Circulatory disorders in the heart are an increasing challenge to the sustainable development of salmonid aquaculture worldwide. The main pathogens causing these problems are piscine orthoreoviruses (PRVs), which often cause Heart and Skeletal Muscle Inflammation (HSMI). In addition to pathogens, acute or chronic toxic exposures to nano- and micrometre-sized plastics and oil spills are a significant risk to aquatic life. The aim of the present study was to develop novel cardiac cell cultures from salmonids to reduce and replace the use of animals to investigate the effects of viral and environmental stressors on cardiac cells. In addition, gene expression signatures in these cardiac primary cultures of salmonids (SCPCs) after infection with PRV-1, PRV-3, piscine myocarditis virus (PMCV) and salmonid alphavirus 3 (SAV-3) were investigated to determine whether innate immune responses could be used as markers to confirm virus replication.
Materials and methods:
A novel method was used to culture SCPCs, which are constantly beating for up to eight weeks. Three-week-old SCPCs from Atlantic salmon, brown trout and rainbow trout were infected with the viruses and then cultured at 8°C and 15°C for a further four weeks. The virus-infected cultures were sampled immediately after infection and at 3, 7, 14, 21 and 28 days post infection. In addition, SCPCs and salmonid larvae were exposed to nano- and microplastics or crude oil. The number of contractions of the cultures was counted and samples of the cells were taken. Cell and media samples were used for virus detection, and cell samples were used to measure 10 genes involved in antiviral and pro-inflammatory responses by RT-qPCR.
The heart cell cultures were more sensitive to the effects of environmental stressors than heart in vivo. While the in vivo studies did not show any change in heart rate, the in vitro studies showed an increase in the number of contractions after exposure. Preliminary results from the virus exposures show that antiviral genes were upregulated when cardiac cells were actively replicating viruses and that these genes are likely to play an important role in the cardiac immune response.
SCPCs may therefore be a valuable tool for monitoring host-pathogen or host-environment interactions involving cardiac cells of fish species used in aquaculture.
Acknowledgements: This project was funded by the Federal Ministry of Education and Research in Germany.