| Abstract |
Epitheliocystis is a bacterial disease characterized by the intracellular presence of
replicating bacteria within inclusions in the gills and epithelial tissues of fish. This disease has
been observed in various species of freshwater and marine fish worldwide and has been
associated with different causative agents, including both chlamydial and non-chlamydial
bacteria. In aquaculture settings, epitheliocystis-related mortalities primarily affect young fish,
with mortality rates ranging from gradual and low to sudden and high. Recent findings suggest
that this condition plays a significant role in multifactorial mixed gill infections leading to fish
mortalities, highlighting the importance of gaining a deeper understanding of the underlying
mechanisms governing these intracellular infections. The study of this disease is hindered by
limited knowledge regarding the functioning of gill cells in different host species and, more
importantly, the lack of viable in vitro cultivation methods for the bacterial agents involved.
In this project, a first objective was to investigate the dynamics of local epizootics and
identify the environmental factors contributing to mortalities. To achieve this, a one-year
monitoring study was conducted at a single aquaculture farm site, focusing on three distinct
fish species: gilthead seabream (Sparus aurata), meagre (Argyrosomus regius), and greater
amberjack (Seriola dumerili). The study aimed to assess the presence of various pathogens
associated with epitheliocystis, which are known to induce structural changes in the
surrounding gill tissue, including both proliferative and non-proliferative reactions. Factors
such as fish size and temperature are discussed in respect to parameters such as pathogen
prevalence, outbreak onset and consequent mortality rates. In greater amberjack we observed
different trends of prevalence for different groups of pathogens, and we related fish mortalities
to a novel beta proteobacterial agent responsible for the proliferative reactions. Higher
mortality rates were related to lower fish age, suggesting more severe outcomes in younger
fish.
Following this first screening, greater amberjack was subjected to extensive analysis
(immunohistochemistry, histology, micro-CT and ultrastructure) that revealed significant
differences between acute proliferative reactions that were attributed to the novel beta
proteobacteria, Ca. Ichthyocystis seriolae and non-proliferative chronic lesions caused by Ca.
Parilichlamydia sp. The two agents that coinfect the same host, target different cells, with the
beta proteobacteria infecting chloride cells and the chlamydial agent infecting mucous cells.
Ultrastructural analysis showed different bacterial morphologies in different stages of
intracellular infection and, moreover, peculiar distinctive interactions with cell structures and
organelles. Host immune cellular response was also observed to be completely different for the
two infecting agents, with proliferative lesions displaying granulocytes and phagocytic cells,
while infected mucous cells were observed to interact mostly with macrophages. Infected mast
cells were also observed, which may have a role as dormant bacterial carriers.
Whole Genome Sequencing (WGS) analysis (including long and short read technologies)
was finally performed for Ca. Ichthyocystis seriolae, whose genome displays clear signs of
genome reduction, typical of an obligate intracellular pathogen. Comparative analysis of gene
families and genomic islands revealed differences and similarities with other species of the
genus. Different proteins were related to disease characteristics observed previously by
histology and transmission electron microscopy, TEM. Special attention was given to virulence factors and toxins that are related to the pathogenic potential of the novel bacterium.
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