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

Protective Immunization of Atlantic Salmon (Salmo salar L.) against Salmon Lice (Lepeophtheirus salmonis) Infestation

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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

Host specificity and clade dependent distribution of putative virulence genes in Moritella viscosa

Moritella viscosa is the aetiological agent of winter-ulcer disease in farmed salmonids in the North Atlantic. Previously, two major (typical and variant) genetic clades have been demonstrated within this bacterial species, one of which is almost solely related to disease in Atlantic salmon (Salmo salar). In the present study infection trials demonstrated that ‘typical’ M. viscosa isolated from Norwegian Atlantic salmon was highly virulent in this fish species but resulted in lower levels of mortality in rainbow trout. ‘Variant’ M. viscosa isolated from rainbow trout resulted in modest mortality levels in both Atlantic salmon and rainbow trout. To investigate the possible genetic background for inter-strain virulence differences, 38 M. viscosa isolates of diverse geographical origin and host species and a number of other Moritella spp. were investigated for the presence/absence of putative virulence related homologs. All isolates were positive for DNA sequences coding for; the Type VI secretion ATPase (clpV), hemolysin co-regulated protein (hcp), bacterioferritins (bfrA and bfrB), lectin (hemG), phospholipase D (pld), multifunctional autoprocessing repeats-in-toxin (martxA), aerolysin (aer), invasin (inv), and cytotoxic necrotizing factor (cnf), with the exception of one isolate in which cnf could not be confirmed. The product of an ABC transporter metal-binding lipoprotein (mat) was consistently detected although 11 isolates, all phylogenetically related, appear to produce a truncated version. A putative insecticidal toxin complex (mitABC) was detected almost exclusively in ‘typical’ Atlantic salmon isolates, and our data indicate that this complex of genes is expressed and co-transcribed. Transmission electron microscopy investigation revealed pili and flagella surface structures on nine M. viscosa representing both typical and variant isolates. Our results provide strong support for the existence of host specificity/high virulence in ‘typical’ M. viscosa related to Atlantic salmon. The gene distribution also provides further support for the genetic division within M. viscosa, and constitutes a basis for further study of the importance of the mitABC complex in winter-ulcer pathogenesis

DNA-Fragments Are Transcytosed across CaCo-2 Cells by Adsorptive Endocytosis and Vesicular Mediated Transport

<div><p>Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >10³ fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to <em>in vivo</em> conditions it could be hypothesized that this transport mechanism has a function in the immune system.</p> </div

Differentiation of CaCo-2 cells.

<p>A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm²) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.</p

Effect of inhibition of endocytosis on transcytosis of DNA-fragment (5 nM).

<p>Transcytosis of DNA fragments and Lucifer yellow (LY) in the apical to basolateral direction were quantified as described in Materials and Methods and tested statistically with a linear mixed-effects model. Inhibition factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). Control for ice-treatment is 37 °C and control for bafilomycin A1 (BafA1)-treatment is with vehicle (DMSO). TEER = trans-epithelial electric resistance.</p

Trans-epithelial electric resistance (TEER) in the presence of compounds increasing Lucifer yellow (LY)-transport.

<p>TEER was measured at the end of the experiment. Deviation (dev) is calculated as the absolute value of the difference between the two experiments divided by 2. The number <i>n</i> is the number of independent experiments and number <i>m</i> is the total number of wells analysed (observations). CpG ODN = short oligonucleotide rich in CG dinucleotide motifs. Control for Cytochalasin D (CytD)-treatment is with vehicle (DMSO).</p

Competition for transcytosis of DNA-fragment (5 nM) by nucleic acids and anionic compounds.

<p>Transcytosis of DNA fragments in the apical to basolateral direction with (treated) and without (control) competitor were quantified after 90 minutes of incubation as described in Materials and Methods and tested statistically with a linear mixed-effects model. Factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). pUC19 lin = linearized pUC19 plasmid. CpG and GpC ODN = short oligonucleotides rich in CpG and GpC dinucleotide motifs, respectively.</p

Transcytosis of Lucifer yellow (LY) in the presence of compounds affecting transcytosis of DNA-fragment.

<p>Transcytosis of LY in the apical to basolateral direction with (treated) and without (control) competitor was quantified after 90 minutes of incubation as described in Materials and Methods and tested statistically with a linear mixed-effects model. Factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). pUC19 lin = linearized pUC19 plasmid. CpG ODN = short oligonucleotide rich in CG dinucleotide motifs. Control for Cytochalasin D (CytD)-treatment is with vehicle (DMSO).</p