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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 Resale or republication not permitted without written consent of the publisherDis Aquat Org 76: [27][28][29][30][31][32][33][34][35][36][37][38] 2007 ized ultrastructurally (Hoffman et al. 1969, Wolke et al. 1970, Nylund et al. 1998), antigenically (Groff et al. 1996 or, most recently, molecularly (Draghi et al. 2004, Meijer et al. 2006 as chlamydia or chlamydia-like bacteria. Horn & Wagner (2001) have proposed the term 'environmental chlamydia' to encompass these diverse, and sometimes medically relevant, chlamydiae isolated from environmental sources, while others suggest a more general term, 'novel chlamydia', to include those chlamydiae not members of the Chlamydiaceae (Corsaro & Greub 2006). In epitheliocystis, infection of branchial epithelium results in formation of intracytoplasmic basophilic granular inclusions that distend the cytoplasm of individual epithelial cells. Host responses to infection vary from non-proliferative, i.e. with litt...

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Arctic charr Salvelinus alpinus production facilities, nonproduction water sources and effluents in the United States and Canada were sampled to determine if chlamydiae associated with epitheliocystis were present in water and were associated with inclusions of epitheliocystis in gill tissue. Gills from 607 fish from 13 sites were processed for histopathologic examination and DNA extraction. Water was collected from 21 locations for DNA testing. Eighteen fish from one location had inclusions of epitheliocystis with proliferative and inflammatory gill lesions. Inclusions were stained using the Gimenez technique and, at the ultrastructural level, consisted of intracytoplasmic membrane-bound vacuoles containing reticulate and intermediate bodies in a fibrillar matrix. PCR using Order Chlamydiales-specific primers performed on DNA extracts from 12 of 13 infected fish yielded amplicons that were identical to (GQ302988) or differed at one base from (GQ302987) the 16S ribosomal RNA gene signature sequence of 'Candidatus Piscichlamydia salmonis', which is the chlamydia that was previously identified in epitheliocystis inclusions of farmed Atlantic salmon. In situ hybridization using a ~1.5 kb riboprobe corresponding to the 'Candidatus Piscichlamydia salmonis' 16S rRNA genetic sequence (AY462244) confirmed its presence within Arctic charr gill inclusions. DNA isolated from water samples was tested by Chlamydiales-specific PCR and yielded 54 partial 16S rRNA genetic sequences spanning the signature region; however, no 16S rRNA genetic sequences associated with epitheliocystis were identified. This is the first report of 'Candidatus Piscichlamydia salmonis' associated with epitheliocystis in Arctic charr, the first identification of 'Candidatus Piscichlamydia salmonis' from a freshwater production location, and the first reported occurrence in North America.

in wild non-salmonid fish (Stephens, Newman,