Standardized tissue sampling guidelines for histopathological and molecular analyses of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

In ecotoxicology, evaluation of toxicities and no observed effect concentrations (NOEC) of test compounds in experimental fish is commonly based on molecular-, biochemical- and analytical chemistry analyses of organ/tissue samples and the assessment of (histo-) pathological lesions. Standardization of organ/tissue sampling locations, sample numbers, and sample processing contributes to warrant the reproducibility and inter- and intra-study comparability of analysis results. The present article provides the first comprehensive tissue sampling guidelines specifically adapted to rainbow trout (Oncorhynchus mykiss) as a frequently used fish species in ecotoxicological studies. A broad spectrum of ~40 different organs and tissues is covered. Appropriate sampling locations, sample sizes and sample numbers for subsequent routine histopathological evaluation (all organs/tissue) and for molecular analyses (~30 organs/tissues) are described in detail and illustrated with schematic drawings and representative macroscopic and histological images. These field-proven sampling guidelines were developed based on the pertinent literature and practical experience in ecotoxicological fish studies. They are intended to serve as a standard reference for any routine ecotoxicological study using rainbow trout as a test system. A broad application of the featured tissue sampling procedures will help to improve the reproducibility of analyses and to reduce inter- and intra-study variability induced by sampling bias and (normal) inter-sample morphological variation, and will therefore provide a robust basis for reliable characterization of toxicity and NOEC identification of diverse test substances and aquatic pollutants.

Proliferative Kidney Disease and Proliferative Darkening Syndrome are Linked with Brown Trout (Salmo trutta fario) Mortalities in the Pre-Alpine Isar River.

For many years, brown trout (Salmo trutta fario) mortalities within the pre-alpine Isar River in Germany were reported by the Bavarian Fisheries Association (Landesfischereiverband Bayern e.V.) and local recreational anglers during August and September. Moribund fish seemed to be affected by proliferative darkening syndrome (PDS). In addition, proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae was discussed. To investigate this phenomenon, the present field study monitored brown trout mortalities by daily river inspection in 2017 and 2018. Moribund brown trout (n = 31) were collected and examined using histology, immunohistochemistry, qPCR, and quantitative stereology. Our investigations identified 29 (93.5%) brown trout affected by PKD. Four brown trout (12.9%) displayed combined hepatic and splenic lesions fitting the pathology of PDS. The piscine orthoreovirus 3, suspected as causative agent of PDS, was not detectable in any of the samples. Quantitative stereological analysis of the kidneys revealed a significant increase of the renal tissue volumes with interstitial inflammation and hematopoietic hyperplasia in PKD-affected fish as compared to healthy brown trout. The identified T. bryosalmonae strain was classified as part of the North American clade by phylogenetical analysis. This study highlights PKD and PDS as contributing factors to recurrent autumnal brown trout mortalities.

Piscine Orthoreovirus 3 Is Not the Causative Pathogen of Proliferative Darkening Syndrome (PDS) of Brown Trout (Salmo trutta fario).

The proliferative darkening syndrome (PDS) is a lethal disease of brown trout (Salmo trutta fario) which occurs in several alpine Bavarian limestone rivers. Because mortality can reach 100%, PDS is a serious threat for affected fish populations. Recently, Kuehn and colleagues reported that a high throughput RNA sequencing approach identified a piscine orthoreovirus (PRV) as a causative agent of PDS. We investigated samples from PDS-affected fish obtained from two exposure experiments performed at the river Iller in 2008 and 2009. Using a RT-qPCR and a well-established next-generation RNA sequencing pipeline for pathogen detection, PRV-specific RNA was not detectable in PDS fish from 2009. In contrast, PRV RNA was readily detectable in several organs from diseased fish in 2008. However, similar virus loads were detectable in the control fish which were not exposed to Iller water and did not show any signs of the disease. Therefore, we conclude that PRV is not the causative agent of PDS of brown trout in the rhithral region of alpine Bavarian limestone rivers. The abovementioned study by Kuehn used only samples from the exposure experiment from 2008 and detected a subclinical PRV bystander infection. Work is ongoing to identify the causative agent of PDS.