Mucosal and Systemic Immune Responses to Salmon Gill Poxvirus Infection in Atlantic Salmon Are Modulated Upon Hydrocortisone Injection.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.

First record of experimentally induced salmon gill poxvirus disease (SGPVD) in Atlantic salmon (Salmo salar L.).

Salmon gill poxvirus (SGPV) infection is a common denominator in many cases of complex gill disease in the Norwegian salmon farming industry and may, as a single agent infection, result in salmon poxvirus disease (SGPVD). Experiences from the field suggest that stress may be a decisive factor for the induction of SGPVD. Here we investigated the effect of stress hormone treatment on SGPV kinetics and disease development. In our experiment, Atlantic salmon were divided into four groups. Two groups of fish received an intraperitoneal injection of hydrocortisone dissolved in a fatty vehicle, whereas fish in the other two groups received a sham injection of the vehicle. After 24 h, one group with hydrocortisone injection and one with sham injection were exposed to dead SGPV-infected fish. Plasma cortisol level, virus kinetics, virus localization, and pathological gill were monitored for 4 weeks post-exposure. Hydrocortisone injected fish displayed higher plasma cortisol and SGPV loads than non-hydrocortisone treated fish. Signs of SGPVD and ensuing mortality appeared only in fish exposed to the virus and injected with hydrocortisone around 2 weeks post-exposure. No clinical signs of disease or mortality were recorded in the other groups. Further, gill histopathology in diseased fish correlated well with SGPV load, with the infection apparently confined to gill epithelial cells. The current findings suggest elevated plasma cortisol being a prerequisite for the development of SGPVD and recommend minimization of stressful farming activities, particularly if SGPV infection has been previously identified.

Development and characterization of two cell lines from gills of Atlantic salmon.

Gill disease in Atlantic salmon, Salmo salar L., causes big losses in the salmon farming industry. Until now, tools to cultivate microorganisms causing gill disease and models to study the gill responses have been lacking. Here we describe the establishment and characterization of two cell lines from the gills of Atlantic salmon. Atlantic salmon gill cell ASG-10 consisted of cells staining for cytokeratin and e-cadherin and with desmosomes as seen by transmission electron microscopy suggesting the cells to be of epithelial origin. These structures were not seen in ASG-13. The cell lines have been maintained for almost 30 passages and both cell lines are fully susceptible to infection by infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), Atlantic salmon reovirus TS (TSRV) and Pacific salmon paramyxovirus (PSPV). While infectious salmon anemia virus (ISAV) did not cause visible CPE, immunofluorescent staining revealed a sub-fraction of cells in both the ASG-10 and ASG-13 lines may be permissive to infection. ASG-10 is able to proliferate and migrate to close scratches in the monolayer within seven days in vitro contrary to ASG-13, which does not appear to do have the same proliferative and migratory ability. These cell lines will be useful in studies of gill diseases in Atlantic salmon and may represent an important contribution for alternatives to experimental animals and studies of epithelial-mesenchymal cell biology.

Lugol's solution eradicates Staphylococcus aureus biofilm in vitro.

OBJECTIVES: The aim of the study was to evaluate the antibacterial efficacy of Lugol's solution, acetic acid, and boric acid against Staphylococcus aureus biofilm. METHODS: The efficacy of Lugol's solution 1%, 0.1%, and 0.05%, acetic acid 5% or boric acid 4.7% for treatment of Staphylococcus aureus biofilm in vitro was tested using 30 clinical strains. Susceptibility in the planktonic state was assessed by disk diffusion test. Antiseptic effect on bacteria in biofilm was evaluated by using a Biofilm-oriented antiseptic test (BOAT) based on metabolic activity, a biofilm bactericidal test based on culturing of surviving bacteria and confocal laser scanning microscopy combined with LIVE/DEAD staining. RESULTS: In the planktonic state, all tested S. aureus strains were susceptible to Lugol's solution and acetic acid, while 27 out of 30 tested strains were susceptible to boric acid. In biofilm the metabolic activity was significantly reduced following exposure to Lugol's solution and 5% acetic acid, while boric acid exposure led to no significant changes in metabolic activities. In biofilm, biocidal activity was observed for Lugol's solution 1% (30/30), 0.1% (30/30), and 0.05% (26/30). Acetic acid and boric acid showed no bactericidal activity in this test. Confocal laser scanning microscopy, assessed in 4/30 strains, revealed significantly fewer viable biofilm bacteria with Lugol's solution (1% p < 0.001, 0.1% p = 0.001 or 0.05% p = 0.001), acetic acid 5% for 10 min (p = 0.001) or 30 min (p = 0.015), but not for acetic acid for 1 min or boric acid. CONCLUSION: Lugol's solution 1.0% and 0.1% effectively eradicated S. aureus in biofilm and could be an alternative to conventional topical antibiotics where S. aureus biofilm is suspected such as external otitis, pharyngitis and wounds.

