Heidrun Plarre

Transmission of infectious salmon anaemia virus (ISAV) in farmed populations of Atlantic salmon (Salmo salar)

Summary.In the present study, 24 smolt production sites were screened for the presence of infectious salmon anaemia virus (ISAV) with the help of a specific real-time RT PCR assay, and 22 of these sites had smolts that were positive. If these smolt production sites are representative for the prevalence of ISAV in Norwegian smolts, then most marine production sites must be considered to be positive for ISAV. In addition, 92 European ISAV isolates have been genotyped based on the hemagglutinin-esterase gene (HE), and their distribution pattern was analysed. This pattern has been coupled to information about the origin of smolt, eggs, and broodfish in those cases where it has been possible to obtain such information, and with information about ISAV in neighbouring farms. The pattern suggests that an important transmission route for the ISAV could be that the salmon farming industry in Norway is circulating some of the isolates in the production cycle, i.e. some sort of vertical or transgenerational transmission may occur. It has also been shown that avirluent ISAV isolates are fairly common in Norwegian farmed salmon. Based on this, it is hypothesized that the change from avirulent to virulent ISAV isolates is a stochastic event that is dependent on the replication frequency of the virus and the time available for changes in a highly polymorphic region (HPR) of the HE gene to occur. This, and the possibility that only avirluent ISAV isolates are vertically transmitted, may explain why ISA most often occurs at marine sites and why no more than about 15 farms get ISA every year in Norway.

Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.

'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.

Evolution of infectious salmon anaemia virus (ISA virus)

Infectious salmon anaemia virus, ISA virus (genus Isavirus, family Orthomyxoviridae), emerged in Norwegian salmon culture in the mid-80s. The genome consists of eight segments coding for at least 10 proteins. ISA viruses show many of similarities to influenza A viruses but differ in many important aspects such as the number of hosts, the host population structure and the route of transmission. The only known hosts and reservoirs for ISA viruses are salmonids found in countries surrounding the North Atlantic. In this study, four different segments of the genome of about 100 ISA viruses have been sequenced in an attempt to understand the evolution of ISA viruses and how these viruses are maintained in and transmitted between populations of farmed Atlantic salmon. The four gene segments code for the nucleoprotein (NP), the putative acid polymerase (PA), the fusion protein (F) and the haemagglutinin-esterase (HE). Analysis of these four genes showed that the substitution rates of the internal proteins (NP and PA) are lower than those of the two surface proteins (F and HE). All four segments are evolving at a lower rate than similar genes in influenza A viruses. The ISA virus populations consist of avirulent viruses and pathogenic strains with variable virulence in Atlantic salmon. Recombination resulting in inserts close to the proteolytic-cleavage site of the precursor F0 protein and deletions in the stalk region of the HE protein seem to be responsible for the transition from avirulent ISA viruses to pathogenic strains. It is also shown that reassortment is a frequent event among the dominating ISA viruses in farmed Atlantic salmon. The pattern that is obtained after phylogenetic analysis of the four gene segments from ISA viruses suggests that the variation is limited to a few distinct clades and that no major changes have occurred in the ISA virus population in Norway since the first viruses were isolated. Calculation of the time of most recent common ancestor (TMRCA) suggests that the Norwegian ISA viruses separated from the European subtype found in North America between 1932 and 1959. The TMRCA data also suggest that the ISA viruses in Chile were transmitted from Norway in the period from 1995 to 2007, depending on which of the four genes were used in the analysis.

Genomic characterization and phylogenetic position of two new species in Rhabdoviridae infecting the parasitic copepod, salmon louse (Lepeophtheirus salmonis).

