James W. Wynne

Tilapia species, native to Africa, is now a major aquaculture commodity globally and an invasive species in freshwater waterways in some countries. Tilapia lake virus (TiLV) has been proposed as a potential biocontrol agent for invasive tilapia in Australia. Australian genotypes of Mozambique tilapia (Oreochromis mossambicus) were highly susceptible to TiLV, while spotted tilapia (Pelmatolapia mariae) showed greater resistance. To investigate the mechanisms underlying this resistance, RNA sequencing was performed on anterior kidney and spleen samples from TiLV-challenged and uninfected control fish. Differential gene expression analysis revealed species-specific transcriptional responses to TiLV in the anterior kidney, whereas the spleen exhibited a more conserved transcriptional profile. Gene set enrichment analysis identified antiviral and adaptive immune pathways associated with resistance, driven by strong upregulation of putative antiviral genes in P. mariae, including rsad2, mxb, mx-like, ifit5, and ifit1. In addition, key transcription factors involved in antiviral signalling, including stat1a, irf3, stat2, irf7, and irf9, were consistently upregulated in P. mariae but not in O. mossambicus. Transcriptomic analysis and the poor survival of O. mossambicus highlight early and effective innate immune activation as a key determinant of resistance, as shown by P. mariae. These findings reveal putative mechanisms of antiviral resistance in tilapia, with implications for aquaculture disease management and the feasibility of TiLV as a biocontrol agent.

Single-chain trimers (SCTs) are valuable tools for T cell epitope screening, providing a stable, scalable alternative to traditional peptide–MHC complexes. However, their application in non-model species is limited by sparse epitope-prediction resources and a lack of species-specific reagents. Here, we present a novel prototype SCT structure and cloning strategy that could provide a practical and cost-effective approach for screening large peptide libraries in non-model species. In this study, we constructed a novel SCT using Atlantic salmon (Salmo salar) genes and an antigenic peptide from chicken ovalbumin, SIINFEKL. Using simple molecular techniques and end-point PCR, our cloning strategy allows a peptide tag identical to the epitope peptide to be engineered at the N-terminus of the SCT protein. In this SCT prototype, one peptide is displayed for engagement with specific T cell receptors (TCRs), enabling the isolation of antigen-specific T cells. The tag peptide, positioned downstream of a cleavage site, is released and identified by proteomic analysis, without existing knowledge of the epitope sequence. To evaluate our cloning strategy, the SCT construct was expressed using a Pichia pastoris secretory expression system, and four strains were screened for production efficiency. All four strains secreted recombinant SCT, with the BG44 strain showing the highest normalised secretion efficiency. Moreover, LC–MS analysis identified nine unique peptides and achieved 37% SCT sequence coverage in the BG44 strain. Following TEV protease cleavage, a 34-residue amino acid fragment was released containing the SIINFEKL peptide, which was confirmed by LC–MS analysis. Although ex vivo functional validation is still required, this study presents a novel SCT design that provides a flexible platform for large peptide library screening and T cell identification in non-model species.

Amoebic gill disease (AGD), caused by Neoparamoeba perurans , is a challenge for Atlantic salmon aquaculture. Research has, therefore, focused on detecting/monitoring N. perurans loads within/around fish pens. Recently, molecular methods to detect N. perurans have been used to reduce labour‐intensive sampling, inconsistency, and stress on stock while complementing gill scoring and histology methods for AGD assessment. Molecular detection depends on reliable and cost‐effective sample collection. Recent studies have demonstrated the potential for utilising naturally occurring filter‐feeding animals as a mechanism for collecting DNA from the environment (i.e., ‘natural samplers’) for use in downstream molecular assays. This opens the possibility of sampling with minimal training or expertise, offering the potential to easily integrate this sampling within normal farm operations. We evaluated, through aquarium‐based experiments, the utility of the Pacific oyster ( Magallana gigas ) to collect N. perurans DNA from the water column. In aquaria inoculated with N. perurans , total water column amoeba load decreased significantly over time in the presence of oysters. Despite this decrease, no correlation between the level of amoeba accumulation within oysters and the decrease in the water column was observed. N. perurans was detected in every oyster organ type tested (mantle, gill, palp, and digestive gland), though with high variation. The detection of N. perurans DNA within the digestive gland indicates that oysters ingested amoeba. Oysters were found to be viable environmental DNA (eDNA) samplers for N. perurans , providing a useful collection method.

