Molecular tools to identify pathogenic microorganisms and evaluate risk of disease

PhD student: Cyril Henard

Thesis defended: 23 May 2025

THE PROJECT 
The global seafood supply for human consumption is currently equally supported by fishery catches and aquaculture production. However, fisheries management (e.g. overfishing) and aquaculture production systems exposed to climate change (i.e. flow-through) make the aquatic product supply uncertain in the near future. From this statement, another type of aquaculture production based on water recirculation (i.e. recirculated aquaculture system: RAS) has witnessed a growing interest in the past few decades. Compared to flow-through systems, the RAS optimises water utilisation, stabilises farming parameters (e.g. temperature, light, water chemistry) and improves overall production performance. In this context of increasing attention towards RAS, the European Union’s Horizon 2020 project entitled RASOPTA aims to support the development of RAS regarding three main aspects: water quality, off-flavours and fish health and welfare. Ultimately, the ambition of this project is to provide an all-in-one diagnostic tool based on the combination of water environmental DNA (eDNA) and the Biomark HD Fluidigm platform.

THE PURPOSE 
The PhD project took place in the work package “fish health and welfare” of the RASOPTA project. Nowadays, most of the plant and animal productions are monospecific. This statement applies to aquaculture’s fish production where salmonids production is prevalent in Europe (e.g. rainbow trout).  Therefore, the surveillance and management of pathogenic microorganisms (e.g. viruses, bacteria and parasites) is primordial to secure the aquaculture’s productions and ensure animal welfare. In RAS, the recirculation of water (commonly 90%) may exacerbate the risk of outbreaks as most of the fish pathogens rely on horizontal transmission, using water as a principal vector. To monitor the abundance of fish pathogens in the water, a water eDNA approach was conducted and compared to disease progression (i.e. mortality, clinical signs, pathogen burden) and fish immune response (i.e. gene expression). Three different infection models were tested with different stressors (e.g. handling, crowing) to evaluate if the detection profile of the pathogen would be altered.

RESULTS
Three different infection models were explored during the PhD. The two-host model employed were the zebrafish (Danio rerio) and the rainbow trout (Oncorhynchus mykiss). The three-pathogen model employed were the bacteria Vibrio anguillarum which causes vibriosis in zebrafish, the parasite Ichthyophthirius multifiliis which causes white spot disease in rainbow trout and the bacteria Yersinia ruckeri which causes enteric red mouth disease in rainbow trout. The water eDNA approach successfully detected the different pathogens in the three experiments along with disease progression. However, we could not demonstrate from our experiments that the water eDNA signal from the pathogens could be used as an early warning tool for disease prevention. The different stressors induced differences in the immune gene expression of the host and the outcome regarding mortality. Nonetheless, the water eDNA profile from the pathogens remains unchanged. In the three experiments, correlations were established between the fish immune gene regulation and the water eDNA profile from the pathogens.

THE FUTURE 
This passive sampling strategy based on water eDNA would be a valuable surveillance tool for pathogen detection in RAS and could be extended to flow-through systems. However, for disease diagnosis, the gold standards (e.g. histopathology, PCR, qPCR) analysis from fish samples remain the most reliable methods. In addition, the specificity of the pathogen (e.g. life cycle of the parasite) must be considered to perform a realistic disease risk assessment. Future studies should investigate the combination of both water eDNA and eRNA, also called environmental nucleic acids (eNA), to provide early pathogen detection.