Egil Karlsbakk

Paranucleospora theridion n. gen., n. sp. (Microsporidia, Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis, Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.

A NEW GENUS GADIMYXA WITH THREE NEW SPECIES (MYXOZOA, PARVICAPSULIDAE) PARASITIC IN MARINE FISH (GADIDAE) AND THE TWO-HOST LIFE CYCLE OF GADIMYXA ATLANTICA N. SP

The myxozoans Gadimyxa atlantica n. sp. and G. sphaerica n. sp., and G. arctica n. sp. (Myxozoa, Parvicapsulidae), are described from Gadus morhua L. and Arctogadus glacialis (Peters) (Gadidae), respectively. They develop coelozoic in bisporic plasmodia in the urinary systems. Two morphological forms of spores were found in all 3 species, i.e., wide and (sub)spherical forms. Both spore types are bilaterally symmetrical along the suture line. The wide spores, semicircular in frontal view and elliptical in apical view, have 2 spherical polar capsules, which open in the sutural or median plane mid on the flat side of the spore. Mean widths of the wide spores of G. atlantica, G. sphaerica, and G. arctica are 7.5, 10.0, and 10.0 μm, respectively. The older, more thick-walled, (sub)spherical spores with binucleate sporoplasm are 8.0, 5.3, and 7.3 μm in mean width, respectively. The mean diameters of the polar capsules of (sub)spherical spores are 2.4, 1.7, and 2.2 μm, respectively. The (sub)spherical forms of Gadimyxa are most similar to Ortholinea within the Ortholineidae, but they differ in the development of the spores and in the arrangement of the polar capsules. The polychaetes Spirorbis spp. (Spirorbidae) act as invertebrate hosts of G. atlantica. The previously described actinospores of the tetractinomyxon type develop to myxospores in Gadus morhua within 8 wk. This is the second known myxozoan 2-host life cycle in the marine environment. Phylogenetic analyses based on partial small subunit rDNA sequences places Gadimyxa spp. among Parvicapsula spp. in the Parvicapsulidae.

Occurrence of Francisella piscicida in farmed and wild Atlantic cod, Gadus morhua L., in Norway

Francisellosis, caused by the bacterium Francisella piscicida, has become one of the most serious diseases in Atlantic cod production in Norway. The major aim of this study was to determine the distribution of F. piscicida in farmed and wild fish in areas with cod farming along the Norwegian coast, and its occurrence in cod from areas without cod farming. Two real-time PCR assays, targeting the 16S rRNA gene and the FopA gene of F. piscicida, were developed since sensitive and specific diagnostic tools are required for detecting asymptomatic carriers of the bacterium. A total of 422 wild cod from 13 sampling areas and 955 farmed cod from 10 areas along the coast of Norway were examined. Using the real-time polymerase chain reaction (PCR) assays, F. piscicida was detected in wild populations of cod from all counties examined south of Sogn og Fjordane in southern Norway (overall prevalence 13%, n = 221). Wild cod north of Sogn og Fjordane were negative for the bacterium (n = 201). Farmed cod from most parts of Norway were F. piscicida positive. The apparent absence of the bacterium in wild populations of cod in the northern parts of Norway and its widespread occurrence in wild cod from southern parts of Norway is believed to relate to differences in seawater temperatures.

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.

Growth and emigration of third-stage larvae of Hysterothylacium aduncum (Nematoda : Anisakidae) in larval herring Clupea harengus

The growth and emigration of Hystherothylacium aduncum in laboratory-reared herring larvae Clupea harengus was studied. Experimental infections of 36-day-old herring larvae resulted in 126 hosts infected with 306 H. aduncum larvae. Regression analyses showed a significant worm emigration from the rectum to the head of the fish, accompanied by an increase in worm body length. The emigration was independent of worm intensity, which suggests an ontogenetic process. Some worms departed from this pattern by moving posteriorly or by penetrating into the muscle, and in 5 cases, the larvae were observed to leave living fish. This individual variation has not been observed in previous studies and might be explained by host signals related to condition or development stage. Indirect evidence suggested parasite-induced mortality in the tanks due to the emigrations because only 4 of the 126 infected fish survived 8 days postinfection; the emigration of H. aduncum affected vital organs, such as the heart and brain, and the larvae penetrating or leaving the hosts tissues can cause extensive damage to the delicate herring larvae.

