In-depth health surveillance and clinical nutrition in farmed Atlantic salmon: a strategic attempt to detect and mitigate an HSMI outbreak.

Fish health personnel have limited tools in combatting viral diseases such as heart and skeletal muscle inflammation (HSMI) in open net-pen farmed Atlantic salmon. In this study, we aimed to predict HSMI by intensified health monitoring and apply clinical nutrition to mitigate the condition. We followed a commercial cohort (G1) of Atlantic salmon that was PRV-1 naïve when transferred to a sea cage at a location where HSMI outbreaks commonly occur. The fish in the other cages (G2-G6) at the location had a different origin than G1 and were PRV-1 positive prior to sea transfer. By continuous analysis of production data and sequentially (approximately every fourth week) performing autopsy, RT-qPCR (for PRV-1 and selected immune genes), blood and histological analysis of 10 fish from G1 and G2, we identified the time of PRV-1 infection in G1 and predicted the onset of HSMI prior to any clinical signs of disease. Identical sequences across partial genomes of PRV-1 isolates from G1 and G2 suggest the likely transfer from infected cages to G1. The isolates were grouped into a genogroup known to be of high virulence. A commercial health diet was applied during the HSMI outbreak, and the fish had low mortality and an unaffected appetite. In conclusion, we show that fish health and welfare can benefit from in-depth health monitoring. We also discuss the potential health value of clinical nutrition as a mean to mitigate HSMI.

Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leaves.

Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the biosynthesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry (MS)-based proteomics has been performed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of peroxisomes from Arabidopsis leaves given a 48-h dark treatment. Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identified a total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the full function of plant peroxisomes.

Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane.

To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 µg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.