Mitigating the impact of PRV-3 infection in rainbow trout production by high-throughput diagnostic platforms

Piscine orthoreovirus (PRV) has been associated with serious and emerging diseases across salmonid culture worldwide in recent years. Three different genotypes have recently been described, each of which has its individual host niche and associated disease. PRV-1 is the causative agent of heart and skeletal muscle inflammation in Atlantic salmon in Norway, PRV-2 is associated with erythrocytic inclusion body syndrome in coho salmon in Japan, and most recently PRV-3 has been associated with severe disease outbreaks and mortality in farmed rainbow trout in Norway and Denmark.

In 2017, severe disease outbreaks associated to PRV-3 occurred at two separate rainbow trout farms in Denmark. As a result, we conducted a surveillance program to examine the spread of the virus within the aquaculture industry and to investigate the spread and potential virulence markers, which is reported in Paper I. Approximately 72% of the examined farms were infected with PRV-3, although, only 26% had disease outbreak associated with the detection. All of the farms with PRV-3 associated disease outbreaks were recirculating aquaculture systems (RAS). Examination of two of the ten genomic segments of PRV-3 did not reveal any mutations linked to disease cases, and all the isolates were nearly or completely identical, including isolates from 1995.

As most disease cases had been observed during the winter, the study reported in Paper II examined the effect of water temperature on PRV-3 infection under experimental conditions. Here we found that while temperature alone did not trigger a disease outbreak within the time frame of the study, low water temperature did enhance virus replication and caused severe heart pathology. On the other hand, high water temperature seemed to have a protective effect as the fish maintained at a high temperature did not develop heart pathology.

Previous studies have reported multiple infections in relation to PRV-3 associated disease. Therefore, the study reported in Manuscript I set out to investigate the pathobiome of RAS farmed rainbow trout by both traditional diagnostic methods and 16s rRNA gene sequencing in a pilot study. The pilot study found that at any given point in time, RAS farmed fish were subject to multiple infections, both viral and bacterial. Furthermore, 16s rRNA gene sequencing of the gills revealed the presence of two putative gill pathogens, Canditatus Branchiomonas cysticola and Candidatus Piscichlamydia salmonis.

To further elaborate on this, the main study set out to develop and test a high-throughput qPCR method for the simultaneous detection of 22 different pathogens and putative pathogens including the ones found in the pilot study. Using the high-throughput qPCR method, we followed a cohort for seven months. Here, we found the presence of PRV-3 and Ca. B. cysticola at all time-points, but at high levels during a disease outbreak in which 2.2 tonnes of fish were lost. The cohort had been moved to a new unit a few days prior to the disease outbreak, indicating that stress from handling may have been the final trigger for disease. Lastly, we find that environmental sampling in the form of water samples is a poor proxy for fish health in RAS farms as multiple bacterial pathogens were found in the water but not in the fish and vice versa for PRV-3.

In conclusion, the study shows that PRV-3 is widespread in Danish aquaculture and that it has been present since at least 1995. Furthermore, we could not identify any virulence markers, and due to the similarity of the isolates examined we conclude that the spread of PRV-3 must have occurred by trade and transportation of live fish. While low water temperature does affect the virus kinetics and increases the severity of heart pathology, low temperature in combination with PRV-3 infection alone is not enough to trigger mortality under experimental conditions. Field studies have shown that co-infections are common in RAS farmed rainbow trout, and here we found PRV-3 together with a putative gill pathogen at high levels during a disease outbreak using high-throughput qPCR. Importantly, stress from handling may have had a significant role.