18S rRNA Metagenomic Analysis of Nodular Gill Disease in Swiss Rainbow Trout (Oncorhynchus mykiss)

Nodular gill disease (NGD) is a serious proliferative gill condition that affects farmed salmonids, particularly in Europe. While the cause of NGD remains unknown (and maybe multifactorial), various amoebae are often isolated from the gills of affected fish and can in some cases be seen associated with lesions by histopathology. The present study aimed to quantify the abundance of different amoeba species directly from the gills of rainbow trout affected by NGD and healthy controls. An 18S rRNA amplicon metagenomic approach was employed to profile the diversity and abundance of micro-eukaryotes (including amoebae) while suppressing the amplification of host DNA using a salmonid-specific C3 spacer blocking primer. The 18S rRNA metagenomics approach identified a diversity of micro-eukaryotes on the gills of rainbow trout, including the phylum's Amoebozoa, Diatomea, Platyhelminthes and Ciliophora. Rainbow trout clinically affected by NGD had a significantly higher abundance of a specific sequence (zOTU2) classified as Vannella sp. compared to healthy controls. A quantitative PCR assay was then developed and validated which accurately quantified the abundance of this Vannella sp. sequence from a NGD outbreak in a Swiss rainbow trout farm. Additional PCR and Sanger sequencing analysis of the zOTU2 sequence demonstrated that this sequence is most likely derived from Vannella mustalahtiana. Our study highlights the potential role of Vannella mustalahtiana in NGD in Switzerland and further describes a specific and validated diagnostic PCR assay for accurate detection of this Vannella species.