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.

Nodular gill disease (NGD) is an infectious condition characterized by proliferative gill lesions leading to respiratory problems, oxygen deficiency and mortality in fish. Globally, NGD primarily impacts freshwater salmonids in intensive aquaculture systems. In recent years, numerous outbreaks of severe gill disease have affected more than half of the larger rainbow trout (Oncorhynchus mykiss) farms in Switzerland, mainly during spring and early summer. Mortality has reached up to 50% in cases where no treatment was administered. Freshwater amoeba are the presumed aetiologic agent of NGD. The gross gill score (GS) categorising severity of gill pathology is a valuable first‐line diagnostic tool aiding fish farmers in identifying and quantifying amoebic gill disease (AGD) in farmed marine salmonids. In this study, the GS was adapted to the NGD outbreak in farmed trout in Switzerland. In addition to scoring disease severity, gill swabs from NGD‐affected rainbow trout were sampled and amoeba were cultured from these swabs. Morphologic and molecular methods identified six amoeba strains: Cochliopodium sp., Naegleria sp., Vannella sp., Ripella sp., Saccamoeba sp. and Mycamoeba sp. However, the importance of the different amoeba species for the onset and progression of NGD still has to be evaluated. This paper presents the first description of NGD with associated amoeba infection in farmed rainbow trout in Switzerland.

Epitheliocystis, an intracellular bacterial infection in the gills and skin epithelium, has been frequently reported in Atlantic salmon (Salmo salar) during freshwater production in a number of countries. This study describes the prevalence and intensity of a natural epitheliocystis infection present in the gills of two strains of Atlantic salmon reared in either a flow‐through (FT) or a recirculation aquaculture system (RAS) in Ireland. Repeated sampling of gills prior to and throughout seawater transfer, histology and quantitative real‐time PCR were used to determine infection prevalence and intensity. Despite no clinical gill disease, and minor histopathological changes, epitheliocystis lesions were identified in histology at all time points. Specific PCR confirmed the presence of Candidatus Clavichlamydia salmonicola in both strains and its number of copies was correlated with intensity of epitheliocystis lesions. A significant interaction between hatchery system and fish strain on the prevalence and intensity of gill epitheliocystis was found both using histological and molecular methods. Specifically, fish from FT had higher prevalence and intensity than RAS reared fish and within FT, the Irish cohort were more affected than Icelandic.

Microbial gill diseases caused by either opportunistic or specific pathogens are an emerging area of concern for aquaculture producers in part due to their sometimes complex and/or cryptic nature. Many antimicrobial treatments used in aquacultural settings are broad spectrum in nature. The effect of such therapeutics upon reduction and recolonization of commensal or pathogenic microbiota post-treatment has received little attention to date. Commensal bacteria are an integral component of the barrier function of mucosal surfaces in animals. This study evaluated the effect of several commercially relevant antimicrobial treatments upon the diversity and composition of branchial bacteria of Atlantic salmon. Here we exposed Atlantic salmon smolt to a number of commercially relevant antimicrobial treatments including chemotherapeutants (chloramine-t and hydrogen peroxide) and antibiotics (oxytetracycline and florfenicol) in vivo. Subsequently we examined the change in bacterial load, 16S rRNA gene expression, and taxonomic diversity post-treatment upon the gills. Results revealed a decrease in cultivable bacterial colonies after antimicrobial treatment, and a downstream decrease in bacterial richness and abundance post-treatment, with colonization of several prominent pathogenic taxa including Vibrio and Tenacibaculum. Temporal tracing over a 14-day period demonstrated that the bacteriome of gill mucus is sensitive to change, and altered by antimicrobial treatment and handling. This study identified candidate antimicrobial treatments which could be implemented in future studies to illustrate the effect of dysbiosis on microbial gill diseases.