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Diseases of Aquatic Organisms

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DAO 76:27-38 (2007)  -  doi:10.3354/dao076027

Characterization of a Neochlamydia-like bacterium associated with epitheliocystis in cultured Arctic charr Salvelinus alpinus

Andrew Draghi II1, Julie Bebak2,6,*, Vsevolod L. Popov3, Alicia C. Noble2, Steven J. Geary1,4, A. Brian West1, Philip Byrne5, Salvatore Frasca Jr.1,4

1Department of Pathobiology and Veterinary Science and 4Center of Excellence for Vaccine Research, University of Connecticut, Storrs, Connecticut 06269-3089, USA
2The Conservation Fund’s Freshwater Institute, 1098 Turner Road, Shepherdstown, West Virginia 25443, USA
3Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas 77555-0609, USA
5Fisheries and Oceans Canada, Charlottetown, Prince Edward Island C1A 5T1, Canada
6Present address: US Department of Agriculture Agricultural Research Service, Aquatic Animal Health Research Laboratory, 990 Wire Road, Auburn, Alabama 36832, USA
*Corresponding author. Email:

ABSTRACT: Infections of branchial epithelium by intracellular gram-negative bacteria, termed epitheliocystis, have limited culture of Arctic charr Salvelinus alpinus. To characterize a bacterium associated with epitheliocystis in cultured charr, gills were sampled for histopathologic examination, conventional and immunoelectron microscopy, in situ hybridization, 16S ribosomal DNA (rDNA) amplification, sequence analysis and phylogenetic inference. Sampling was conducted at the Freshwater Institute (Shepherdstown, West Virginia, USA) during outbreaks of epitheliocystis in April and May 2002. Granular, basophilic, cytoplasmic inclusions in charr gill were shown to stain with Macchiavello, Lendrum’s phloxine-tartrazine and Gimenez histochemical techniques. Ultrastructurally, inclusions were membrane-bound and contained round to elongate reticulate bodies that were immunoreactive to an antibody against chlamydial lipopolysaccharide, suggesting the presence of similar epitopes. DNA extracted from gills supported amplification of the most polymorphic and phylogenetically relevant region of the 16S rRNA gene, which had 97 to 100% identity with several uncultured clinical Neochlamydia spp. (order Chlamydiales) Clones WB13 (AY225593.1) and WB258 (AY225594.1). Sequence-specific riboprobes localized to inclusions during in situ hybridization experiments. Taxonomic affiliation was inferred by distance- and parsimony-based phylogenetic analyses of the 16S sequence, which branched with Neochlamydia hartmannellae in the order Chlamydiales with high confidence. This is the first molecular characterization of a chlamydia associated with epitheliocystis in Arctic charr and the fourth Neochlamydia spp. sequence to be associated with epitheliocystis. Presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae.

KEY WORDS: Arctic charr · Environmental chlamydiae · Epitheliocystis · In situ hybridization · Neochlamydia · Salvelinus · Ultrastructure · 16S ribosomal RNA gene

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