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.
Highlights
A suspicious species-specific die-off of brown trout (Salmo trutta fario) has been reported from pre-alpine river systems in Austria, Southern Germany, and Switzerland resulting in drastically decreased population densities in the impacted regions [1, 2]
Our approach of first screening immune responses along a timeline to identify synchronously affected stages in different specimens which were subsequently ultra-deep sequenced revealed contigs similar to PRV genome fragments, pointing at a piscine reovirus as a likely causing agent of Proliferative Darkening Syndrome” (PDS). This was further confirmed by intensive gap-filling Sanger re-sequencing across these contigs where 51.0% of the total PRV reference genome was successfully sequenced (Table 1, designed primers are shown in S1 Table)
The analysis of these identified sequence segments resulted in similarities between 73% and 100% to PRV and piscine orthoreovirus types detected previously in S. salar from Norway and West Canada [35, 36, 37, 38] in Onchorhynchus kisutch (Japan and North America) [39, 40], in O. kisutch from Chile (NCBI GenBank record, unpublished) and in O. mykiss from Norway (NCBI GenBank record, unpublished)
Summary
A suspicious species-specific die-off of brown trout (Salmo trutta fario) has been reported from pre-alpine river systems in Austria, Southern Germany, and Switzerland resulting in drastically decreased population densities in the impacted regions [1, 2]. In the affected river sections first external signs of PDS in brown trout include behavioral changes (decreased appetite and increasing listlessness), followed by emaciation, exophthalmia, gasping and the development of black sub-cutaneous spots [5] observable in the late summer (mid-August to late September). Die-offs are only observed in late summer and only if brown trout have already been exposed to water from the PDS-affected river section in late spring, between the beginning of May and the end of June [6]. It appears likely that brown trout already become exposed to the causative agent of PDS during spring, which irreversibly leads to their die-off in the late summer [6]. The initial histopathological changes in the liver include inflammation, appearance of multifocal lesions and hepatocyte degeneration. The course of PDS can be divided into three stages: (i) The initial stage following infection or contact with the causative agent, with no external signs of PDS (phenotypically healthy) and no pathological changes in internal organs; (ii) The clinical stage with no external signs of PDS (phenotypically healthy) but with pathological changes in internal organs, and (iii) the symptomatic stage with external signs of PDS (phenotypically sick) and severe pathological changes in internal organs terminated by the death of the organism
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