A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS)

<p>Abstract</p> <p>Background</p> <p>Cardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS.</p> <p>Results</p> <p>Using high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. <it>In situ </it>hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus.</p> <p>Conclusions</p> <p>Although causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.</p

qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.publishedVersio

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus reservoirs

Effects of cnidarian biofouling on salmon gill health and development of amoebic gill disease

This study examines the potential implications of biofouling management on the development of an infectious disease in Norwegian farmed salmon. The hydroid Ectopleura larynx frequently colonises cage nets at high densities (thousands of colonies per m2) and is released into the water during regular in-situ net cleaning. Contact with the hydroids’ nematocysts has the potential to cause irritation and pathological damage to salmon gills. Amoebic gill disease (AGD), caused by the amoeba Paramoeba perurans, is an increasingly international health challenge in Atlantic salmon farming. AGD often occurs concomitantly with other agents of gill disease. This study used laboratory challenge trials to: (1) characterise the gill pathology resulting from the exposure of salmon to hydroids, and (2) investigate if such exposure can predispose the fish to secondary infections–using P. perurans as an example. Salmon in tanks were exposed either to freshly ‘shredded’ hydroids resembling waste material from net cleaning, or to authentic concentrations of free-living P. perurans, or first to ‘shredded’ hydroids and then to P. perurans. Gill health (AGD gill scores, non-specific gill scores, lamellar thrombi, epithelial hyperplasia) was monitored over 5 weeks and compared to an untreated control group. Nematocysts of E. larynx contained in cleaning waste remained active following high-pressure cleaning, resulting in higher non-specific gill scores in salmon up to 1 day after exposure to hydroids. Higher average numbers of gill lamellar thrombi occurred in fish up to 7 days after exposure to hydroids. However, gill lesions caused by hydroids did not affect the infection rates of P. perurans or the disease progression of AGD. This study discusses the negative impacts hydroids and current net cleaning practices can have on gill health and welfare of farmed salmon, highlights existing knowledge gaps and reiterates the need for alternative approaches to net cleaning

Effect of boric acid on the nuclear division and germination of <i>Saprolegnia</i> spores.

<p>Confocal laser scanning microscopy images of <i>Saprolegnia</i> spores stained with the nucleic acid dye DAPI. a1–a4) Spore germination in non-treated water control group. Note the movement of the nucleus towards the newly developing germ tube following 2 and 4 h incubation (a1 and a2). Development of multinuclear hyphae indicating growth and viability is shown in image a3 and a4. b1–b4) Gradual reduction of fluorescence intensity of <i>Saprolegnia</i> spores treated with boric acid following 2, 4, 6 and 8 h of incubation, b1, b2, b3 and b4 respectively. No nuclear division was observed in the treated group.</p

Effect of boric acid on <i>Saprolegnia</i> mitochondria using fluorescence microscopy.

<p>Fluorescence microscope image showing the concentration of tetramethyl rhodamine (TMRE) staining in healthy non-treated <i>Saprolegnia</i> hyphae (a1) compared to boric acid treated hyphae (b1) where the depolarized mitochondria exhibit reduced red fluorescence. Figure (a2) is showing ROS level in the non-treated control compared to treated <i>Saprolegnia</i> hyphae (b2). TMRE and ROS staining are merged in a3 and b3.</p

Viability of treated <i>Saprolegnia</i> spores at different time post treatment.

<p>Mitochondrial activity and viability in <i>Saprolegnia</i> spores following boric acid (1 g/L) and bronopol (100 mg/L) treatment was compared to non-treated control using the MTS assay. The diagram shows the percent of viable spores relative to the non-treated ones calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110343#s2" target="_blank">methodology</a> section. Spores viability was significant reduced (p<0.001) in BA and bronopol treated samples at all time points (4–24 h) relative to non-treated control.</p

Effect of boric acid on the integrity of <i>Saprolegnia</i> spore membranes.

<p>Fluorescence microscopy analysis of Propidium Iodide (PI) uptake by <i>Saprolegnia</i> spores. a) non-treated spores kept in water, were able to germinate and to form hyphae that only flourecent green with SYTO 9 (a1) without PI uptake (a2). b) Boric acid treated spores, neither germinate (b1) nor absorb the PI dye (b2). c) Non-viable, bronopol treated <i>Saprolegnia</i> spores showing uptake of SYTO 9 (c1) and PI dye (c2).</p