Molecular characterization of chlamydia-like bacteria associated with epitheliocystis in farmed salmonids, Atlantic salmon (Salmo salar) and Arctic char (Salvelinus alpinus), and environmental chlamydiae from aquatic environments

Epitheliocystis is the cause of morbidity and mortality in fish important to the aquaculture industry, such as Atlantic salmon ( Salmo salar) and Arctic char (Salvelinus alpinus). Preliminary histopathologic and electron microscopic evaluation of infected fish suggests that the agents in both hosts are chlamydiae. These agents have yet to be cultured and ribosomal RNA sequence data has yet to be determined. The objective of this research is to determine if these bacteria are chlamydiae, and to describe their occurrence in aquaculture facilities. Histologic, transmission electron microscopic and antigenic techniques along with ribosomal operon sequence data were used to characterize these bacteria.^ Bacteria associated with epitheliocystis were found to be chlamydiae in salmon (Piscichlamydia salmonis, AY462244.1) and char (Neochlamydia sp., AY225594.1). Histologically, branchial epithelium contained cytoplasmic basophilic, granular inclusions typical of chlamydiae, which were found to be reactive to special stains, e.g. Macchiavello, Lendrum's, and Gimenez, in char but, unreactive in salmon. Utrastructurally, life stages typical of the order Chlamydiales were present in both hosts. Antigenically, both Piscichlamydia salmonis in salmon and Neochlamydia from char were reactive to anti-chlamydial LPS antibody. The 16S rRNA gene sequence amplified from salmon was shown by phylogenetic analysis to branch with members of the order Chlamydiales , but separate from currently identified families. The 16S signature region amplified from char was identical to a Neochlamydia sp., previously identified in a cat with clinical eye disease. This bacterium groups in the order Chlamydiales within the family Parachlamydiaceae.^ Eighteen char from one farm collected during a survey of aquaculture facilities had epitheliocystis. PCR amplification of the 16S rRNA signature region from these gill sections yielded twelve sequences nearly identical (98--100%) to Piscichlamydia salmonis and 1 with 88% identity to Chlamydiales bacterium (AY082465.1). Fifty-four 16S signature region sequences amplified from water were likely members of the order Chlamydiales, and 74 had >85% identity to members of the Planctomycetes, Verrucomicrobia, and Chlamydiae superphylum. No sequences associated with epitheliocystis were identified in water. Characterization of these agents in fish and their production systems is the first step in understanding the pathogenesis, epidemiology, treatment and diagnostic options for epitheliocystis. ^