Proliferative gill inflammation (PGI) is an important cause of loss in seawater-farmed Atlantic salmon in Norway. Several microbes have been associated with PGI, including the commonly but not exclusively observed inclusions (epitheliocysts) within the gill lamellae related to infection with 'Candidatus Piscichlamydia salmonis'. Atlantic salmon transferred in the spring of 2004 to 12 seawater farms situated in mid-and southwest Norway were sampled throughout that year. Outbreaks of PGI, as evaluated by clinical examination, histology, and mortality data, were diagnosed in 6 of 7 farms in southwest Norway but not in the 5 farms studied in mid-Norway. Generally, mortality started 3 to 5 mo after seawater transfer and outbreaks lasted at least 1 to 3 mo. 'Ca. P. salmonis' was detected by real-time PCR only in fish from PGI-affected farms and our results indicate an association between 'Ca. P. salmonis' load and PGI severity. Likewise, although widely distributed in all 12 farms studied, epitheliocyst prevalence and number per fish as observed by histology appears associated with PGI prevalence and severity. However, the occurrence of epitheliocysts showed no association with molecular detection of 'Ca. P. salmonis', suggesting that at least 1 other organism is responsible for many of the observed inclusions. A microsporidian, Desmozoon lepeophtherii, was identified at high prevalence regardless of fish and farm PGI status, but at higher loads in fish with PGI. Our results support a multifactorial etiology for PGI in which 'Ca. P. salmonis', an unidentified epitheliocyst agent, and the microsporidian are contributing causes. No evidence for the involvement of Atlantic salmon paramyxovirus in PGI development was identified in the present study. High water temperatures and ectoparasites probably exacerbated mortality. KEY WORDS: Proliferative gill inflammation · Epitheliocysts · 'Candidatus Piscichlamydia salmonis' · Desmozoon lepeophtherii · 'Candidatus Clavochlamydia salmonicola' · Atlantic salmon paramyxovirusResale or republication not permitted without written consent of the publisher Dis Aquat Org 91: 201-211, 2010 to months (Kvellestad et al. 2004, Skjelstad et al. 2007. Mortality varies and losses of 15 to 20% or higher have been reported (Flesjå et al. 2004, Kvellestad et al. 2004. Estimation of economic losses is difficult, but one of the largest farming companies in Norway considered PGI to be the disease responsible for most losses in terms of both numbers of fish and biomass in 2007(Skjelstad et al. 2007). Gill disorders were also the most important cause of losses in Ireland in 2005(Rodger 2007. Previous studies have indicated a multifactorial etiology behind PGI, following identification of various microorganisms occurring alone or in combination with others, including Atlantic salmon paramyxovirus (ASPV) (Kvellestad et al. 2003(Kvellestad et al. , 2005, salmon gill poxvirus , various ectoparasites (Kvellestad et al. 2005), as well as lamellar bacterial inclusions (epitheliocystis) (Nylund ...

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 .

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.

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.

Body malformation due to shortness of the vertebral column, in most cases of unknown cause, has been observed in fish for more than 100 yr. It periodically occurs with high prevalence in farmed Atlantic salmon Salmo salar in Norway, and this paper describes the results of macroscopic, radiographic and histologic examination of parr and seawater-transferred fish. The vertebral bodies in both age groups did not acquire the length that they normally should due to a growth disturbance leading to the condition of platyspondyly and shortness in the column. The pathologic changes became visible at different ages in both groups and the process apparently starts in intervertebral tissues. There was proliferation of connective tissue and blood vessels, and sometimes infiltration with inflammatory cells, around affected vertebrae, especially in seawater-transferred fish. This is the first description of inflammation in abnormally short-spined fish, and it may indicate an infectious etiology, at least in farmed seawater-transferred salmon.

Melanized focal changes in skeletal muscle of farmed Atlantic salmon (Salmo salar) are a major quality problem. The aetiology is unknown, but infection with Piscine orthoreovirus (PRV) has been associated with the condition. Here, we addressed the pathogenesis of red and melanized focal changes and their association with PRV. First, a population of farmed fish (PRV‐negative prior to sea transfer) was sequentially investigated throughout the seawater period. The fish were autopsied and tested for PRV infection. Muscular changes were described by macroscopy and histology, and a classification system was established. Second, in an experimental infection trial, PRV was injected intramuscularly to induce changes. The farmed fish was gradually infected with PRV. Red focal changes occurred throughout the observation period with a low prevalence regardless of PRV status. Melanized changes were highly diverse and their prevalence increased during the trial. Changes of low macroscopic grade and histological category were more prevalent in PRV‐negative fish. Diffuse granulomatous melanized changes only occurred after PRV infection. No muscular changes were observed in the experimentally challenged fish. Our studies do not indicate that PRV infection causes red focal changes, but seems important in the development of granulomatous melanized changes.

