A Novel Betaproteobacterial Agent of Gill Epitheliocystis in Seawater Farmed Atlantic Salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although ‘Candidatus Piscichlamydia salmonis’, previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as ‘Candidatus Branchiomonas cysticola’, was the cyst-forming agent in these samples. While histology and ultrastructure of ‘Ca. B. cysticola’ cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae

International variation in the definition of ‘main condition' in ICD-coded health data

Hospital-based medical records are abstracted to create International Classification of Disease (ICD) coded discharge health data in many countries. The ‘main condition' is not defined in a consistent manner internationally. Some countries employ a ‘reason for admission' rule as the basis for the main condition, while other countries employ a ‘resource use' rule. A few countries have recently transitioned from one of these approaches to the other. The definition of ‘main condition' in such ICD data matters when it is used to define a disease cohort to assign diagnosis-related groups and to perform risk adjustment. We propose a method of harmonizing the international definition to enable researchers and international organizations using ICD-coded health data to aggregate or compare hospital care and outcomes across countries in a consistent manner. Inter-observer reliability of alternative harmonization approaches should be evaluated before finalizing the definition and adopting it worldwid

International variation in the definition of 'main condition' in ICD-coded health data

Hospital-based medical records are abstracted to create International Classification of Disease (ICD) coded discharge health data in many countries. The 'main condition' is not defined in a consistent manner internationally. Some countries employ a 'reason for admission' rule as the basis for the main condition, while other countries employ a 'resource use' rule. A few countries have recently transitioned from one of these approaches to the other. The definition of 'main condition' in such ICD data matters when it is used to define a disease cohort to assign diagnosis-related groups and to perform risk adjustment. We propose a method of harmonizing the international definition to enable researchers and international organizations using ICD-coded health data to aggregate or compare hospital care and outcomes across countries in a consistent manner. Inter-observer reliability of alternative harmonization approaches should be evaluated before finalizing the definition and adopting it worldwide

Epitheliocystis in Atlantic salmon, Salmo salar L., farmed in fresh water in Ireland is associated with 'Candidatus Clavochlamydia salmonicola' infection

Intracellular inclusions containing chlamydia-like organisms are frequently observed in the gill epithelial cells of Atlantic salmon, Salmo salar L., cultured in fresh water in Ireland. In this study, the causative agent was identified in four separate freshwater sites, using 16s rRNA sequencing, as 'Candidatus Clavochlamydia salmonicola'. Histopathology and real-time (RT) PCR were used to further assess infections. The prevalence of infection ranged from 75-100% between sites and infection intensity was highly variable. No significant lesions were associated with these infections. As a diagnostic tool, RT-PCR proved marginally more sensitive than histopathology. The fate of 'Candidatus Clavochlamydia salmonicola' in Atlantic salmon post-seawater transfer was investigated in a 12-week marine longitudinal study. Both RT-PCR and histopathological examination indicate that the organism disappears from the gills 4-6 weeks post-transfer

'Candidatus Branchiomonas cysticola' is a common agent of epitheliocysts in seawater-farmed Atlantic salmon Salmo salar in Norway and Ireland

The prevalence and geographical distribution of the recently described endosymbiont 'Candidatus Branchiomonas cysticola' in Atlantic salmon Salmo salar gill epithelial cell cysts was investigated in seawater-farmed fish suffering proliferative gill inflammation (PGI). To this end, we developed a specific and sensitive real-time PCR assay for detection of the bacterium. 'Ca. B. cysticola' was found to be highly prevalent in Atlantic salmon gills sampled over 7 yr and from 17 geographically distant seawater locations in Norway and Ireland. 'Ca. B. cysticola' was found in significantly greater quantities in fish with large numbers of epitheliocysts, and fluorescence in situ hybridization confirmed its localisation within cysts. 'Ca. Piscichlamydia salmonis', a bacterium previously linked to epitheliocysts, was identified at relatively low levels of infection, apparently independent of epitheliocyst prevalence. These results suggest that 'Ca. B. cysticola' is the main cyst-forming bacterium in seawater-farmed Atlantic salmon in the studied countries. Our results also suggest a relationship between load of 'Ca. B. cysticola' and extent of pathological changes. Taken together with a previously described association between epitheliocyst load and severity of PGI in Norwegian salmon, the results could indicate a role for 'Ca. B. cysticola' in gill diseases such as PGI