Effects of perfluorinated alkyl acids on cellular responses of MCF-10A mammary epithelial cells in monolayers and on acini formation in vitro.

Perfluorinated alkyl acids (PFAAs) are stable chemicals detected in tissue and serum from various species, including humans, and have been linked to adverse health outcomes. Experimental PFAA exposure in rodents has been associated with changes in mammary gland development. The estrogen receptor (ER)-negative human breast epithelial cell line, MCF-10A, can be grown as monolayer, but also has the ability to form three-dimensional acini in vitro, reflecting aspects of mammary glandular morphogenesis. Cells were exposed to five different PFAAs, perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA), both in monolayer and acini cultures. In monolayer cultures only the higher concentrations of PFOS, PFNA and PFDA (400-500µM) caused a significant increase in cell death, whereas PFOA and PFUnDA had no effect. Normal acini maturation was negatively impacted by PFOS, PFNA and PFDA already at the lowest concentration tested (0.6µM). Observed effects included loss of organization of the cell clusters and absence of a hollow lumen. Overall, this study demonstrated that PFAAs can interfere with cellular events related to normal development of glandular breast tissue through ER-independent mechanisms.

Piscine orthoreovirus can infect and shed through the intestine in experimentally challenged Atlantic salmon (Salmo salar L.).

Piscine orthoreovirus (PRV) is a ubiquitous virus in Norwegian salmon farms associated with the disease heart and skeletal muscle inflammation (HSMI). Experimental challenge has shown that the virus replicates in circulating red blood cells of Atlantic salmon prior to infecting heart myocytes. The infection route from water to blood is however still unknown. The related mammalian orthoreovirus primarily infects the lungs and gastrointestinal (GI) tract and is proposed to spread mainly through the faecal-oral route. To investigate the role of the salmonid GI tract in PRV-infection, oral and anal administration of virus was compared to intraperitoneal (i.p.) injection. When administered anally, PRV was transferred to blood 4 days post challenge (dpc) and levels peaked at 42 dpc, similar to i.p. injected fish. PRV was detected in heart and faeces with corresponding kinetics, and inflammatory heart lesions consistent with HSMI were observed from 49 dpc. The orally intubated group showed slower virus kinetics in both blood and heart, and no signs of HSMI. Compared to the oral and i.p. administration routes, leakage of virus inoculate by anal intubation was minor and challenge was restricted to the mid- and distal intestine. These findings show that anal intubation is an efficacious method for PRV delivery to the GI tract and demonstrates that PRV can establish infection through the intestine with the potential for transmission via faeces.

Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia.

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.

Mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia.

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4-24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp.

Boric acid inhibits germination and colonization of Saprolegnia spores in vitro and in vivo.

Saprolegnia infections cause severe economic losses among freshwater fish and their eggs. The banning of malachite green increased the demand for finding effective alternative treatments to control the disease. In the present study, we investigated the ability of boric acid to control saprolegniosis in salmon eggs and yolk sac fry. Under in vitro conditions, boric acid was able to decrease Saprolegnia spore activity and mycelial growth in all tested concentrations above 0.2 g/L, while complete inhibition of germination and growth was observed at a concentration of 0.8 g/L. In in vivo experiments using Atlantic salmon eyed eggs, saprolegniosis was controlled by boric acid at concentrations ranging from 0.2-1.4 g/L during continuous exposure, and at 1.0-4.0 g/L during intermittent exposure. The same effect was observed on salmon yolk sac fry exposed continuously to 0.5 g/L boric acid during the natural outbreak of saprolegniosis. During the experiments no negative impact with regard to hatchability and viability was observed in either eggs or fry, which indicate safety of use at all tested concentrations. The high hatchability and survival rates recorded following the in vivo testing suggest that boric acid is a candidate for prophylaxis and control of saprolegniosis.

Deciphering microbial landscapes of fish eggs to mitigate emerging diseases.

Animals and plants are increasingly suffering from diseases caused by fungi and oomycetes. These emerging pathogens are now recognized as a global threat to biodiversity and food security. Among oomycetes, Saprolegnia species cause significant declines in fish and amphibian populations. Fish eggs have an immature adaptive immune system and depend on nonspecific innate defences to ward off pathogens. Here, meta-taxonomic analyses revealed that Atlantic salmon eggs are home to diverse fungal, oomycete and bacterial communities. Although virulent Saprolegnia isolates were found in all salmon egg samples, a low incidence of Saprolegniosis was strongly correlated with a high richness and abundance of specific commensal Actinobacteria, with the genus Frondihabitans (Microbacteriaceae) effectively inhibiting attachment of Saprolegniato salmon eggs. These results highlight that fundamental insights into microbial landscapes of fish eggs may provide new sustainable means to mitigate emerging diseases.

