Coldwater-associated ulcers, i.e. winter ulcers, in seawater-reared Atlantic salmon Salmo salar L. have been reported in Norway since the late 1980s, and Moritella viscosa has been established as an important factor in the pathogenesis of this condition. As routine histopathological examination of winter ulcer cases in our laboratory revealed frequent presence in ulcers of long, slender rods clearly different from M. viscosa, a closer study focusing on these bacteria was conducted. Field cases of winter ulcers during 2 sampling periods, 1996 and 2004-2005, were investigated and long, slender rods were observed by histopathological examination in 70 and 62.5% of the ulcers examined, respectively, whereas cultivation on marine agar resulted in the isolation of yellowpigmented colonies with long rods from 3 and 13% of the ulcers only. The isolates could be separated into 2 groups, both identified as belonging to the genus Tenacibaculum based on phenotypic characterization and 16S rRNA sequencing. Bath challenge for 7 h confirmed the ability of Group 1 bacterium to produce skin and cornea ulcers. In fish already suffering from M. viscosa-induced ulcers, co-infection with the Group 1 bacterium was established within 1 h. Ulcers from field cases of winter ulcers and from the transmission experiments tested positive by immunohistochemistry with polyclonal antiserum against the Group 1 bacterium but not the Group 2 bacterium. Our results strongly indicate the importance of the Group 1 bacterium in the pathogenesis of winter ulcers in Norway. The bacterium is difficult to isolate and is therefore likely to be underdiagnosed based on cultivation only. KEY WORDS: Winter ulcers · Tenacibaculum · Atlantic salmon · Transmission experiment · Moritella viscosa · Cod · Halibut Resale or republication not permitted without written consent of the publisherDis Aquat Org 94: [189][190][191][192][193][194][195][196][197][198][199] 2011 winter ulcers (Lunder et al. 1995, Benediktsdóttir et al. 1998; however, demonstration of pathogenicity for this bacterium under experimental conditions has so far been unsuccessful (Lunder et al. 1995). Many culture-based investigations of winter ulcers have resulted in the identification of mixed cultures of mainly Vibrio spp. (Benediktsdóttir et al. 1998, H. Nilsen & A. B. Olsen unpubl.).Worldwide, members of the Cytophaga-Flavobacterium-Bacterioides group are reported to cause mortality and economical losses because of skin ulcers and eroded fins of both freshwater and seawater fish species (Wakabayashi et al. 1986, Kent et al. 1988, Bernardet 1989, Bernardet & Kerouault 1989, Bernardet et al. 1990). The bacteria appear as yellow colonies with long, slender filamentous rods and are relatively difficult to culture. Four species are considered pathogenic to salmonids: Flavobacterium branchiophilum (bacterial gill disease in freshwater), F. columnare (freshwater above 14°C), F. psychrophilum (mainly rainbow trout in freshwater) and Tenacibaculum maritimum (prev. Flexibacter maritimus) in...

In intensive aquaculture systems, high mortalities are frequently observed during the early life stages of marine fish. The aim of this study was to investigate differences in the susceptibility of turbot Scophthalmus maximus, halibut Hippoglossus hippoglossus and cod Gadus morhua to various strains of Vibrio anguillarum (serotypes O1, O2α and O2β), V. salmonicida and V. splendidus. The bath challenge experiments were performed using a multidish system, with 1 egg well -1. Unchallenged eggs and larvae were used as controls. Larvae in challenged groups that suffered high mortality rates were examined by immunohistochemistry. The overall results with respect to mortality showed that the O2α serotype was pathogenic to all 3 species, while the O1 serotype was pathogenic to halibut and cod. The immunohistochemical examinations revealed differences in histopathology. The O1 serotype produced more severe and highly developed infections than the O2α serotype. In larvae exposed to the O1 serotype, necrosis and bacterial cells were seen in the dermis, gastrointestinal tract, brain and eye area, while in larvae exposed to the O2α serotype, bacteria were usually limited to the gastrointestinal tract. These results suggest either that there are undetermined species differences in host immunity or that these pathogens are host-specific even in the early life stages of fish. The O2β strain did not cause an increased mortality to halibut and turbot.

