Introduction
Specific and sensitive tools for detecting and monitoring pathogens in the aquatic environment can be crucial for preventing the spread of infectious agents in aquaculture facilities. Monitoring waterborne pathogens through water filtration and subsequent detection using target-specific (RT-) qPCRs and/or sequencing is an approach that can reduce or even replace the need to euthanize fish for monitoring purposes in aquaculture, allowing earlier biosecurity- and disease control measures.
Material and methods
We first performed an in vitro work where we tested various filtration- and nucleic acid extraction methods for selected pathogens prior to target-specific (RT-) qPCRs to simplify and facilitate on-site water filtration without compromising detection probability. This was tested in combination with different filtration rates, lysis buffers, and filters for freshwater and seawater spiked with relevant salmon pathogens. The suitability and feasibility of the methodology was then tested in the field, where a fish group was followed over time from the hatchery to offshore Atlantic salmon aquaculture facilities until slaughter. Additionally, the third generation sequencing approach (MinION Oxford Nanopore Technologies) was used to correlate shotgun metagenomics sequencing for microbiome analyses to events in the production cycle.
Results
The results showed that a "sandwich" filtration method combining two different filters allows for the detection of various pathogens with a single filtration step and a single nucleic acid extraction step, followed by (RT-) qPCRs. This reduces both the costs and time required for analysis. Moreover, we showed that this methodology is feasible and effective under field conditions. This means that aquaculture facilities could implement these methods on-site, ensuring timely and accurate pathogen monitoring. Additional shotgun metagenomics sequencing for microbiome analyses with the MinION allowed a microbial screening technique which would lead to future real-time acquisition and analysis of data in the field. This dual qPCR-sequencing approach will provide a unique system to gain knowledge on the impact of real-time monitoring to detect various pathogenic specimens (e.g., viruses, bacteria, parasites).