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.

Individual monitoring of immune responses in rainbow trout after cohabitation and intraperitoneal injection challenge with Yersinia ruckeri.

Yersinia ruckeri, the causative agent of enteric red mouth disease (ERM), is a widely studied pathogen in disease models using rainbow trout. This infection model, mostly based on intraperitoneally injection or bath immersion challenges, has an impact on both components (innate and adaptive) of the fish immune system. Although there has been much attention in studying its host-pathogen interactions, there is still a lack of knowledge regarding the impact of a cohabitation challenge. To tackle this we used a newly established non-lethal sampling method (by withdrawing a small amount of blood) in rainbow trout which allowed the individual immune monitoring before (non-infected) and after infection with Yersinia ruckeri either by intraperitoneal (i.p.) injection or by cohabitation (cohab). A range of key immune genes were monitored during the infection by real-time PCR, and results were compared between the two infection routes. Results indicated that inflammatory (IL-1ß1 and IL-8) cytokines and certain antimicrobial peptides (cathelicidins) revealed a different pattern of expression between the two infected groups (i.p. vs cohab), in comparison to adaptive immune cytokines (IL-22, IFN-γ and IL-4/13A) and ß-defensins. This suggests a different involvement of distinct immune markers according to the infection model, and the importance of using a cohabitation challenge as a more natural disease model that likely simulates what would occur in the environment.

Individual Monitoring of Immune Response in Atlantic Salmon Salmo salar following Experimental Infection with Infectious Salmon Anaemia Virus (ISAV).

Monitoring the immune response in fish over the progression of a disease is traditionally carried out by experimental infection whereby animals are killed at regular intervals and samples taken. We describe here a novel approach to infectiology for salmonid fish where blood samples are collected repeatedly in a small group of PIT-tagged animals. This approach contributes to the reduction of animals used in research and to improved data quality. Two groups of 12 PIT-tagged Atlantic salmon (Salmo salar) were i.p infected with Infectious Salmon Anaemia Virus (ISAV) or culture medium and placed in 1 m3 tanks. Blood samples were collected at 0, 4, 8, 12, 16, 21 and 25 days post infection. The viral load, immune and stress response were determined in individual fish by real-time quantitative PCR (QPCR) on the blood cells, as well as the haematocrit used as an indicator of haemolysis, a clinical consequence of ISAV infection. "In-tank" anaesthesia was used in order to reduce the stress related to chase and netting prior to sampling. The data were analysed using a statistical approach which is novel with respect to its use in fish immunology. The repeated blood collection procedure did not induce stress response as measured by HSP70 and HSP90 gene expression in the un-infected animals. A strong increase in viraemia as well as a significant induction of Mx and γIP gene expression were observed in the infected group. Interleukin 10 was found induced at the later stage of the infection whereas no induction of CD8 or γ IFN could be detected. These results and the advantages of this approach are discussed.

A method to measure an indicator of viraemia in Atlantic salmon using a reporter cell line.

RTG-P1 is a transgenic fish cell line producing luciferase under the control of the IFN-induced Mx rainbow trout gene promoter. This cell line was used to measure viraemia of Salmonid alphavirus (SAV), the cause of Salmon Pancreas Disease (SPD), a serious disease in farmed Atlantic salmon. Two SAV genotype 1 (SAV1) isolates were used in this study, F93-125 (tissue-culture adapted, from Ireland) and 4640 (from a field case in Scotland). The kinetics and magnitude of luciferase activity were monitored versus the time of infection. During a direct infection experiment, the induction of luciferase significantly increased 16- and 4-fold after incubation for 6 days with F93-125 at 15 and 20°C, respectively. Filtration and heat treatment experiments demonstrated that the luciferase induction in RTG-P1 was dependent on viral replication. Unlike many cell lines used in fish viral diagnostic, RTG-P1 is not sensitive to salmonid serum, therefore, viraemia could be successfully monitored on serum collected from fish during a cohabitation challenge with 4640 isolate. A peak of viraemia could be detected 16 days post IP inoculation of the shedders. This novel cost-effective method to measure viraemia does not rely on development of cytopathic effect (CPE) in culture, is compatible with non-lethal blood collections in fish and can be used to assign emerging diseases to a viral aetiology.

Development of an in vitro system to measure the sensitivity to the antiviral Mx protein of fish viruses.

Mx is a structural protein, induced by type I interferon (IFN), with direct antiviral properties. In fish the inherent contribution of Mx protein to viral protection is unknown. The transgenic Chinook salmon embryonic (CHSE)-TOF cell line was genetically modified to express the rainbow trout Mx (rbtMx1) protein under the control of the tetracycline derivative, doxycycline (DOX). Two clones CHSE-TOF-MX8 and CHSE-TOF-MX10 were isolated and characterised by qPCR. The level of resistance to Infectious Pancreatic Necrosis Virus (IPNV), Salmon Alphavirus (SAV), Infectious Haematopoietic Necrosis Virus (IHNV) and Epizootic Haematopoietic Necrosis Virus (EHNV) of the CHSE-TOF, CHSE-TOF-MX8 and CHSE-TOF-MX10 cell lines cultivated with and without DOX was measured. A novel method was established to measure accurately the level of sensitivity of any given viral isolate to Mx protein. IPNV and SAV viruses were highly sensitive to the presence of rbtMx1 in the cells whereas IHNV and EHNV showed partial resistance suggesting contrasting viral evasion strategies between these categories of viruses.

Ubiquitin E3 ligase atrogin-1 (Fbox-32) in Atlantic salmon (Salmo salar): sequence analysis, genomic structure and modulation of expression.

E3 ubiquitin ligases are central for the selection of proteins targeted for degradation by the ubiquitin proteasome pathway. In this study atrogin-1 (Fbox-32), a major E3 ligase in muscle, has been characterized in Atlantic salmon (Salmo salar). The protein sequence is highly conserved between teleosts and mammals and is characterized by the presence of five conserved motifs related to the identification of protein targets. The genomic structure is conserved between teleosts and mammals and contains 9 exon and 8 introns. The phylogenetic relationship between atrogin-1 and two other closely related ubiquitin E3 ligases FBXO25 and MuRF1 showed atrogin-1 and FBXO25 grouped together with MuRF1 being more distant. The mRNAs were expressed in multiple tissues, atrogin-1 and MuRF1 were most abundant in white muscle and heart whereas FBXO25 had greatest expression in brain, white muscle and heart. The transcriptional modulation of these E3 ligases was examined in starved fish and fish following different immune stimulations. Expression of atrogin-1 and MuRF1 was increased following food deprivation, implementing these two genes in degradation of muscle protein during starvation. During viral infection atrogin-1 expression was not altered, whereas it was increased following stimulation with LPS, indicating an onset of catabolic processes during inflammatory responses.