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

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 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...

Molecular Characterization of “Candidatus Similichlamydia latridicola” gen. nov., sp. nov. (Chlamydiales: “Candidatus Parilichlamydiaceae”), a Novel Chlamydia-Like Epitheliocystis Agent in the Striped Trumpeter, Latris lineata (Forster)

“…Epithelial hyperplasia, epithelial lifting along the lamellae, and lamellar fusion in the striped trumpeter were observed, although the filling of the interlamellar spaces was not as severe as that in previous reports. This differs from the nearly complete filling of the interlamellar spaces reported in both the largemouth bass (28) and the Arctic charr (15,27), which resulted in a severely compromised respiratory system. Striped trumpeter fish were recently reported to be affected by epitheliocystis (6).…”

“…Epitheliocystis is now known to affect Ͼ80 different species of fish (7,10), and intriguingly, from all of these reports, it appears that the host responses considered to be severe or as having a hyperinfection are all from farmed fish (14)(15)(16)(27)(28)(29)(30)(31)(32). Molecular diagnostics confirmed that this CLO, from both cultured and wild sources, is a member of the order Chlamydiales, based on the Ͼ80% sequence similarity to other species in the order (18).…”

“…These CLOs are Gram-negative, intracellular bacteria that may cause cyst-like lesions in the gill lamellae (9,11,12). The lesions may lead to epithelial hyperplasia and inflammation of the infected tissues, increased mucus production, and respiratory distress, sometimes ending in death (12)(13)(14)(15). Most reported losses in aquaculture attributed to epitheliocystis occur during the larval or juvenile culture stage (16).…”

Severe mortality in mesocosm-reared sharpsnout sea bream Diplodus puntazzo larvae due to epitheliocystis infection

“…It is caused by Gram-negative bacteria, recently characterized as Chlamydia (Order: Chlamydiales), forming intracellular inclusions (Draghi et al 2007). These inclusions progressively enlarge, resulting in hypertrophied cells that resemble spherical cysts.…”

A report on the 2012 mass summer mortalities of black rockfish, Sebastes schlegeli in the Southeast Sea, Korea

“…2). (Draghi et al, 2004(Draghi et al, & 2007Nowak and Lapatra, 2006;Polkinghorne et al, 2010). (Kranz, 1989;Wada et al, 1991;Wolke, 1992;Park and Huh, 1994;Montero et al, 1999;Wolf and Wolfe, 2005) Oh, 1999 &Sohn et al, 2000;Kim et al, 2008).…”