Several new viruses have emerged during farming of salmonids in the North Atlantic causing large losses to the industry. Still the blood feeding copepod parasite, Lepeophtheirus salmonis, remains the major challenge for the industry. Histological examinations of this parasite have revealed the presence of several virus-like particles including some with morphologies similar to rhabdoviruses. This study is the first description of the genome and target tissues of two new species of rhabdoviruses associated with pathology in the salmon louse. Salmon lice were collected at different Atlantic salmon (Salmo salar) farming sites on the west coast of Norway and prepared for histology, transmission electron microscopy and Illumina sequencing of the complete RNA extracted from these lice. The nearly complete genomes, around 11 600 nucleotides encoding the five typical rhabdovirus genes N, P, M, G and L, of two new species were obtained. The genome sequences, the putative protein sequences, and predicted transcription strategies for the two viruses are presented. Phylogenetic analyses of the putative N and L proteins indicated closest similarity to the Sigmavirus/Dimarhabdoviruses cluster, however, the genomes of both new viruses are significantly diverged with no close affinity to any of the existing rhabdovirus genera. In situ hybridization, targeting the N protein genes, showed that the viruses were present in the same glandular tissues as the observed rhabdovirus-like particles. Both viruses were present in all developmental stages of the salmon louse, and associated with necrosis of glandular tissues in adult lice. As the two viruses were present in eggs and free-living planktonic stages of the salmon louse vertical, transmission of the viruses are suggested. The tissues of the lice host, Atlantic salmon, with the exception of skin at the attachment site for the salmon louse chalimi stages, were negative for these two viruses.

Genotyping of Candidatus Syngnamydia salmonis (chlamydiales; Simkaniaceae) co-cultured in Paramoeba perurans (amoebozoa; Paramoebidae)

Candidatus Syngnamydia salmonis (Chlamydiales, Simkaniaceae) was described as an epitheliocystis-causing bacterium from the gills of Atlantic salmon (Salmo salar) in Norway. A bacterium showing 99.2% 16S rRNA identity to Cand. S. salmonis is able to multiply in Paramoeba perurans and based on the classification criteria this bacterium could represent the same species as Cand. S. salmonis. Sequencing the genome of the cultured bacterium has made it possible to fulfill the minimal standards for genetic characterization of species within the order Chlamydiales. The complete rRNA genes, the amino acid sequences of SucA, PepF, Adk, HemL, DnaA, FtsK and FabI, are presented in addition to the morphology of the Chlamydia-like morphs in the cytoplasm of P. perurans.

Presence of selected pathogens on the gills of five wrasse species in western Norway

The objective of this study was to identify gill pathogens in Labridae (wrasse) species used as cleaner fish to control salmon louse in western Norwegian aquaculture. Wrasse are often moved over long distances, raising issues of fish health, welfare and pathogen transmission. Histological examination and real-time RT-PCR analysis of the gills from Centrolabrus exoletus, Ctenolabrus rupestris, Labrus bergylta, L. mixtus and Symphodus melops revealed several pathogens: a new species of Ichthyobodo, Paramoeba perurans, microsporidia, trichodinids, Hatschekia spp., Candidatus Similichlamydia labri and 2 putative new species of Chlamydiae. Cand. S. labri or closely related bacteria were present on most wrasse specimens. Epitheliocysts on the gills of L. mixtus contained large inclusions (120 µm) with actiniae radiating from the inclusion membrane. A possible member of the Candidatus family Parilichlamydiaceae was present at a high prevalence on the gills of L. mixtus, L. bergylta and C. rupestris. Sequencing the 16S rRNA gene showed 93.9% similarity to Cand. S. labri and 96.8% similarity to Cand. Parilichlamydia carangidicola from the gills of Seriola lalandi. This bacterium probably represents a new species within the order Chlamydiales, family Cand. Parilichlamydiaceae. The other Chlamydiae detected on gills of S. melops could represent a new species in Cand. genus Syngnamydia. Ichthyobodo sp. and Paranucleospora theridion were detected on the gills of nearly all individuals, while Paramoeba spp. were detected on the gills of L. bergylta and L. mixtus. Trichodinids, microsporidia and parasitic copepods had low prevalence. Viral haemorrhagic septicaemia virus was not detected.