Is the Intestinal Bacterial Community in the Australian Rabbitfish Siganus fuscescens Influenced by Seaweed Supplementation or Geography?

We recently demonstrated that dietary supplementation with seaweed leads to dramatic improvements in immune responses in S. fuscescens, a candidate species for aquaculture development in Asia. Here, to assess whether the immunostimulatory effect was facilitated by changes to the gut microbiome, we investigated the effects of those same seaweed species and four commercial feed supplements currently used in aquaculture on the bacterial communities in the hindgut of the fish. Since we found no correlations between the relative abundance of any particular taxa and the fish enhanced innate immune responses, we hypothesised that S. fuscescens might have a core microbiome that is robust to dietary manipulation. Two recently published studies describing the bacteria within the hindgut of S. fuscescens provided an opportunity to test this hypothesis and to compare our samples to those from geographically distinct populations. We found that, although hindgut bacterial communities were clearly and significantly distinguishable between studies and populations, a substantial proportion (55 of 174 taxa) were consistently detected across all populations. Our data suggest that the importance of gut microbiota to animal health and the extent to which they can be influenced by dietary manipulations might be species-specific or related to an animals' trophic level.

Host-Parasite Interaction of Atlantic salmon (Salmo salar) and the Ectoparasite Neoparamoeba perurans in Amoebic Gill Disease.

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.

The Effect of Antimicrobial Treatment upon the Gill Bacteriome of Atlantic Salmon (Salmo salar L.) and Progression of Amoebic Gill Disease (AGD) In Vivo.

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.

Evaluation of the Infectious Potential of Neoparamoeba perurans Following Freshwater Bathing Treatments.

Freshwater bathing for 2-3 h is the main treatment to control amoebic gill disease of marine-farmed Atlantic salmon. Recent in vitro studies have demonstrated that amoebae (Neoparamoeba perurans) detach when exposed to freshwater and that some eventually reattach to culture plates when returned to seawater. Here, we evaluated the potential for gill-detached N. perurans to survive a commercially relevant treatment and infect AGD-naïve fish and whether holding used bathwater for up to 6 h post treatment would lower infectivity. AGD-affected fish were bathed in freshwater for 2 h. Naïve salmon were exposed to aliquots of the used bathwater after 2, 4, 6 and 8 h. The inoculation was performed at 30 ppt for 2 h, followed by gradual dilution with seawater. Sampling at 20 days post inoculation (dpi) and 40 dpi confirmed rapid AGD development in fish inoculated in 2 h used bathwater, but a slower AGD development following exposure to 4 h bathwater. AGD signs were variable and reduced following longer bathwater holding times. These results suggest that viable amoebae are likely returned to seawater following commercial freshwater treatments, but that the risk of infection can be reduced by retention of bathwater before release.

Microbiome Profiling Reveals a Microbial Dysbiosis During a Natural Outbreak of Tenacibaculosis (Yellow Mouth) in Atlantic Salmon.

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.

Bacteriomic Profiling of Branchial Lesions Induced by Neoparamoeba perurans Challenge Reveals Commensal Dysbiosis and an Association with Tenacibaculum dicentrarchi in AGD-Affected Atlantic Salmon (Salmo salar L.).

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.

The interaction between temperature and dose on the efficacy and biochemical response of Atlantic salmon to hydrogen peroxide treatment for amoebic gill disease.

Hydrogen peroxide (H2 O2 ) 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, H2 O2 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 H2 O2 ; 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 H2 O2 to treat AGD. We demonstrated that a 20-min bath treatment of H2 O2 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, H2 O2 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 H2 O2 treatment temperatures can adversely affect the host and do not improve the effectiveness of the treatment.