The branchial microbiome and Neoparamoeba perurans infection

Amoebae are unicellular protists distributed throughout terrestrial and aquatic environments. Commonly known as bacterivores or detritivores, members of the Amoebozoa group can parasitise higher vertebrate hosts and cause infectious disease. Furthermore, the virulence of such amoebic infections can in some cases be mediated by the presence of specific bacterial cofactors at the host-pathogen interface. Amoebic gill disease (AGD) remains one of the most significant diseases affecting the productivity of Atlantic salmon (Salmo salar L.) aquaculture, incurring significant costs to the Australian salmonid industry. The aetiological agent Neoparamoeba perurans is a free-living marine amoeba, which colonise gill mucosal surfaces eliciting often fatal branchialitis in affected fish. Although Koch's postulates have been established for AGD, N. perurans is a multi-organism complex of amoeba, a kinetoplastid endosymbiont and associated bacterial consortia. Determination of virulence factors that underpin AGD pathogenesis is therefore complicated by the potential interplay between these organisms. Additionally, commensal or pathogenic microbes that simultaneously colonise the host gill could potentially influence the course of AGD. Bacteria and N. perurans inhabit the same ecological niche, sharing resources and space on the gill surface, although the dynamics of microbial communities in the context of AGD remain largely unknown. It was hypothesised that the type and abundance of bacterial taxa present may ultimately affect amoebic-host interactions. Therefore, the aim of this thesis was to characterise the gill mucus community in the context of AGD pathogenesis. To investigate whether non-culturable bacteria may influence the course of AGD, methods and bioinformatic pipelines to accurately profile branchial bacterial communities required initial refinement and validation. Chapter 2 compared sampling techniques and preservatives and tissue collection strategies. Results indicated that non-terminal mucus swabbing of the gill surface provided a robust bacteriomic representation of whole gill filaments. This study also demonstrated that the bacterial communities across different gill arches were not homogenous, and that both the diversity and richness of these communities upon the posterior holobranch were significantly decreased. Development of a suitable method to effectively reduce gill bacterial loads was required to manipulate the bacteriome prior to AGD challenge. Chapter 3 used antimicrobial treatments to reduce the bacterial gill load, and assess subsequent community change over a two-week timeframe. Chemical therapeutant baths and orally administered antibiotic elicited a perturbation event characterised by a significant bacterial dysbiosis on the gill surface. The post-treatment impacts of antimicrobial usage resulted in large scale bacterial imbalance, and promoted the proliferation of potentially pathogenic genera. A subsequent in vivo challenge trial (chapter 4) exposed antimicrobial treated fish gills to wild-type N. perurans, to identify changes in progression of AGD and examine community dynamics on the gill. Results indicated that AGD developed in amoebae exposed groups irrespective of antimicrobial treatment and subsequent duration of dysbiosis. In addition, infection load and disease signs were marginally more advanced in fish treated with chloramine-t following challenge with N. perurans. Furthermore, the bacterial community that developed with AGD onset was prevalent in known pathogenic taxa (including Aliivibrio, Tenacibaculum and Pseudomonas) which increased in abundance concurrent to AGD severity. Subsequently, chapter 5 investigated potential linkage of bacterial taxa and AGD affected gill tissue. Bacterial community profiling was applied to branchial lesions in contrast to adjunct, unaffected filaments. Diversity of gill lesion material was decreased significantly, and dominated by the Flavobacterium Tenacibaculum dicentrarchi. This bacterium was moderately correlated in abundance with N. perurans offering new insights on the association between Tenacibaculum and N. perurans during AGD progression. Taken together, these studies provide an approach to reflect bacterial load and diversity present upon gill mucosal surfaces, and how AGD progression impacts this dynamic. We demonstrate how these communities can be modulated to gain insights into the interactive dynamics of N. perurans and branchial bacteria during the development of amoebic branchialitis. The research presented in this thesis advances our pathobiological perspective of AGD and informs future research that seeks to further elucidate microbial co-factors that underpin the pathogenesis of AGD.