRV-1 protein production in erythrocytes halts while virus RNA can persist for months. Furthermore, PRV infection in Coho salmon and rainbow trout are associated with anemia, and in Atlantic salmon lower hemoglobin levels are observed. Here we summarize and discuss the recently published findings on PRV infection, replication and effects on salmonid erythrocytes, and discuss how PRV can be a useful tool for the study of innate immune responses in erythrocytes, and help reveal novel immune functions of the red blood cells in fish.

Erythrocyte heat shock protein responses to chronic (in vivo) and acute (in vitro) temperature challenge in diploid and triploid salmonids

This research investigated how ploidy level (diploid versus triploid) affects the heat shock protein (HSP) response in erythrocytes under different thermal stress regimes, both in vivo and in vitro, in Atlantic salmon (Salmo salar) and brook charr (Salvelinus fontinalis) in order to address the question of why triploids typically have reduced thermal tolerance. A preliminary study confirmed that identical volumes of diploid and triploid erythrocytes (which equates to a smaller number of larger cells for triploids compared to diploids) did not differ in total protein synthesis rates. After chronic (100d) acclimation of fish to 5, 15 and 25°C, triploid erythrocytes had lower HSP70, HSP90, heat shock factor 1 (HSF1) and ubiquitin (free and total) levels than diploids in both species. Furthermore, Atlantic salmon erythrocytes showed significantly higher protein breakdown (based on conjugated ubiquitin levels) in triploids than diploids after acute heat stress in vitro, but no significant difference was detected between ploidies after acute cold stress. These results indicate that: 1) triploid erythrocytes synthesize more total protein per cell than diploids as a result of increased cell size; 2) triploids have sufficient total HSP levels for survival under low stress conditions; and 3) the lower basal titres of HSPs in triploids may be a handicap when combating acute stress. Taken together, this suggests that triploids are limited in their ability to withstand thermal stress because of a reduced ability to maintain proteostasis under stressful conditions.

Cell-surface properties of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chilean farms.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture, but the factors determining its virulence have not yet been elucidated. In this work, cell-surface-related properties of the isolates responsible for outbreaks in Atlantic salmon were investigated. We also briefly examined whether pathogenicity against fish varied for V. ordalii strains with differing cell-surface properties. Hydrocarbon adhesions indicated the hydrophobic character of V. ordalii, although only 4 of 18 isolates induced haemagglutination in Atlantic salmon erythrocytes. A minority of the studied isolates (6 of 18) and the type strain ATCC 33509T produced low-grade biofilm formation on polyethylene surface after 2 h post-inoculation (hpi), but no strains were slime producers. Interestingly, V. ordalii isolates showed wide differences in hydrophobicity. Therefore, we chose 3 V. ordalii isolates (Vo-LM-03, Vo-LM-18 and Vo-LM-16) as representative of each hydrophobicity group (strongly hydrophobic, relatively hydrophobic and quasi-hydrophilic, respectively) and ATCC 33509T was used in the pathogenicity studies. All tested V. ordalii strains except the type strain resisted the killing activity of Atlantic salmon mucus and serum, and could proliferate in these components. Moreover, all V. ordalii isolates adhered to SHK-1 cells, causing damage to fish cell membrane permeability after 16 hpi. Virulence testing using rainbow trout revealed that isolate Vo-LM-18 was more virulent than isolates Vo-LM-03 and Vo-LM-16, indicating some relationship between haemagglutination and virulence, but not with hydrophobicity.

Piscine orthoreovirus (PRV) replicates in Atlantic salmon (Salmo salar L.) erythrocytes ex vivo.

Piscine orthoreovirus (PRV) is a reovirus that has predominantly been detected in Atlantic salmon (Salmo salar L.). PRV is associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and recently erythrocytes were identified as major target cells. The study of PRV replication and pathogenesis of the infection has been impeded by the inability to propagate PRV in vitro. In this study we developed an ex vivo cultivation system for PRV in Atlantic salmon erythrocytes. PRV was successfully passaged to naïve erythrocytes using lysates of blood cells from infected salmon. During cultivation a significant increase in viral load was observed by RT-qPCR and flow cytometry, which coincided with the formation of cytoplasmic inclusions. The inclusions resembled viral factories and contained both PRV protein and dsRNA. In addition, the erythrocytes generated an antiviral immune gene activation after PRV infection, with significant up-regulation of IFN-α, RIG-I, Mx and PKR transcripts. Supernatants from the first passage successfully transmitted virus to naïve erythrocytes. This study demonstrates that PRV replicates in Atlantic salmon erythrocytes ex vivo. The ex vivo infection model closely reflects the situation in vivo and can be used to study the infection and replication mechanisms of PRV, as well as the antiviral immune responses of salmonid erythrocytes.