Proliferative gill inflammation (PGI) causes significant losses in farmed Atlantic salmon Salmo salar L. in Norway, especially during the first months following seawater transfer. The aetiology is apparently multifactorial, including infection with chlamydia-like bacteria and Atlantic salmon paramyxovirus (ASPV). In the present study, gills from diseased fish from 3 farms on the western coast of Norway were sampled. The pathological changes were briefly described and the aetiological significance of ASPV studied by immunofluorescent staining of cryosections and by immunohistochemistry on sections of formalin-fixed and paraffin-embedded tissue. The pathological changes were macroscopically characterized by palour of the gills, and histologically by inflammation, circulatory disturbances, cell death and epithelial cell proliferation. ASPV was demonstrated in fish from all farms studied, as immunostaining consistent with ASPV was obtained in lamellar epithelial and endothelial cells of pathologically altered tissues. It is concluded that ASPV is at least a contributing cause of PGI. As far as we know, this is the first demonstration of fish disease related to infection with a paramyxovirus. KEY WORDS: Atlantic salmon · Salmo salar · Proliferative gill inflammation · PGI · Epitheliocystis · Atlantic salmon paramyxovirus · ASPV · Immunofluorescence · Immunohistochemistry Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [47][48][49][50][51][52][53][54] 2005 parasites may also be inconsistently observed at gill surfaces (Kvellestad et al. 2004), their role as primary aetiological factors is doubtful. Recently, a previously unknown virus, Atlantic salmon paramyxovirus (ASPV), was isolated from the gills of fish with PGI (Kvellestad et al. 2003), suggesting a possible viral involvement in development of this disease. However, due to the fact that the above-mentioned agents are inconsistently detected and their aetiological significance has not been fully investigated, we currently use the descriptive term 'proliferative gill inflammation' for this disease in order to reflect the unsolved aetiology.This paper briefly describes the gill pathology of PGI as it occurred in fish selected from 3 seawater farms. A rabbit hyperimmune serum against ASPV was produced, characterized and used to detect viral antigen in gills by an indirect immunofluorescence (IIF) test on cryosections and immunohistochemistry (IHC) on sections of formalin-fixed and paraffin-embedded tissue. MATERIALS AND METHODSVirus and cell cultures. ASPV from the third to fifth passages (Kvellestad et al. 2003) was used. Rainbow trout gill (RTgill-W1) cells (Bols et al. 1994, ATCC CRL-2523 were grown at 14°C in Leibovitz L-15 supplemented with 5% (v/v) foetal bovine serum, 2 mM Lglutamine and 24 µg ml -1 gentamicin. Cells were incubated at 14°C after inoculation with virus without removal of the inoculum.Production of polyclonal anti-ASPV serum in rabbit. Virus for immunization of rabbit was propagated a...

Abstract. The present study is mainly focusing on mortality variations of fish due to changing A1-chemistry of mixing zones. An artificial mixing zone was made by pumping water from a limed stream and an acidic tributary into a mixing channel. Atlantic salmon (Salmo salar L.) parr were exposed to the mixed water, limed stream water, and acidic tributary water. Mortality, blood haematocrit and plasma C1--concentration were recorded. Neither mortality, nor changes in haematocrit and plasma C1-were observed when fish were exposed to limed water, while in both acidic and mixed water, mortalities and loss of plasma CI-were observed. The highest mortality rates were found within the initial part (0 to 20 s) of the mixing zone. Blood haematocrit increased only in fish exposed to acidic tributary water. Our results shows that changes in Al-chemistry and subsequent Al-polymerization occur when acidic tributary water is mixed with limed stream water. We have also demonstrated that the toxicity which can arise in mixing zones are greater than in the original acidic water before mixing. The variations in mortality observed are associated with the quality and quantity of Al-polymerization as well as ageing of the polymers.