Epitheliocystis has been associated with heavy mortality and reduced growth of survivors in farmed Atlantic salmon (Salmo salar) (22). Ultrastructural studies of the epitheliocystis agent found in Atlantic salmon have revealed it to be an intracellular gram-negative coccoid bacterium with distinct developmental stages typical of bacteria of the order Chlamydiales (22). Epitheliocystis has been described in other salmonid hosts, e.g., juvenile steelhead trout (Oncorhynchus mykiss) (28) and cultured lake trout (Salvelinus namaycush) (3), as well as in a number of nonsalmonid species, including bluegill (Lepomis macrochirus) (16), striped bass (Morone saxatilis) (32), white perch (Morone americanus) (32), sea bream (Sparus aurata) (25), grey mullet (Liza ramada) (25), and cultured white sturgeon (Acipenser transmontanus) (14). Morphological studies of epitheliocystis agents in sea bream (S. aurata) have provided evidence for two distinct chlamydia-like developmental cycles associated with proliferative and nonproliferative host reactions (5). Although transmission electron microscopic examinations of intracellular inclusions have demonstrated that the agents of epitheliocystis in both salmonid and nonsalmonid hosts are gram-negative bacteria with developmental stages typical of members of the order Chlamydiales (5,14,22,28,32), the genetic relatedness of these bacteria has yet to be determined.Sequence data from the rRNA operon have revised phylogenetic relationships between Chlamydia species and chlamydia-like bacteria (CLB) (7,8,9). Reclassifying chlamydial species on the basis of 16S rRNA gene sequence identity, 16S and 23S ribosomal DNA (rDNA) sequences, and phenotypic characterization is considered by some to be the best means of taxonomically categorizing chlamydiae (24). Based on this approach, species within the family Chlamydiaceae have 16S rRNA gene sequences that are Ͼ90% identical (26), whereas chlamydia-like bacteria, defined as obligate intracellular bacteria having reticulate (RBs) and elementary bodies (EBs) characteristic of chlamydia, have been shown to have Ͼ80% 16S rRNA

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...

Background There is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR), a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites, has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC. Methods Wild-type and PXR−/− mice were studied at various time points of development in an experimental model of NEC. In addition, we studied the ability of the secondary bile acid lithocholic acid (LCA), a known PXR agonist in liver, to activate intestinal PXR and reduce NEC-related intestinal inflammation. Results We found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine, with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition, PXR−/− mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover, LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC. Conclusion These findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention.

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.

BackgroundProgrammed death cell 1 (PD-1) is an inhibitor of T cell activation and is also functionally linked to glycolysis. We hypothesized that PD-1 expression is defective in activated T cells from children with type 1 diabetes (T1D), resulting in abnormal T cell glucose metabolism.MethodsIn this pilot study, we enrolled children with new onset T1D within 2 weeks of diagnosis (T1D), unaffected siblings of T1D (SIBS), unaffected, unrelated children (CTRL), children with new onset, and untreated Crohn disease (CD). We repeated the assays 4–6 months post-diagnosis in T1D (T1D follow up). We analyzed anti-CD3/-CD28-stimulated peripheral blood mononuclear cells (PBMC) subsets for PD-1 expression by flow cytometry at baseline and after 24 h in culture. We measured cytokines in the culture medium by multiplex ELISA and glycolytic capacity with a flux analyzer.ResultsWe enrolled 37 children. T cells derived from subjects with T1D had decreased PD-1 expression compared to the other study groups. However, in T1D follow-up T cells expressed PD-1 similarly to controls, but had no differences in PBMC cytokine production. Nonetheless, T1D follow up PBMCs had enhanced glycolytic capacity compared to T1D.ConclusionsActivated T cells from T1D fail to upregulate PD-1 upon T-cell receptor stimulation, which may contribute to the pathogenesis of T1D. T1D follow up PBMC expression of PD-1 normalizes, together with a significant increase in glycolysis compared to T1D. Thus, insulin therapy in T1D children is associated with normal PD1 expression and heightened glycolytic capacity in PBMC.