Comparison of NADH-dependent cytochrome b5 reductase activity and in vitro methemoglobin induction by sodium nitrite in Oncorhynchus mykiss, Salmo salar, and Salvelinus fontinalis

Methemoglobin is hemoglobin containing ferric iron. Methemoglobin cannot bind to oxygen and at high concentrations causes tissue hypoxia. Brook trout (Salvelinus fontinalis) develop significantly greater methemoglobinemia than Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss) following general anesthesia with benzocaine or tricaine methanesulfonate. The objective of this study was to compare the activity of the major methemoglobin reducing enzyme, NADH-dependent cytochrome b5 reductase (CB5R), in brook trout erythrocytes to the activity of CB5R in Atlantic salmon and rainbow trout erythrocytes. Methemoglobin levels were compared using co-oximetry following in vitro incubation of erythrocytes with sodium nitrite (NaNO(2)). The CB5R activity was measured using a ferricyanide assay. There was significantly greater methemoglobin at time 0 in brook trout erythrocytes than in rainbow trout or Atlantic salmon erythrocytes (2.79 ± 0.29 %, 2.19 ± 0.23 %, 2.08 ± 0.14 %), (P < 0.001). There was significantly greater methemoglobin induction by NaNO(2) in brook trout erythrocytes (33.14 ± 3.32 %) than in rainbow trout or Atlantic salmon erythrocytes (28.73 ± 2.92 % and 24.85 ± 1.40 %, respectively), (P < 0.001). The CB5R activity was significantly less in brook trout erythrocytes (median of 3.05 μmol/min/μl) than in rainbow trout erythrocytes (median of 6.73 μmol/min/μl). The CB5R activity in Atlantic salmon erythrocytes (median 4.09 μmol/min/μl) was not significantly different than in brook or rainbow trout erythrocytes. Total methemoglobin at any one time is a balance between induction by oxidants and reduction by antioxidants. Lower CB5R activity in brook trout erythrocytes may contribute to a species-specific sensitivity to methemoglobin induction; however, there are likely additional factors.

Regulatory volume response following hypotonic stress in Atlantic salmon erythrocytes

The purpose of this study was to examine regulatory volume decrease (RVD) in Atlantic salmon red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured electronically, and changes in intracellular Ca(2+) concentration were visualized using fluorescence microscopy and fluo-4-AM. Cells displayed an increase in osmotic fragility and an inhibition of volume recovery following hypotonic shock when they were exposed to a high taurine Ringer or when placed in a high K(+) medium. Interestingly, RVD in cells from fish collected during the summer depended more on taurine efflux, whereas fall cells relied more on the loss of K(+). In addition, RVD in fall cells was prevented with the K(+) channel inhibitor quinine, whereas the ionophore gramicidin decreased osmotic fragility and potentiated volume recovery. Further, hypotonic shock (0.5X Ringer) for both summer and fall cells caused an increase in cytosolic Ca(2+), which resulted from influx of this ion because it was not observed when extracellular Ca(2+) was chelated with EGTA (10 nM free Ca(2+)). Cells exposed to a low Ca(2+) hypotonic Ringer also had a greater osmotic fragility and failed to recover from hypotonic swelling. Finally, inhibition of phospholipase A(2) with ONO-RS-082 blocked volume recovery. In conclusion, Atlantic salmon RBCs displayed volume decrease in response to hypotonic shock, which depended on a swelling-induced influx of Ca(2+) and an increase in the efflux of K(+) and taurine.

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 °C and not at 28 °C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HE’s 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.

