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Identification of a piscine reovirus-related pathogen in Proliferative Darkening Syndrome infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline

ABSTRACT: The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100 % in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.

ORGANISM(S): Salmo salar Oncorhynchus mykiss Salmo trutta fario

PROVIDER: GSE70257 | GEO | 2015/06/26

SECONDARY ACCESSION(S): PRJNA288087

REPOSITORIES: GEO

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Project description:The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) in PDS-impacted pre-alpine rivers of central Europe. The mortality rate for PDS is near 100% and consequently the survival of brown trout populations in the impacted regions is threatened. The progression of PDS occurs in two stages, a subclinical stage and a symptomatic stage, over a time period of more than 3 months starting in spring and culminating with the die-off of brown trout in late summer. Based on experimental evidence it is hypothesized that PDS is caused by an infectious agent. To substantiate this working hypothesis as well as to discern the type of pathogen likely to be responsible for PDS, microarray analysis were conducted using a salmonid-specific cDNA microarray to assess the hepatic immune response of brown trout during the progression of PDS in 7-day intervals over a time period of 98 days.The microarray analysis revealed that brown trout suffering from PDS exhibit increased hepatic expression of important anti-viral genes both during the subclinical stage of PDS, namely the Barrier-to-autointegration factor, and during the symptomatic stage of PDS, namely the interferon regulatory factor 1 as well as the Guanylate-binding protein 1. Additionally, during the symptomatic stage of PDS there is strong hepatic up-regulation of the chemokine CCL19, complement components C1QC and C6, Proteasome activator complex subunits 1 and 2 as well as a variety of both MHC class I and MHC class II transcripts. In conclusion, the increased expression of hepatic immune response genes involved in a variety of immune system processes that mediate defense against infection identified by the microarray analysis during the progression of PDS support the working hypothesis that PDS is caused by an infectious agent. Furthermore, the up-regulation of antiviral immune response genes during both the subclinical stage and the symptomatic stage of PDS suggests that this disease is caused by a pathogenic virus. On May 29, 2008, brown trout (Salmo trutta fario) of the same age class (1+) with an individual weight ranging between 25-85 grams were obtained from a single hatchery (Schwäbischer Fischereihof Salgen, Fachberatung für Fischerei Schwaben, Germany) and randomly allocated to one of two different experimental stations that are both located along the Iller river, named here simply the control station (location by Oberstdorf, Germany) and the treatment station (location by Kempten, Germany). At both experimental stations brown trout were held in tanks (n=750 at the treatment station and n=70 at the control station) that were supplied with water from the Iller river in a flow-through system. Sampling at both experimental stations started on May 29, 2008, which was also the day on which the fish were first transferred to their exposure tanks (referred to as 0 day post exposure; d.p.e), and ended on the 5th of September 2008. At the treatment station 3 fish were sampled each day (always at 2pm), whereas at the control station 3 fish were sampled in 7-day intervals (always at 12 noon). Fish were anaesthetized by a blow to the head and organ tissue of interest (liver, kidney, spleen, gill, muscle, stomach and foregut tissue) were immediately harvested from sacrificed fish, snap-frozen in liquid nitrogen and subsequently stored at -80°C. Livers from the three brown trout (n=3) that were sampled concurrently from both treatment and control group (n=2) in 7-day intervals starting 7 d.p.e (7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91 and 98 d.p.e; n=14) were recruited for the use in the microarray analysis. Microarray analyses were performed using a direct comparison two-channel design in which equimolar amounts of liver sample of one brown trout from both treatment and control group were co-hybridized on the same microarray. For each time point (n=14) microarray co-hybridizations were repeated in triplicate (n=3) and included one dye-swap in order to reduce dye-bias. Thus a total of 42 microarray slides were used in this study (3 biological replicate microarrays x 14 time points = 42 microarrays).

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Identification of a piscine reovirus-related pathogen in Proliferative Darkening Syndrome infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline

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