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus reservoirs.KEY WORDS: Heart and skeletal muscle inflammation · HSMI · Transmission · Reovirus · Wild marine fish species · Farmed fish · PCR Resale or republication not permitted without written consent of the publisherDis Aquat Org 97: [255][256][257][258] 2012 Research, Bergen. Sampling was financed and performed by the project 'Viral haemorrhagic septicaemia virus (VHSV) in wild and farmed fish in Norway' (NFR-190245). Briefly, the fish were captured by trawling and 1 g samples of spleen, kidney and brain were pooled and directly frozen in 9 ml Leibovitz (L-15) culture medium supplemented with 4 mM L-glutamine and 100 ng ml -1 gentamicin (all from SigmaAldrich). Each pool consisted of material from 2 to 5 individuals. Samples were immediately transferred to −80°C for storage. RNA was extracted from 100 µl of mixed tissue homogenate with the NucliSENS easyMAG nucleic acid extraction system (bioMérieux) according to the manufacturer's recommendations. A total of 1627 fish (379 pools) from 37 different species were sampled and screened by means of a PRV-specific RT-qPCR assay (Palacios et al. 2010). RESULTSThe majority of the 379 pools of samples tested negative for the PRV-specific real-time PCR, but pools from 4 different fish species yielded positive results (Table 1) that had relatively high threshold cycle number (C t )-values (great silver smelt Argentina silus, C t = 37; capelin Mallotus villosus, C t = 38; Atlantic horse mackerel Trachurus trachurus, C t = 37; and Atlantic herring Clupea harengus, C t = 31). DISCUSSIONThe fish species that tested positive with the PRV-specific real-time PCR are common species found along the Norwegian coast at different periods of the year. Argentina silus is a smelt of the Argentinidae family. It is a pelagic species that is common along the Norwegian coastline. The positive pool originated from A. silus that were caught by trawl in northern Trøndelag. Trachurus trachurus is a pelagic species that is common in west and northern parts of Norway during late summer and autumn. Only one T. trachurus was caught and it was in the Andøyfjord. Mallotus villosus is a small fish of the smelt family Osmeridae. This is an arctic species, but local stocks are also found along the coast of the northern part of Norway. The positive pool of M. villosus was from the Altafjord. Clupea harengu...

Viral hemorrhagic septicemia virus (VHSV) infects a wide range of marine fish species. To study the occurrence of VHSV in wild marine fish populations in Norwegian coastal waters and fjord systems a total of 1927 fish from 39 different species were sampled through 5 research cruises conducted in 2009 to 2011. In total, VHSV was detected by rRT-PCR in twelve samples originating from Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangius merlangus) and silvery pout (Gadiculus argenteus). All fish tested positive in gills while four herring and one silvery pout also tested positive in internal organs. Successful virus isolation in cell culture was only obtained from one pooled Atlantic herring sample which shows that today's PCR methodology have a much higher sensitivity than cell culture for detection of VHSV. Sequencing revealed that the positive samples belonged to VHSV genotype Ib and phylogenetic analysis shows that the isolate from Atlantic herring and silvery pout are closely related. All positive fish were sampled in the same area in the northern county of Finnmark. This is the first detection of VHSV in Atlantic herring this far north, and to our knowledge the first detection of VHSV in silvery pout. However, low prevalence of VHSV genotype Ib in Atlantic herring and other wild marine fish are well known in other parts of Europe. Earlier there have been a few reports of disease outbreaks in farmed rainbow trout with VHSV of genotype Ib, and our results show that there is a possibility of transfer of VHSV from wild to farmed fish along the Norwegian coast line. The impact of VHSV on wild fish is not well documented.