First cases of amoebic gill disease (AGD) in Norwegian seawater farmed Atlantic salmon, Salmo salar L., and phylogeny of the causative amoeba using 18S cDNA sequences

Amoebic gill disease (AGD) was observed in seawater farmed Atlantic salmon at four geographically distant locations on the western coast of Norway. To the best of our knowledge, these are the first detected AGD outbreaks in Norway. The outbreaks lasted for 7-12 weeks in late autumn 2006 and were for the most part concurrent. The crude, cumulative mortality was in the range of 12-20% at three farms and 82% at a fourth. The histopathology showed uniform parasomal amoebae in lesions characteristic for AGD. Another gill disease, proliferative gill inflammation (PGI), was also present to a variable degree and the distinction between the two gill problems is discussed. Seawater temperatures were 3.5 degrees C higher than average before disease outbreaks, which subsided in early winter. The geographical and time pattern of these outbreaks strongly indicates simultaneous infection from the marine environment. Two contiguous 18S cDNA sequences, obtained by reverse transcriptase PCR from gill tissue with AGD-related lesions, showed highest similarity (99.2%) to a newly recognized species designated Neoparamoeba perurans and maximum likelihood analysis demonstrates that they represent Norwegian strains of this Neoparamoeba lineage

Phylogenetic relationship of ‘<i>Ca.</i> Branchiomonas cysticola’ with the <i>Betaproteobacteria</i>.

<p>A 16 S rRNA-based TREEPUZZLE tree is shown. The branching order near the root of the tree varies between different treeing methods and can thus not be reliably resolved. TREEPUZZLE support and bootstrap values for maximum parsimony, maximum likelihood and Neighbor-Joining (1000 resamplings) are indicated at the inner nodes. GenBank accession numbers are given in the square brackets. Bar, 10% estimated evolutionary distance.</p

<i>In situ</i> identification and localization of ‘<i>Ca.</i> Branchiomonas cysticola’ within cysts.

<p>Overlays of digital interference contrast (DIC) and fluorescence images of gill tissue sections are shown. (<b>A</b>) Hybridization with the ‘<i>Ca.</i> B. cysticola’ specific probe BraCy-129 labelled with Cy3 (red) in combination with a bacterial probe mix targeting most <i>Bacteria</i> labelled in Fluos (green). The combined fluorescence signals of both probes appear yellow. Scale bar represents 10 µm. (<b>B</b>) Hybridization with the ‘<i>Ca.</i> B. cysticola’ specific probe BraCy-129 labelled in Cy3 (red), the bacterial probe mix labelled in Cy5 (blue), and probe BTWO23A targeting a subset of the <i>Betaproteobacteria</i> labelled in Fluos (green). The combined fluorescence signals of all three probes appear white. Scale bar represents 20 µm.</p

Absence of ‘<i>Ca.</i> Piscichlamydia salmonis’ in cysts.

<p>Fluorescence <i>in situ</i> hybridization of sections of gill tissues using (<b>A</b>) a general bacterial probe mix labelled with Fluos (green), and (<b>B</b>) the ‘<i>Candidatus</i> P. salmonis’ specific probe Psc-523 labelled with Cy3 (red) simultaneously. The faint red signal represents autofluorescence of the gill tissues. (<b>C</b>) Digital interference contrast image showing cysts (arrow) in the epithelial cells. (<b>D</b>) The overlay of all three images demonstrates the presence of bacteria in the cysts, which hybridize with the bacterial probe mix but not with the ‘<i>Ca.</i> Piscichlaymdia salmonis’ specific probe and thus appear green. Additional staining with the DNA stain 4′,6-diamidino-2-phenylindole (DAPI) confirmed that all bacteria present in the gill tissues hybridized with the general bacterial probe mix. Scale bar represents 20 µm.</p