Vaughan, Lloyd

Summary Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae . Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange‐spotted grouper ( Epinephelus coioides ) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper ( Epinephelus lanceolatus ) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca . Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae , including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca . S. epinephelii provides new opportunities to study the biology of distantly‐related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae .

Abstract New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

Summary Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout ( Salmo trutta ) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site‐specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected ‘ Candidatus Piscichlamydia salmonis’ and ‘ Candidatus Clavochlamydia salmonicola’ infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.