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes

BackgroundInfectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.ResultsHaemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-α in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 – 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (<3.5 relative fold increase) of STAT1 and/or ISG15 and Mx genes, whereas erythrocytes exposed to UV-inactivated virus lacked any cytokine induction.ConclusionISAV-induced haemagglutination by a highly pathogenic virus strain results in virus uptake and productive infection of Atlantic salmon erythrocytes accompanied by significant induction of IFN-α. This study also highlights the critical role of ISAV strain variation in the initial stages of the virus-cell interaction during haemagglutination, and possibly in the pathogenesis of ISA. Moreover, the study shows for the first time that fish erythrocytes immunologically respond to ISAV infection.

Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon

Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon (Salmo salar) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout (Oncorhynchus mykiss) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus.

Calcium and regulated volume decrease in Atlantic salmon and American alligator erythrocytes

A cell’s ability to regulate its volume is a property that has been conserved in a diversity of cell types; however, the mechanisms by which this compensation operates are not clearly understood. The goal of this study was to elucidate the role of Ca2+ in regulated volume recovery (RVD) in Atlantic salmon and American alligator erythrocytes. Mean cell volume was determined by electronic sizing using a Coulter counter, and changes in intracellular Ca2+ concentration were visualized using fluorescence microscopy and fluo-4. Hypotonic shock led to an increase in cytosolic Ca2+ in salmon. This increase resulted from Ca2+ influx because it was not observed in cells bathed in a hypotonic, low Ca2+ medium (10 nM, buffered with EGTA). Furthermore, cells of both species exposed to a low Ca2+ environment failed to recover from cell swelling, indicating that extracellular Ca2+ was needed for RVD. Interestingly, the Ca2+ ionophore A23187 inhibited RVD in Na-Ringer and when Na+ was replaced with NMDG+, indicating this inhibition was not due to Na+ influx. Further, although the Ca2+ channel antagonists gadolinium and lanthanum inhibited RVD in alligator, they enhanced volume recovery in salmon. Finally, pilot studies with hexokinase and ONO-RS-082 suggest that RVD in alligator depends on the activation of P2 receptors and the formation of arachidonic acid, respectively. In conclusion, our results indicate extracellular Ca2+ is necessary for RVD in both species. Future studies will examine how Ca2+ elicits volume recovery. (Supported by Lake Forest College.)

Pathogenicity of Vibrio harveyi to salmonids

Out of 19 Vibrio harveyi isolates obtained from a diversity of hosts and geographical locations, 14 were pathogenic to rainbow trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon, Salmo salar L., with mortalities of up to 100% following intraperitoneal injections of 106 cells fish−1. The extracellular products (ECPs) of only five pathogenic isolates were harmful to fish. Both pathogenic and non‐pathogenic cultures produced ECPs containing caseinase, gelatinase, phospholipase, lipase and haemolysins. Vibrio harveyi VIB 645, which was the most pathogenic isolate, produced ECPs with a maximal effect on salmonids from preparations obtained by using cellophane overlays on tryptone soya agar supplemented with 1% (w/v) sodium chloride with incubation at 28 °C for 24 h. This preparation contained the highest titre of haemolytic activity to Atlantic salmon (1:256) and rainbow trout (1:32) erythrocytes.

Multiplication and haemadsorbing activity of infectious salmon anaemia virus in the established Atlantic salmon cell line.

Infectious salmon anaemia virus (ISAV), which previously had never been isolated in any of the commercially available established fish cell lines, was successfully propagated in the continuous cell line Atlantic salmon (AS). The yield of infectious ISAV increased with the incubation time of virus-inoculated cells, demonstrated by in vivo infectivity trials in groups of Atlantic salmon. Trypsin treatment of the virus was not necessary for primary infection of AS cells with salmon-grown ISAV. The infection was non-cytopathic, but it was possible to detect virus-infected cells by a haemadsorption centre assay using Atlantic salmon erythrocytes. Pleomorphic enveloped virus particles were seen by transmission electron microscopy of infected AS cells. Elongated forms were observed, but spherical particles with diameters of 90-130 nm were commonest. Growth of ISAV was inhibited by actinomycin D but not by 5-bromo-2-deoxyuridine treatment, which indicates that ISAV may be an aquatic orthomyxovirus.