Tenacibaculosis remains a major health issue for a number of important aquaculture species globally. On the west coast of Canada, yellow mouth (YM) disease is responsible for significant economic loss to the Atlantic salmon industry. While Tenacibaculum maritimum is considered to be the primary agent of clinical YM, the impact of YM on the resident microbial community and their influence on the oral cavity is poorly understood. Using a 16s rRNA amplicon sequencing analysis, the present study demonstrates a significant dysbiosis and a reduction in diversity of the microbial community in the YM affected Atlantic salmon. The microbial community of YM affected fish was dominated by two amplicon sequence variants (ASVs) of T. maritimum, although other less abundant ASVs were also found. Interestingly clinically unaffected (healthy) and YM surviving fish also had a high relative abundance of T. maritimum, suggesting that the presence of T. maritimum is not solely responsible for YM. A statistically significant association was observed between the abundance of T. maritimum and increased abundance of Vibrio spp. within fish displaying clinical signs of YM. Findings from our study provide further evidence that YM is a complex multifactorial disease, characterized by a profound dysbiosis of the microbial community which is dominated by distinct ASVs of T. maritimum. Opportunistic taxa, including Vibrio spp., may also play a role in clinical disease progression.

Amoebic gill disease is a parasitic condition that commonly affects marine farmed Atlantic salmon. The causative agent, Neoparamoeba perurans, induces a marked proliferation of the gill mucosa and focal superficial necrosis upon branchial lesions. The effect that amoebic branchialitis has upon gill associated commensal bacteria is unknown. A 16S rRNA sequencing approach was employed to profile changes in bacterial community composition, within amoebic gill disease (AGD)-affected and non-affected gill tissue. The bacterial diversity of biopsies with and without diseased tissue was significantly lower in the AGD-affected fish compared to uninfected fish. Furthermore, within the AGD-affected tissue, lesions appeared to contain a significantly higher abundance of the Flavobacterium, Tenacibaculum dicentrarchi compared to adjunct unaffected tissues. Quantitative PCR specific to both N. perurans and T. dicentrarchi was used to further examine the co-abundance of these known fish pathogens. A moderate positive correlation between these pathogens was observed. Taken together, the present study sheds new light on the complex interaction between the host, parasite and bacterial communities during AGD progression. The role that T. dicentrarchi may play in this complex relationship requires further investigation.

Hydrogen peroxide (H2O2) is a commonly used treatment for a range of parasitic diseases of marine finfish, including amoebic gill disease (AGD). While this treatment is partially effective at reducing parasite load, H2O2 can have detrimental effects on the host under certain conditions. Treatment temperature and dose concentration are two factors that are known to influence the toxicity of H2O2; however, their impact on the outcome of AGD treatment remains unclear. Here, we investigated the effects of treatment temperature (8, 12 or 16°C) and dose concentration (750, 1,000, 1,250 mg/L) on the efficacy of H2O2 to treat AGD. We demonstrated that a 20‐min bath treatment of H2O2 at all doses reduced both parasite load and gross gill score significantly. Parasite load and gross gill score were lowest in the 1,000 mg/L treatment performed at 12°C. At the high dose and temperature combinations, H2O2 caused moderate gill damage and a significant increase in the plasma concentration of electrolytes (sodium, chloride and potassium). Taken together, our study demonstrates that higher H2O2 treatment temperatures can adversely affect the host and do not improve the effectiveness of the treatment.

Marine farmed Atlantic salmon (Salmo salar) are susceptible to recurrent amoebic gill disease (AGD) caused by the ectoparasite Neoparamoeba perurans over the growout production cycle. The parasite elicits a highly localized response within the gill epithelium resulting in multifocal mucoid patches at the site of parasite attachment. This host-parasite response drives a complex immune reaction, which remains poorly understood. To generate a model for host-parasite interaction during pathogenesis of AGD in Atlantic salmon the local (gill) and systemic transcriptomic response in the host, and the parasite during AGD pathogenesis was explored. A dual RNA-seq approach together with differential gene expression and system-wide statistical analyses of gene and transcription factor networks was employed. A multi-tissue transcriptomic data set was generated from the gill (including both lesioned and non-lesioned tissue), head kidney and spleen tissues naïve and AGD-affected Atlantic salmon sourced from an in vivo AGD challenge trial. Differential gene expression of the salmon host indicates local and systemic upregulation of defense and immune responses. Two transcription factors, znfOZF-like and znf70-like, and their associated gene networks significantly altered with disease state. The majority of genes in these networks are candidates for mediators of the immune response, cellular proliferation and invasion. These include Aurora kinase B-like, rho guanine nucleotide exchange factor 25-like and protein NDNF-like inhibited. Analysis of the N. perurans transcriptome during AGD pathology compared to in vitro cultured N. perurans trophozoites, as a proxy for wild type trophozoites, identified multiple gene candidates for virulence and indicates a potential master regulatory gene system analogous to the two-component PhoP/Q system. Candidate genes identified are associated with invasion of host tissue, evasion of host defense mechanisms and formation of the mucoid lesion. We generated a novel model for host-parasite interaction during AGD pathogenesis through integration of host and parasite functional profiles. Collectively, this dual transcriptomic study provides novel molecular insights into the pathology of AGD and provides alternative theories for future research in a step towards improved management of AGD.

