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An outbreak of IHN in rainbow trout farmed in seawater in Croatia

Cage farming of rainbow trout (Oncorhynchus mykiss) in seawater in Croatia started several years ago. Fry intended for growing in the sea cages was imported from two different hatcheries and situated in the rainbow trout farm in the hinterland for several months. The water temperature on the farm was 10 oC the same as the sea temperature in spring 2020 when batches were transferred into sea cages. Shortly mortalities have started and swabs were taken from the kidneys of moribund fish, placed into transport media and delivered for analysis. Upon receipt, swabs were streaked onto bacteriological media and viral RNA was extracted on KingFisher Duo Prime Purification System using MagMAX CORE Nucleic Acid Purification Kit following manufacturer’s instructions. RNA was tested for viral hemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) using RT qPCR. All samples tested negative for VHSV and positive for the presence of IHNV. Bacterial colonies also appeared on the solid media and were identified as Vibrio spp. using API20E. The affiliation to Vibrio anguillarum was confirmed by PCR using amiB gene amplification and 16S rRNA protocols and sequencing. After obtaining preliminary results sampling of fish from the cages was performed. Sampled rainbow trouts weighing 50-300 grams showed loss of scales, dark gills, haemorrhages in the mouth, on the fin basis and skin of opercula and vent. Necropsy revealed haemorrhages in the fat tissue, pyloric caeca, peritoneum and intestines, swollen and dark spleen and liver. Organ pools were tested on the cell cultures that resulted by virus isolation and identification by ELISA followed by RT qPCR. „Mid-G“ region of the G gene of isolated IHNV was amplified and sequenced. The obtained sequence showed the affiliation to the IHNV genotype E. Phylogenetic analysis of sequences available at the NCBI database and sequences from previous IHNV outbreaks in Croatia showed the presence of ”Croatian clade”. It means that sequences from this outbreak clustered with sequences from other IHNV outbreaks in Croatia from 2005, 2014 and 2015. It may be concluded that the virus has been circulated in the country rather than being imported from other countries.

rainbow trout, Infectious hematopoietic necrosis, seawater

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