Characterization of 'Candidatus Syngnamydia Salmonis
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Arch Microbiol DOI 10.1007/s00203-014-1038-3 ORIGINAL PAPER Characterization of ‘Candidatus Syngnamydia salmonis’ (Chlamydiales, Simkaniaceae), a bacterium associated with epitheliocystis in Atlantic salmon (Salmo salar L.) Stian Nylund · Andreas Steigen · Egil Karlsbakk · Heidrun Plarre · Linda Andersen · Marius Karlsen · Kuninori Watanabe · Are Nylund Received: 30 October 2013 / Revised: 12 September 2014 / Accepted: 13 September 2014 © The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Two Chlamydiales have previously been found Introduction to infect Atlantic salmon (Salmo salar L.), Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia Hoffman coined the term epitheliocystis (Hoffman et al. salmonicola. Both develop intracellularly in cyst-like inclu- 1969) for cyst-like epidermal lesions in fish caused by bac- sions in gill cells, generally referred to as epitheliocysts. teria ultrastructurally resembling chlamydiae (as Bedso- Here, we present evidence for the association of a novel nia-like Chlamydia-like; Bedsonia a synonym of Chla- = species of Chlamydiales with epitheliocystis in Atlantic mydia). The bacteria occur intracellularly in cyst-like salmon. Based on its partial 16S rRNA gene sequence, it inclusions; usually resulting in significantly enlarged is a new member of the family Simkaniaceae, and a 95.7 % infected cells (epitheliocystis cells). Additional evidence identity to the type species Candidatus Syngnamydia vene- for a chlamydial aetiology of epitheliocystis in various fish zia suggests inclusion in the candidate genus Syngnamydia. species came from further ultrastructural observations, The presence of the bacterium in epitheliocysts in gills of including documentation of a two-stage replication cycle Atlantic salmon was demonstrated by RNA–RNA hybridi- including vegetative reticulate bodies (RBs) and infectious zation. Ultrastructurally, the novel bacterium produces elementary bodies (EBs; Paperna et al. 1981; Nylund et al. pleomorphic reticulate bodies and elementary bodies (EBs) 1998). Genetic studies of these agents of epitheliocystis with a characteristic morphology. The EBs are short rods have confirmed that they usually are members of the order with a terminal disc-like cap area, a sub-apical spherical Chlamydiales and established that the genetic diversity vacuole-like electron-lucent structure and a post-equatorial among them is large (Draghi et al. 2004; Meijer et al. nucleoid. We propose the name Candidatus Syngnamydia 2006; Draghi et al. 2007; Karlsen et al. 2008; Polking- salmonis for this new agent from epitheliocysts in seawa- horne et al. 2010; Camus et al. 2013; Steigen et al. 2013; ter-reared salmon. Stride et al. 2013a, b). Two Chlamydiales have previously been found to infect and produce epitheliocyst in Atlantic Keywords Epitheliocystis · Chlamydia · Simkaniaceae · salmon (Salmo salar), ‘Candidatus Piscichlamydia sal- Syngnamydiae · Atlantic salmon monis’ and ‘Candidatus Clavichlamydia1 salmonicola’ (Draghi et al. 2004; Karlsen et al. 2008). However, a betaproteobacterium, Candidatus Branchiomonas cysti- Communicated by Andreas Brune. cola, has also recently been detected in cysts in the gills of Atlantic salmon (Toenshoff et al. 2012; Mitchell et al. * S. Nylund · A. Steigen ( ) · E. Karlsbakk · H. Plarre · 2013). L. Andersen · M. Karlsen · K. Watanabe · A. Nylund Department of Biology, University of Bergen, P.O. Box 7803, Chlamydiae are difficult to culture in vitro and knowl- 5020 Bergen, Norway edge on the genetic diversity within the phylum mainly e-mail: [email protected] E. Karlsbakk Institute of Marine Research, P.O. Box 1870, 5817, Nordnes, 1 First spelled Clavochlamydia by Karlsen et al. (2008), amended to Bergen, Norway Clavichlamydia in Bergey’s manual vol 4 (2010) p 866 (Horn 2011). 1 3 Arch Microbiol relies on 16S rRNA gene sequences. A 16S rRNA Gills collected from population PIII were also used for in gene-based system for classification of Chlamydiales situ hybridization (ISH). has been recommended that suggests percentage sequence identity limits for the classification into taxa RNA extraction, reverse transcription RT-PCR (Everett et al. 1999). Using these thresholds for classi- and real-time RT-PCR fication, nine families have been proposed (Horn 2008, 2011; Lagkouvardos et al. 2014). Sequence data that RNA was extracted from gills and transcribed into cDNA as exist from other uncultivable Chlamydiales from fish do, previously described by Devold et al. (2000). PCR was run however, suggest an even higher diversity at family level with primers 16SIGF, 806R and 16SB1 (Draghi et al. 2004), (Horn 2008; Polkinghorne et al. 2010; Corsaro and Work using cDNA as template for amplification of the nearly 2012; Camus et al. 2013; Steigen et al. 2013; Stride et complete 16S rRNA gene of the novel chlamydia. Real- al. 2013a, b). The three Chlamydiae associated with epi- time PCR was run as described by Hodneland and Endresen theliocystis in salmonids, Candidatus Piscichlamydia (2006) with primers and probes directed against Candida- salmonis, Candidatus Clavichlamydia salmonicola, tus Piscichlamydia salmonis (Nylund et al. 2008) and the and Neochlamydia sp. represent three different families novel chlamydia on cDNA template. The real-time assay (Horn 2008). targeting the new chlamydia consisted of specific prim- During the autumn of 2006, we investigated Atlan- ers SCh-F (5′-GGGTAGCCCGATATCTTCAAAGT-3′), tic salmon from a farm in Western Norway where the fish SCh-R (5′-CCCATGAGCCGCTCTCTCT-3′) and a showed signs of respiratory distress and had prominent gill TaqMan® FAM™ dyed minor groove binder (MGB) probe lesions. PCR testing and sequencing revealed that a suite of (Fam-5′-TCCTTCGGGACCTTAC-3′-MGB). infectious agents were present on the gills of these salmon, including a novel epitheliocystis associated chlamydia Sequencing and sequence analysis with affinities to the familySimkaniaceae. The bacterium was subsequently detected in salmon from other farms in Sequencing of purified PCR products and plasmids was Norway. done using an ABI Prism BigDye Terminator Cycle Here, we present morphological and genetic data Sequencing Ready Reaction kit, v3.1 (Applied Biosystems, describing the novel chlamydia and demonstrate that its Perkin-Elmer) according to producer’s recommendations. RNA is present in epitheliocysts in infected gills. We sug- Sequencing was done in both directions, and sequences gest a new provisional taxon, ‘Candidatus Syngnamydia used for phylogenetic studies originated from direct salmonis’ in the family Simkaniaceae, for this new parasitic sequencing of PCR products. Sequencing was performed at bacterium infecting Atlantic salmon. the sequencing facility at the University of Bergen (http:// www.seqlab.uib.no). An alignment of 56 16s rRNA gene sequences from Materials and methods the phylum Chlamydiae, retrieved from the GenBank or obtained from the present study, was made using Vector Material NTI 9.0 software. The alignment included members from all families within order Chlamydiales and several 16S Salmon were collected from three separate seawater farms rRNA gene sequences obtained from fish gills. Phyloge- in Norway (populations PI–PIII) in October 2006 (Table 1). netic analysis was performed using TREE-PUZZLE 5.2 PI was from Northern Norway while PII and PIII were (available at: http://www.tree-puzzle.de), maximum likeli- from Western Norway. All fish suffered from gill disease hood (ML). The best-fit nucleotide substitution model for with associated mortality. the dataset was GTR I G, identified by Modeltest 3.6 + + Gills were collected from all populations and subse- (Posada and Crandall 1998). This model was implemented. quently processed for histology and nucleic acid extraction. Trees were viewed using TreeView (Page 1996). Table 1 Number of gill Population Origin (country) Mean weight (g) Number of positive samples/total number samples found positive for two of samples chlamydia in populations I–III, using real-time RT-PCR `Cand. P. salmonis´ Novel chlamydia I Nordland 264 0/10 10/10 Fish from PIII solely infected II Sogn-og Fjordane 365 25/25 25/25 by the novel Chlamydia were III Hordaland 362 1/10 10/10 used for in situ hybridization 1 3 Arch Microbiol Cloning and in vitro transcription of DIG-labelled RNA wax (60 °C). Paraffin Sects. (8 μm) were cut on a Leica probes RM2255 microtome and floated on a DEPC water bath at 50 °C until creases disappeared and then collected on pol- Digoxigenin-labelled RNA probes against the novel chla- ylysine-covered slides. The slides were dried at 40 °C and mydia, and Candidatus P. salmonis and Candidatus B. then kept at 34 °C over night. The slides were dewaxed by cysticola were made as previously described (Karlsen immersion in Histoclear (National Diagnostics) twice for et al. 2008). A DNA fragment (769 bp) coding for the par- 10 min. The slides were washed twice in 100 % ethanol for tial 16S rRNA gene sequence from both chlamydia was 2 min and rehydrated through an ethanol series in 1 PBS. × amplified using primers 16sSIGF and 806R. A DNA frag- DEPC.H2O: 100 % twice, 75, 50 and 25 % for 5 min each. ment (700 bp) from the 16s DNA gene from Candidatus Sections were washed twice in 1 PBS.DEPC.H O, then × 2 B. cysticola was amplified using primers. PCR products once in a 0.05 M Tris–HCl buffer, pH 7.5. Sections were from Candidatus B. cysticola were used to make the sense/ overlaid with Proteinase K (Promega) for 10 min (10 μg/ anti-sense probes (EUGB 27F: 5′-AGAGTTTGATCMTG- ml) in a buffer containing 0.05 M TrisHCl and CaCl2 at pH GCTCAB-3′), (BProto-R1: 5′-GCA TTTCACCGCTACA- 9.5. Sections were washed with 0.05 M Tris–HCl buffer, CATGG-3′). The fragment was subsequently cloned into then 1 PBS.DEPC.H O, refixed for 20 min in 4 % para- × 2 the PCR4-vector (Invitrogen) that carries the T7 promoter. formaldehyde in PBS.DEPC.H2O in room temperature, Clones with insert in opposite directions were selected as washed three times in 1 PBS.DEPC.H O for 5 min and × 2 templates for transcription of RNA in the presence of DIG- dehydrated 25, 50, 70 and 2 100 % in 1 PBS.DEPC.