28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Molecular Characterization Of The Chlamydia-like Bacterium Associated With Epitheliocystis In Atlantic Salmon (Salmo salar) Salvatore Frasca Jr.1, Andrew Draghi II1, Vsevolod L. Popov2, James B. Stanton3, Corrie C. Brown3, Julie Bebak-Williams4, Gregory J. Tsongalis5, and A. Brian West6 1Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269; 2Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 3Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602; 4Freshwater Institute, Shepherdstown, West Virginia 25443; 5Department of Clinical Chemistry and Molecular Pathology, Hartford Hospital, Hartford, CT 06102; 6Department of Pathology, New York University Medical Center, New York, NY 10016 Epitheliocystis, a term used loosely to describe gill infection by intracellular, gram-negative, chlamydia-like bacteria, is characterized histologically by intracytoplasmic, basophilic, granular inclusions in branchial epithelial cells which limits salmon farming. To characterize these yet uncultured bacteria for future diagnostic and phylogenetic investigations, gill samples were collected for histopathologic, transmission electron microscopic and nucleic acid studies during outbreaks of epitheliocystis in Atlantic salmon (Salmo salar) in Ireland and Norway. Histopathologic studies revealed inclusions typical of epitheliocystis, and transmission electron microscopy demonstrated chlamydial developmental stages within intracytoplasmic vacuoles in host branchial epithelial cells. Immunogold labeling using an antibody to chlamydial lipopolysaccharide was reactive with reticulate bodies of inclusions.  Chlamydial and eubacterial primers were utilized in PCR-based experiments to amplify overlapping segments of 16S SSU ribosomal DNA from gill samples. The PCR products were isolated, cloned, sequenced, and assembled to generate a consensus 16S rDNA sequence.  Preliminary molecular phylogenetic studies using this 16S rDNA sequence indicated that this bacterium branched with other chlamydia-like bacteria, e.g. Simkania sp and Parachlamydia spp.  This sequence had an identity of 80-82% with members of the chlamydiales and suggested a common ancestor with that of the Chlamydia. Intracellular gram-negative bacteria associated with epitheliocystis in fish may represent a new group of chlamydia-like or related bacteria.  Molecular phylogenetic characterization of the agents of epitheliocystis is a first step toward understanding their biology and the pathogenesis of gill infections caused by these organisms.  Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page