New clade of betanodaviruses detected in wild and farmed cod ( Gadus morhua ) in Norway

Betanodaviruses have been isolated and detected in both farmed and wild fish species worldwide. They are classified in five clusters, and all are connected to mortalities in farmed fish. The clusters do not represent specific geographical areas or host species, but one cluster, barfin flounder nervous necrosis virus (BFNNV), is mainly associated with cold water fish species. This study presents the first species-specific clade within the BFNNV cluster. This clade consists of six isolates from wild and farmed Atlantic cod in Norway and is genetically distinct from other betanodaviruses in the North Atlantic. Screening of farmed and wild cod in Norway shows that betanodaviruses are present in wild fish on the west coast of Norway, including migratory cod, but so far we have not detected any betanodavirus-positive wild cod in northern Norway. The presence of significant amounts of betanodaviruses in wild cod represents a serious challenge for the management of viral nervous necrosis in farmed cod in Norway. Betanodavirus-positive farmed cod were present both in western and northern Norway. Mortalities in three cod farms were suspected to be caused by betanodaviruses; however, in two of these, other pathogens may have been responsible for or strongly contributed to the mortalities.

New data on the early development of Hysterothylacium aduncum (Nematoda, Anisakidae).

This note reports on incidental observations of the early development of the third-stage larvae of Hysterothylacium aduncum from gadid fishes. Gravid H. aduncum females were collected from Pollachius virens, Pollachius pollachius. Gadus morhua, and Molva molva in Norwegian waters. The eggs were incubated at 20 per thousand salinity and 5 C. Spontaneous hatching of third-stage larvae was observed 10-25 days after egg deposition. These larvae were long lived and could infect Acartia tonsa copepods, the infections being maintained for up to 34 days. The morphology of the third-stage larvae in the copepods and some traits of the life cycle were similar to those reported in previous studies. However, our results disagree with evidence suggesting that H. aduncum eggs rarely hatch, and hatched larvae have lower survival and a poorer ability to infect the first intermediate host than unhatched ones. It is difficult to account for these discrepancies because information on the early development of Hysterothylacium species is incomplete. However, we tentatively suggest that differences in the early development of H. aduncum may indicate the existence of at least 2 different taxonomic entities in the North Atlantic, which is consistent with previous evidence based on morphological traits.

Potential disease interaction reinforced: double-virus-infected escaped farmed Atlantic salmon, Salmo salar L., recaptured in a nearby river

The role of escaped farmed salmon in spreading infectious agents from aquaculture to wild salmonid populations is largely unknown. This is a case study of potential disease interaction between escaped farmed and wild fish populations. In summer 2012, significant numbers of farmed Atlantic salmon were captured in the Hardangerfjord and in a local river. Genetic analyses of 59 of the escaped salmon and samples collected from six local salmon farms pointed out the most likely source farm, but two other farms had an overlapping genetic profile. The escapees were also analysed for three viruses that are prevalent in fish farming in Norway. Almost all the escaped salmon were infected with salmon alphavirus (SAV) and piscine reovirus (PRV). To use the infection profile to assist genetic methods in identifying the likely farm of origin, samples from the farms were also tested for these viruses. However, in the current case, all the three farms had an infection profile that was similar to that of the escapees. We have shown that double-virus-infected escaped salmon ascend a river close to the likely source farms, reinforcing the potential for spread of viruses to wild salmonids.

'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.

Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

Atlantic cod, Gadus morhua, averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2(-DeltaDeltaCt) method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.