Abstract

Gill disease from intracellular gram-negative chlamydia-like bacteria, loosely termed epitheliocystis, has been associated with epizootic mortality of farmed Atlantic salmon (Salmo salar) in Ireland and Norway. Molecular characterization of the chlamydia-like bacteria associated with epitheliocystis for purposes of developing diagnostic tests and understanding the epizootiology and pathogenesis of infections has not been accomplished primarily due to a collective inability to culture the organisms in vitro. In order to obtain comparative morphologic and molecular data, samples of proliferative gill lesions from epitheliocystis outbreaks at two different times and locations, 1999 Ireland and 2000 Norway, were collected for histopathology, standard transmission and immuno-electron microscopy, in situ hybridization, and nucleic acid extraction, and were compared on the basis of ultrastructure and immunoreactivity to epitheliocystis inclusions from non-proliferative Atlantic salmon gill from Ireland archived in 1995. DNA extracts from 1999 Ireland and 2000 Norway proliferative gill lesions were used to amplify 16S rRNA genetic sequences for molecular phylogenetic analyses. Epitheliocystis inclusions in proliferative gill lesions from 2000 Norway possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and non-vacuolated intermediate bodies, whereas inclusions in non-proliferative gill from 1995 Ireland had 3 developmental stages typical of chlamydia, as well as distinctive head-and-tail cells not seen in the inclusions from proliferative lesions. Immunogold processing using a monoclonal anti-chlamydial LPS primary antibody labeled reticulate and intermediate bodies of inclusions from proliferative and nonproliferative gill sections. 16S rRNA genetic sequences amplified directly from proliferative gill lesions of 1999 Ireland and 2000 Norway samples had 99% nucleotide sequence identity when compared to each other in pair-wise alignments. Cloned amplicons of near-full-length 16S rRNA genetic sequences from Norwegian gill samples were used to transcribe digoxigenin-labeled riboprobes that hybridized with epitheliocystis inclusions in proliferative gill lesions from 1999 Ireland and 2000 Norway samples. A 1487-bp consensus 16S rRNA genetic sequence representing the epitheliocystis bacterium from proliferative gill lesions was constructed from the sequence data amplified from 1999 Ireland and 2000 Norway samples. This consensus 16S rRNA genetic sequence was not identified in BLAST searches of the Genbank database and shared highest percent nucleotide sequence identity with members of the order Chlamydiales, e.g., endosymbionts of Acanthamoeba sp. UWE1 (82%) and UWC22 (81%). Molecular phylogenetic relationships inferred from distance and parsimony optimality criteria using 16S rRNA gene sequences indicated that the epitheliocystis bacterium from proliferative gill lesions grouped with members of the order Chlamydiales, separately from members of the order Rickettsiales. 'Candidatus' Piscichlamydia salmonis, gen. nov., sp. nov., is proposed for the epitheliocystis bacterium associated with proliferative gill lesions in Atlantic salmon, which has a unique 16S rRNA genetic sequence and exhibits developmental stages different from those identified in non-proliferative gill sections.