Viral haemorrhagic septicaemia (VHS) is a viral disease known to cause high mortality in many teleost species, both in wild and farmed populations. The effects of this virus infection on the population dynamics of Atlantic herring Clupea harengus are still unknown; however, high mortalities have been reported in infected Pacific herring C. pallasii. We investigated Atlantic herring from the Norwegian spring-spawning (NSS) stock during the spawning season and found a much higher prevalence of VHS virus (VHSV) than had been identified in previous surveillance studies. Positive results were found in 19% of the samples analysed using viral cell culture on pooled brain, spleen and kidney tissue samples from 5 fish. Real-time RT-PCR performed on the same pooled samples revealed 33% positive samples, while analysis of individual organs resulted in a prevalence of 12−13%. Gills, which were analysed only by real-time RT-PCR, displayed a prevalence of 69%. It is not clear whether the virus detected in the gills reflects an infection or a passive carrier status. Sequencing of positive samples from gills and internal organs revealed high identity levels with VHSV of genotype Ib detected previously in Norwegian herring. The high prevalence identified in our study indicates a need for further research into the significance of VHSV infection on the herring population.

Farming of Atlantic cod Gadus morhua is one of the most rapidly growing sectors of Norwegian aquaculture. Classical vibriosis caused by Vibrio anguillarum is a problem in cod aquaculture. To prevent disease outbreaks, a thorough understanding of the infection route and the impact of the bacteria on the host is important. The intestinal tract, skin and gills have all been proposed as routes of entry for bacterial infections such as vibriosis. We aimed to further develop understanding of V. anguillarum serotype O2α infections in cod larvae by elucidation of a possible route of entry, the pattern of infection and its histopathology. Cod eggs were transferred to a 24-well polystyrene multi-dish with 2 ml of sterile aerated 80% (28 ‰ salinity) seawater. Challenge doses were 10 4 and 10 6 CFU ml -1. Unchallenged larvae were used as controls. Larvae for immunohistochemical examination were sampled daily from each group. In most of the larvae, either no or very few bacteria were observed. Typical findings were clusters of bacteria in the spaces between the primary gill lamellae. None of these bacteria seemed to have adhered to the gills. Intestines of 3 out of 161 larvae examined contained positively immunostained bacteria. Some bacteria appeared attached to the microvilli, but none was observed inside epithelial cells. Only 2 larvae from the low-challenge dose group showed clear signs of histopathology, which occurred in the intestine. It is not possible to draw any conclusions regarding the portal of entry. KEY WORDS: Vibrio anguillarum · Cod larvae · Immunohistochemical studies · Challenge experimentResale or republication not permitted without written consent of the publisher OPEN PEN ACCESS CCESS

Three challenge experiments were carried out on larvae of the great scallop Pecten maximus. Larvae were bath-challenged with Vibrio pectenicida and 5 strains resembling Vibrio splendidus and one Pseudoalteromonas sp. Unchallenged larvae were used as negative controls. The challenge protocol was based on the use of a multidish system, where the scallop larvae (10, 13 and 15 d post-hatching in the 3 experiments, respectively) were distributed to 2 ml wells with stagnant seawater and exposed to the bacterial cultures by bath challenge. Presence of the challenge bacteria in the wells was verified by polymerase chain reaction (PCR). A significantly increased mortality was found between 24 and 48 h in most groups challenged with V. pectenicida or V. splendiduslike strains. The exception was found in larval groups challenged with a Pseudoalteromonas sp. LT 13, in which the mortality rate fell in 2 of the 3 challenge experiments. Larvae from the challenge experiments were studied by immunohistochemistry protocol. Examinations of larval groups challenged with V. pectenicida revealed no bacterial cells, despite a high degree of positive immunostaining. In contrast, intact bacterial cells were found in larvae challenged with V. splendidus. In the case of larvae challenged with the Pseudoalteromonas sp., positive immuno-staining was limited to visible bacteria inside the digestive area and cells of the mucosa. The experiments confirm that V. splendidus and V. pectenicida are pathogenic to scallop larvae, and that the Pseudoalteromonas strain is probably not a primary pathogen, although it cannot be ruled out as a secondary pathogen. KEY WORDS: Pecten maximus · Vibrio splendidus · Immunohistochemistry · Challenge experiments · LarvaeResale or republication not permitted without written consent of the publisher OPEN PEN

In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10 6 and 10 4 CFU ml -1. Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 10 6 CFU ml -1, only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.