Aims Assess bacterial diversity and richness in mucus samples from the gills of Atlantic salmon in comparison to preserved or fixed gill filament tissues. Ascertain whether bacterial diversity and richness are homogeneous upon different arches of the gill basket. Methods and Results Bacterial communities contained within gill mucus were profiled using 16S rRNA gene sequencing. No significant difference in taxa richness, alpha (P > 0·05) or beta diversity indices (P > 0·05) were found between the bacterial communities of RNAlater preserved gill tissues and swab‐bound mucus. A trend of lower richness and diversity indices were observed in bacterial communities from posterior hemibranchs. Conclusions Non‐lethal swab sampling of gill mucus provides a robust representation of bacterial communities externally upon the gills. Bacterial communities from the fourth arch appeared to be the least representative overall. Significance and Impact of the Study The external mucosal barriers of teleost fish (e.g. gill surface) play a vital role as a primary defence line against infection. While research effort on the role of microbial communities on health and immunity of aquaculture species continues, the collection and sampling processes to obtain these data require evaluation so methodologies are consistently applied across future studies that aim to evaluate the composition of branchial microbiomes.

Branchial surfaces of finfish species contain a microbial layer rich in commensal bacteria which can provide protection through competitive colonization and production of antimicrobial products. Upon disturbance or compromise, pathogenic microbiota may opportunistically infiltrate this protective barrier and initiate disease. Amoebic gill disease (AGD) is a globally significant health condition affecting salmonid mariculture. The current study examined whether altering the diversity and/or abundance of branchial bacteria could influence the development of experimentally induced AGD. Here, we challenged Atlantic salmon (Salmo salar) with Neoparamoeba perurans in a number of scenarios where the bacterial community on the gill was altered or in a state of instability. Administration of oxytetracycline (in-feed) and chloramine-T (immersion bath) significantly altered the bacterial load and diversity of bacterial taxa upon the gill surface, and shifted the community profile appreciably. AGD severity was marginally higher in fish previously subjected to chloramine-T treatment following 21 days post-challenge. This research suggests that AGD progression and severity was not clearly linked to specific bacterial taxa present in these systems. However, we identified AGD associated taxa including known pathogenic genus (Aliivibrio, Tenacibaculum and Pseudomonas) which increased in abundance as AGD progressed. Elucidation of a potential role for these bacterial taxa in AGD development is warranted.

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.

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a major health challenge for Atlantic salmon aquaculture globally. While freshwater bathing for 2 hr is effective in reducing infection severity, there is need for more rapid and lower cost alternatives. To this end, a combination of sodium percarbonate (SPC) in freshwater was examined for its treatment efficacy. Initial in vitro studies showed a reduction in amoeba viability when exposed for 30 min to freshwater containing >500 mg/L SPC. Subsequently, AGD‐affected salmon were bathed for 30 min in 16°C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16°C freshwater to mimic industry practice. Treatment at the highest SPC concentration caused extensive gill damage and substantial mortality. Neither occurred to a significant extent at lower SPC concentrations. Gill pathology of surviving fish 10 days post‐treatment (dpt) was comparable to or more severe than pre‐treatment, and significantly (p < .001) more severe than in 2 hr freshwater bathed fish. N. perurans DNA was confirmed by qPCR in all treatment groups at 10 dpt. The data indicate that a 30‐min exposure to SPC in freshwater is not a suitable alternative to existing freshwater treatment of AGD.