Authors

Discussion

Amoebic Gill Disease (AGD) is a severe infection of farmed Atlantic salmon caused by Neoparamoeba perurans and has continued to spread globally. We still know little about how N. perurans spread between farms, production cycles, regions, and countries.  Such an understanding could inform fallowing and control in salmonid aquaculture. Genomic analyses required to achieve N. perurans molecular epidemiology, however, are frustrated by characteristically high levels of intracellular bacterial contamination. A handful of N. perurans genes have been successfully sequenced (18S rRNA, cytochrome oxidase I, ITS, etc) but with an insufficient genetic resolution to be useful to the farmer or regulator. A draft genome of N. perurans was generated at the Llewellyn lab to enable hundreds of new sequencing markers to be developed. These markers can be sequenced directly from gill swabs at low cost to provide epidemiologically relevant information. A highly multiplexed testing strategy is under development with the intent to sequence a series of AGD samples from Ireland and Scotland in 2019-2023 for molecular epidemiological analysis. Gill swab samples from Atlantic salmon affected by AGD were analysed. DNA extractions were conducted on all samples and amplified through qPCR to determine the presence of the 18S rRNA gene of N. perurans. A multiplex PCR was developed using 96 primer pairs across two 48 primer pair reactions to amplify genes across the nuclear, mitochondrial, and kinetoplast genome from qPCR data. PCR products were barcoded and cleaned. Library preparation was conducted as per Nanopore specifications. PCR products were sequenced using the Nanopore MinION system. Basecalling was completed per MinKNOW 23.04.3 protocol. Sequences were aligned with phylogeny estimated and analysed in R, MEGA, and Figtree. Extracted DNA of 164 gill swabs from Atlantic salmon in Scotland and Ireland revealed 120 (73.5%) to be positive for N. perurans) presence at or before 30 amplification cycles of the 18S rRNA gene and quantified (ng/μl) with Ct scores greater than 30 considered negative. The best candidates for DNA sequencing based on DNA quantity, and epidemiological considerations (location & time) were chosen to maximise the success of genome sequencing and allow for phylogenetic investigation inclusive of spatiotemporal changes. Analysis of phylogenetic trees indicated a high level of genetic mixing and reinfection year over year in the Scottish and Irish farmed salmon populations signifying that treatments should remain focused on aquaculture cage hygiene and therapeutic freshwater fallowing as prophylactic measures are unlikely to prevent reintroduction of AGD. To date, there is no effective means of attributing AGD infections to a source. Although incomplete, this work has taken steps towards achieving this by demonstrating that high-resolution genetic information may be readily derived directly from gill swab samples to inform AGD treatment and control.