Deletions in the highly polymorphic region (HPR) of infectious salmon anaemia virus HPR0 haemagglutinin-esterase enhance viral fusion and influence the interaction with the fusion protein.

Since the discovery of a non-virulent infectious salmon anaemia virus (ISAV) HPR0 variant, many studies have speculated on the functional role of deletions within the highly polymorphic region (HPR) of genomic segment 6, which codes for the haemagglutinin-esterase (HE) protein. To address this issue, mutant HE proteins with deletions in their HPR were generated from the Scottish HPR0 template (NWM10) and fusion-inducing activity was measured using lipid (octadecyl rhodamine B) and content mixing assays (firefly luciferase). Segment six HPR was found to have a strong influence on ISAV fusion, and deletions in this near-membrane region predominantly increased the fusion-inducing ability of the resulting HE proteins. The position and length of the HPR deletions were not significant factors, suggesting that they may affect fusion non-specifically. In comparison, the amino acid composition of the associated fusion (F) protein was a more crucial criterion. Antibody co-patching and confocal fluorescence demonstrated that the HE and F proteins were highly co-localized, forming defined clusters on the cell surface post-transfection. The binding of erythrocyte ghosts on the attachment protein caused a reduction in the percentage of co-localization, suggesting that ISAV fusion might be triggered through physical separation of the F and HE proteins. In this process, HPR deletion appeared to modulate and reduce the strength of interaction between the two glycoproteins, causing more F protein to be released and activated. This work provides a first insight into the mechanism of virulence acquisition through HPR deletion, with fusion enhancement acting as a major contributing factor.

Development and reproduction of Saprolegnia species in biofilms.

Saprolegnia spp. can cause mortality and economic losses in freshwater fish and eggs. Biofilm formation is generally regarded as a virulence factor, and biofilms can be an important cause of infection recurrence. Evidence of persistent sources of Saprolegnia infections on fish and eggs in fish farms support the assumption that Saprolegnia spp. might be able to form biofilms. In this study, we aimed to test the ability of Saprolegnia to form biofilms where it can survive, reproduce and resist different chemicals used for its control. Naturally formed biofilms were obtained from laboratory aquaria. Saprolegnia growth within these biofilms was demonstrated with light microscopy and confirmed by isolation. Isolates were identified morphologically and molecularly on the basis of ITS-sequences. Two isolates were identified as Saprolegnia parasitica, a species known to be highly pathogenic for fish, while the other belonged to S. australis. Selected Saprolegnia strains obtained from natural biofilms were then used to establish simple methods for in vitro induction of Saprolegnia biofilm. The ability of Saprolegnia isolates to form biofilms with subsequent production of infective motile zoospores within the biofilm was documented by light and confocal laser scanning microscopy. We demonstrate for the first time that isolates of S. parasitica and S. australis can form biofilm communities together with multiple microorganisms, wherein they grow and reproduce. It is therefore likely that natural biofilms constitute incessant Saprolegnia reservoirs in nature and aquaculture.

Microwell enumeration of viable Saprolegniaceae in water samples.

Existing methods for enumeration of viable Saprolegniaceae propagules in water are laborious, time consuming and prevent examination of large numbers of samples or samples with high spore loads. In the present study a microwell plate (MWP) assay for estimation of Saprolegniaceae in water samples, modified from Hagen (1992), was evaluated. The ability of the assay to recover Saprolegniaceae was assessed by applying it to spore suspensions with four predetermined concentrations, 500-10,000 spores per liter of samples tested. The method also was used to analyze a set of field samples and compare it to a standard filtration method to ascertain its practicability. The MWP assay underestimated the number of spores in the test suspensions with predetermined concentrations. The accuracy of the assay varied with spore concentration, giving the lowest recovery (62.5%) at low spore numbers and the highest (86%) at intermediate concentrations (1000-5000 spores/L) for both isolates and growth media. The findings indicate that spores aggregate with increasing concentration. When applied to field samples the assay clearly distinguished among samples with presumptive differences in spore load and yielded significantly higher counts than the filtration method. The results justify the MWP method foruse in estimation of Saprolegniaceae in water bodies particularly relevant for monitoring of spore load in aquaculture as well as in ecological studies.