First identification of mycobacteriosis in Atlantic mackerel (Scomber scombrus).

Mycobacterium infection in fish is a well-known disease problem globally, mainly in the farming of ornamental fish or fish for food. Less is known about the prevalence, distribution and the effects such infections have on wild fish species. Presumptive mycobacteriosis has previously been observed in Atlantic mackerel (Scomber scombrus). Since 2018, there has been an increase in reports of granulomatous kidney disease in Atlantic mackerel with the suspicion of this being mycobacteriosis. A total of six individuals were sent to the Institute of Marine Research for further examination. They were caught in the Nordic Sea by either commercial fishing vessels or during the International Ecosystem Summer Survey in the Nordic Seas (IESSNS research cruise) between 2018 and 2020. Samples for both histological and molecular analysis were collected. Here, we detect a likely novel Mycobacterium species in tissue samples from Atlantic mackerel with this condition, on the basis of rDNA and protein gene sequences. The same unnamed bacterium seems to have been found in some Pacific marine fishes. The macroscopic and histological manifestation of the disease is described. Over the past years, there has been an increase in reports of mycobacteriosis worldwide and climate change has been suggested as one of the driving forces as these bacteria prefer warm water.

Infection cycle of Marteilia pararefringens in blue mussels Mytilus edulis in a heliothermic marine oyster lagoon in Norway.

Agapollen is a traditional heliothermic marine oyster lagoon in western Norway, representing the northernmost site of any Marteilia sp. protists detected in Europe. The semi-closed lagoon is a unique site to study the life cycle and development of M. pararefringens in naïve mussels. Two baskets with uninfected mussels were deployed in the lagoon outlet in May and October 2018, respectively, and sampled every 6 wk. The parasite was first detected in the mussels by PCR in early July and by histology in late August. By then, M. pararefringens had developed into mature stages, indicating a rapid development during mid-summer. Sporulation occurred during autumn. Mussels deployed in October never became infected, indicating that transmission was restricted to the warmest period of the year. Pronounced pathology was observed in infected mussels, including degenerated digestive tubules and infiltration of haemocytes. Mussel mortality was observed in the baskets, but whether this was due to infections of M. pararefringens or other environmental factors could not be determined. Plankton samples from the lagoon were also collected for PCR analysis. These samples, dominated by copepods, were positive for M. pararefringens in summer. In sorted samples, M. pararefringens was detected in the Acartia spp. and Paracartia grani fractions between July and October. These plankton copepods are therefore potentially involved in the life cycle of M. pararefringens.

High nervous necrosis virus (NNV) diversity in wild wrasse (Labridae) in Norway and Sweden.

Wild goldsinny wrasse Ctenolabrus rupestris, corkwing wrasse Symphodus melops and ballan wrasse Labrus bergylta were collected at 8 sampling sites in Sweden and Norway during summer 2014. Brain tissue from 466 wrasses were analyzed for nervous necrosis virus (NNV) infections by real-time RT-PCR, and positive samples were subjected to sequencing and phylogenetic analysis of partial segments of the RNA2 and RNA1 genes. This study shows that NNV is present in wild ballan, corkwing and goldsinny wrasse along the coastline of Sweden and Norway. The overall prevalence in the sampled labrids was 6.7%. Prevalence was 6.4% in goldsinny, 6.3% in corkwing and 18% in ballan wrasse. The wrasse RNA2 NNV sequences revealed high genetic variability and were divided into 3 clusters within the cold water barfin flounder NNV (BFNNV) and warm water cluster red-spotted grouper NNV (RGNNV) genogroups. Within the BFNNV genogroup, wrasse NNVs clustered in 2 sub-genogroups, with grey mullet NNV (GMNNV) and with Atlantic halibut NNV (AHNNV). These groups were previously dominated by virus originating from Atlantic cod Gadus morhua and Atlantic halibut Hippoglossus hippoglossus from the northeast Atlantic. The presence of NNV in wild wrasse and the surprising high genetic variability observed in this study should be considered before moving wild-caught wrasse between geographically distant sites. The results show that use of wild-caught wrasse as brood fish in wrasse farming represents a risk of introducing NNV into aquaculture.

Relationship between viral dose and outcome of infection in Atlantic salmon, Salmo salar L., post-smolts bath-challenged with salmonid alphavirus subtype 3.

Salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) and adversely affects salmonid aquaculture in Europe. A better understanding of disease transmission is currently needed in order to manage PD outbreaks. Here, we demonstrate the relationship between viral dose and the outcome of SAV3 infection in Atlantic salmon post-smolts using a bath challenge model. Fish were challenged at 12 °C with 3 different SAV3 doses; 139, 27 and 7 TCID50 L-1 of seawater. A dose of as little as 7 TCID50 L-1 of seawater was able to induce SAV3 infection in the challenged population with a substantial level of variation between replicate tanks and, therefore, likely represents a dose close to the minimum dose required to establish an infection in a population. These data also confirm the highly infectious nature of SAV through horizontal transmission. The outcome of SAV3 infection, evaluated by the prevalence of viraemic fish, SAV3-positive hearts, and the virus shedding rate, was positively correlated to the original SAV3 dose. A maximal shedding rate of 2.4 × 104 TCID50 L-1 of seawater h-1 kg-1 was recorded 10 days post-exposure (dpe) from the highest dose group. The method reported here, for the quantification of infectious SAV3 in seawater, could be useful to monitor PD status or obtain data from SAV3 outbreaks at field locations. This information could be incorporated into pathogen dispersal models to improve risk assessment and to better understand how SAV3 spreads between farms during outbreaks. This information may also provide new insights into the control and mitigation of PD.

Summer mortalities and detection of ostreid herpesvirus microvariant in Pacific oyster Crassostrea gigas in Sweden and Norway.

The Pacific oyster Crassostrea gigas has recently expanded its range in Scandinavia. The expansion is presumably a result of northwards larval drift. Massive settlements were recorded in many areas along the Swedish west coast and southern Norway in 2013 and 2014. After the spawning season in 2014, the temperature of the surface water peaked at 24-26°C. After this period, high and sudden mortalities occurred in a Swedish hatchery and in wild populations along the Swedish west coast and south coast of Norway. Surveys and collected data showed that mortalities mainly occurred during 3 wk in September. All size classes were affected, and affected populations displayed a patchy distribution with heavily affected and unaffected populations in close proximity. Flat oysters Ostrea edulis and blue mussels Mytilus edulis were unaffected. Ostreid herpesvirus (OsHV) was detected in moribund Pacific oyster spat as well as in surviving adults. The virus was identified as OsHV-1 µvar. This is the first detection of this variant in Scandinavia, showing that OsHV-1 µvar is present in areas with recent establishments of Pacific oysters, and where there is no aquaculture of this species.

'Cand. Actinochlamydia clariae' gen. nov., sp. nov., a unique intracellular bacterium causing epitheliocystis in catfish (Clarias gariepinus) in Uganda.

BACKGROUND AND OBJECTIVES: Epitheliocystis, caused by bacteria infecting gill epithelial cells in fish, is common among a large range of fish species in both fresh- and seawater. The aquaculture industry considers epitheliocystis an important problem. It affects the welfare of the fish and the resulting gill disease may lead to mortalities. In a culture facility in Kampala, Uganda, juveniles of the African sharptooth catfish (Clarias gariepinus) was observed swimming in the surface, sometimes belly up, showing signs of respiratory problems. Histological examination of gill tissues from this fish revealed large amounts of epitheliocysts, and also presence of a few Ichthyobodo sp. and Trichodina sp. METHODS AND RESULTS: Sequencing of the epitheliocystis bacterium 16S rRNA gene shows 86.3% similarity with Candidatus Piscichlamydia salmonis causing epitheliocystis in Atlantic salmon (Salmo salar). Transmission electron microscopy showed that the morphology of the developmental stages of the bacterium is similar to that of members of the family Chlamydiaceae. The similarity of the bacterium rRNA gene sequences compared with other chlamydia-like bacteria ranged between 80.5% and 86.3%. Inclusions containing this new bacterium have tubules/channels (termed actinae) that are radiating from the inclusion membrane and opening on the cell surface or in neighbouring cells. CONCLUSIONS: Radiation of tubules/channels (actinae) from the inclusion membrane has never been described in any of the other members of Chlamydiales. It seems to be a completely new character and an apomorphy. We propose the name Candidatus Actinochlamydia clariae gen. nov., sp. nov. (Actinochlamydiaceae fam. nov., order Chlamydiales, phylum Chlamydiae) for this new agent causing epitheliocystis in African sharptooth catfish.

