Amoebic gill disease : host immunity and microbial interactions in Atlantic salmon

Amoebic gill disease, caused by the marine protozoan, Neoparamoeba perurans, has emerged as a persistent threat to production and animal welfare in Atlantic salmon mariculture operations worldwide. While partial control over AGD has been afforded by selective breeding programs and therapeutic intervention via freshwater or hydrogen peroxide bathing, the disease entails a significant growth limiting economic burden on the industry. Innovation in methods of AGD control is required yet constrained by a limited understanding of both the parasite-specific mechanisms facilitating virulence and AGD pathogenesis, and the factors associated with disease resistance in host animals. Sequencing and omics technologies have advanced in affordability, accessibility, and resolution, providing valuable tools by which factors influencing pathogen virulence and host resistance can be identified. This thesis aimed to provide a greater understanding of the mechanisms involved in N. perurans virulence, and Atlantic salmon resistance to AGD by leveraging molecular techniques, second and third generation sequencing, and omics technologies.
Anecdotal reports from commercial salmon producers suggest that historically, smaller Atlantic salmon smolts are more susceptible to AGD than larger smolts. To assess this anecdotal evidence, large (> 350 g) and small (< 200 g) commercially sourced, AGD-naïve Atlantic salmon cohorts were experimentally exposed to 50 N. perurans trophozoites L ^-1 without intervention. Progression and severity of AGD in challenged cohorts was evaluated through gill pathology, using gill score and histological examination, and detection of gill-associated amoebae burden using qPCR. Post challenge, large Atlantic salmon cohorts had significantly lower gill N. perurans burdens and reduced AGD-related gill pathology compared to the small cohorts. To determine the potential basis for differences in AGD susceptibility between cohorts, transcriptome analysis was conducted using RNA extracted from whole gill arches. A comparison between the gene expression profiles of large and small smolt cohorts highlighted upregulation of genes consistent with elevated immune activity in large smolts. Combined, these results provide evidence of size-dependent resistance to AGD in AGD-naïve Atlantic salmon facilitated by non-specific immunity.
To identify potential factors contributing to pathogenicity, a comparative transcriptome analysis of two N. perurans isolates with contrasting virulence profiles was conducted. RNA from gill-isolated, virulent (wild type), and in vitro cultured, avirulent (clonal) N. perurans isolates was purified in triplicate and sequenced using an Illumina Hiseq system as 101 base paired-end reads. Raw RNAseq reads were processed and then assembled de novo using Trinity. Trinity predicted genes were annotated and GO terms assigned using the Trinotate pipeline. Differential gene expression analysis and gene set enrichment analysis were conducted at the gene level and combined, revealed upregulated gene expression in virulent N. perurans isolates associated with lipid metabolism, oxidative stress response, protease activity, and cytoskeleton reorganisation. These findings provide a suite of gene targets potentially associated with N. perurans virulence that may serve future research focused on the development of novel therapeutic and prophylactic AGD control measures for use in commercial aquaculture.
Leveraging advances in established methodologies since early AGD research, the protective efficacy of whole cell N. perurans vaccination, and CpG oligonucleotides was re-elevated using large Atlantic salmon smolts (> 300 g) via experimental challenge. Although relative N. perurans gill burden and AGD-associated gill lesion coverage increased between 14- and 42-days post-challenge, neither measure of AGD progression significantly differed between treatments. Sonicated whole cell N. perurans did not induce a significant plasma IgM response in injected fish. Development of an indirect immuno-PCR assay permitted a three-fold increase in the limit of detection of Atlantic salmon plasma IgM compared to an analogous indirect ELISA. However, detection of gill mucus IgM was confounded by false positive signal owing to non-specific binding. While all treatments failed to provide protection against the development of AGD, the general absence of mortality observed during the experimental challenge supports the notion of inherent resistance to AGD observed in larger Atlantic salmon smolts. It is anticipated that the immuno-PCR protocol presented here will provide a foundation for future development toward a sensitive method of detection of Atlantic salmon antibodies in limited sample volume biofluids.
Bacteria are closely associated with N. perurans to the extent that attempts to culture the amoeba axenically have proven universally unsuccessful. Despite this association, the potential role of the N. perurans microbiome in modulation of amoebae virulence and AGD outcome in the host remains unknown. Taking advantage of the affordability and accessibility of Oxford Nanopore Technologies, full-length 16S rRNA gene and metagenomic sequencing were employed to investigate the closely associated microbiome of virulent (wild type) and avirulent (clonal) isolates of N. perurans. While Alteromonas, Thalassospira, Gimesia, and Paraglaciecola species were dominant in the clonal isolate microbiome depending on sequencing method used, the wild type isolate was found to be dominated by Vibrio species, regardless of sequencing method. With metagenomic sequencing, V. cyclitrophicus was identified at the species level as the most abundant taxon across all wild type N. perurans replicates. Assembly and annotation of a draft V. cyclitrophicus genome revealed genes supporting the functional potential for intracellular survival and grazing resistance to N. perurans. The dominance of an opportunistic pathogen in the closely associated microbiome of wild type N. perurans supports the speculative role of dysbiosis and microbial coinfection as potential mediators of AGD outcome.
The work presented in this thesis establishes larger Atlantic salmon smolts as a population with inherent resistance to AGD and provides insights into the potential mechanisms by which AGD is facilitated and overcome. While protective efficacy against AGD was not demonstrated through treatments utilised herein, it is anticipated that this thesis will aid in refinement of management strategies currently employed to control AGD in commercial mariculture.