Vibrio tapetis-like strain isolated from introduced Manila clams Ruditapes philippinarum showing symptoms of brown ring disease in Norway.

The Manila clam Ruditapes philippinarum was introduced to Norway in 1987 and was produced in 2 hatcheries until 1991. Clam seed was planted at 6 sites. Two sites were on the Island of Tysnes, south of Bergen. Surviving adult Manila clams were recovered in 1995 and 1996. In the present study, Manila clams from the original seeding that displayed morphological signs of brown ring disease (BRD) were recovered in June 2003 (n=7) and in June 2004 (n=17). Samples from extrapallial fluid, tissues and haemolymph were inoculated on marine agar. Replicate subcultures on selective media were used to select potential Vibrio tapetis strains, and in total, 190 bacterial strains were isolated. One of these strains clustered within the V tapetis clade and was named NRP 45. DNA:DNA hybridisation with the type strain CECT4600 showed 52.7 and 57.3% DNA:DNA similarity. Hybridisation of NRP 45 and the V tapetis LP2 strain, isolated from corkwing wrasse Symphodus melops, produced 46.6 and 44.4% re-association. Partial gene segments encoding 16S rRNA, gyrase B protein (GyrB) and chaperonin 60 protein (Cpn60) were characterised and compared to CECT 4600. NRP 45 showed 5 differences in the 1416 nucleotides (nt) of the 16S rRNA encoding gene (99.6% similarity), while the GyrB encoding gene had 62 substitutions of 1181 nt compared (94.8% similarity) and the Cpn60 encoding gene had 22 substitutions out of 548 nt compared (96% similarity). This is the first finding of BRD and the first isolation of a V. tapetis-like bacterial strain from a bivalve in Norway.

Fate of infectious salmon anaemia virus (ISAV) in experimentally challenged blue mussels Mytilus edulis.

In order to investigate the potential role of blue mussels Mytilus edulis as a vector of the fish pathogenic infectious salmon anaemia virus (ISAV), we developed an experimental bioaccumulation system in which mussels can accumulate virus during normal filtration. Detection of virus in mussels was performed by means of real-time RT-PCR. ISAV-RNA was detected in the mussels until 72 h post-challenge. Hepatopancreas homogenate from experimentally challenged mussels was injected into salmon. All the fish injected with homogenate prepared immediately after accumulations were strongly ISAV positive 4 wk post-challenge. In the group injected with homogenate prepared 24 h after the challenge, 1 fish out of 25 was weakly ISAV positive. All of the fish that were challenged with mussel homogenate prepared 96 h after accumulation were ISAV negative. Mussels sampled from a tank with experimentally infected salmon demonstrating clinical signs consistent with ISA (infectious salmon anaemia) and mussels collected on net pen cages during ISA outbreaks in Atlantic salmon were all ISAV negative. The results indicate that the ISAV is rapidly inactivated in mussels and that mussels are not a likely reservoir host or vector for ISAV.

Characterisation and subcellular localisation of the GroEL-like and DnaK-like proteins isolated from Fusobacterium nucleatum ATCC 10953.

Fusobacterium nucleatum is associated with periodontitis in humans, and is a central member of the dental biofilm. Heat shock proteins (HSPs) of many different bacteria have been considered to play important roles during inflammations and infections. We have identified and characterised the HSP60 and HSP70, the Escherichia coli GroEL and DnaK homologues, respectively, in F. nucleatum ATCC 10953. The N-terminal 22 amino acid residues of HSP60 exhibited up to 63.6% identity with members of the HSP60 heat shock protein family of some selected bacterial species, while the N-terminal of 25 residues of HSP70 revealed up to 80% identity with members of the HSP70 family. The subcellular localisation of HSP60 and HSP70 was analysed by immunoblotting of bacterial cell fractions and immunoelectron microscopy of whole cells. HSP60 and HSP70 were localised in the cytosol, associated with membranes and extracellular fractions. These results are consistent with localisation for HSPs found in other micro-organisms, which further lead to the suggestion of a potential role in the pathogenesis of infectious diseases.