Atlantic salmon Salmo salar and ballan wrasse Labrus bergylta display different susceptibility to clonal strains of Paramoeba perurans.

Amoebic gill disease (AGD), caused by the marine amoeba Paramoeba perurans, is an important disease of farmed Atlantic salmon Salmo salar L. in Norway. The use of wrasse as cleaner fish in salmon net pens raises questions about interspecies transmission of pathogens such as P. perurans. In this study, cohabitant transmission of clonal isolates of P. perurans between Atlantic salmon and ballan wrasse Labrus bergylta Ascanius was examined, using isolates originating from both salmon and wrasse. The challenges resulted in AGD in both species, although less severely in wrasse. The amoeba isolate originating from ballan wrasse was more virulent than that originating from salmon, suggesting P. perurans strain-related virulence differences. The isolate originating from salmon showed limited proliferation in bath-challenged wrasse and salmon, and limited transfer to cohabitants. Our results support previous observations suggesting that salmon may be more susceptible to P. perurans and AGD than ballan wrasse. Treatment of P. perurans infection in wrasse is challenging, as it is a strictly marine fish species. In this study, brackish water (<15‰ seawater) treatment of AGD affected salmon and wrasse was examined. Both salmon and wrasse were treated for short periods (3 h and 24 h), and treatment of wrasse over longer periods (3-5 d) was also examined. Short exposure to brackish water was not enough to remove P. perurans, although the 24 h treatment reduced amoeba levels. It was not possible to culture or detect P. perurans from wrasse exposed to brackish water for 3 d, suggesting that this treatment would be effective in controlling the parasite.

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.

Prevalence of piscine orthoreovirus and salmonid alphavirus in sea-caught returning adult Atlantic salmon (Salmo salar L.) in northern Norway.

Heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) and pancreas disease (PD) caused by salmonid alphavirus (SAV) are among the most prevalent viral diseases of Atlantic salmon farmed in Norway. There are limited data about the impact of disease in farmed salmon on wild salmon populations. Therefore, the prevalence of PRV and SAV in returning salmon caught in six sea sites was determined using real-time RT-PCR analyses. Of 419 salmon tested, 15.8% tested positive for PRV, while none were positive for SAV. However, scale reading revealed that 10% of the salmon had escaped from farms. The prevalence of PRV in wild salmon (8%) was significantly lower than in farm escapees (86%), and increased with fish length (proxy for age). Sequencing of the S1 gene of PRV from 39 infected fish revealed a mix of genotypes. The observed increase in PRV prevalence with fish age and the lack of phylogeographic structure of the virus could be explained by virus transmission in the feeding areas. Our results highlight the need for studies about the prevalence of PRV and other pathogens in Atlantic salmon in its oceanic phase.

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.

Paranucleospora theridion (Microsporidia) infection dynamics in farmed Atlantic salmon Salmo salar put to sea in spring and autumn.

The microsporidian Paranucleospora theridion (syn. Desmozoon lepeophtheirii) is a parasite of Atlantic salmon Salmo salar and also a hyperparasite of the salmon louse Lepeophtheirus salmonis. The parasite develops 2 types of spores in salmon, cytoplasmic spores in phagocytes and intranuclear spores in epidermal cells. The former type of development is assumed to be propagative (autoinfection), while the epidermal spores transfer the parasite to lice. Development in lice is extensive, with the formation of xenoma-like hypertrophic cells filled with microsporidian spores. We show that salmon are infected in the absence of lice, likely through waterborne spores that initiate infections in the gills. During summer and autumn the parasite propagates in the kidney, as evidenced by peaking normalised expression of P. theridion rRNA. Lice become infected during autumn, and develop extensive infections during winter. Lice mortality in winter and spring is likely responsible for a reservoir of spores in the water. Salmon transferred to sea in November (low temperature) did not show involvement of the kidney in parasite propagation and lice on such fish did not become infected. Apparently, low temperatures inhibit normal P. theridion development in salmon.

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.

Real-time PCR detection of Parvicapsula pseudobranchicola (Myxozoa: Myxosporea) in wild salmonids in Norway.

The myxozoan genus Parvicapsula contains 14 species infecting fish, some of which are known to cause severe disease in farmed and wild salmonids. Parvicapsula pseudobranchicola infections were first reported from seawater-reared Atlantic salmon, Salmo salar, in Norway in 2002 and have since then been an increasing problem. The present study describes a Taqman real-time PCR assay for specific detection of P. pseudobranchicola. The Taqman assay targets the 18S rRNA gene of P. pseudobranchicola and is able to detect as few as ten copies of the target sequence. Using the described assay, P. pseudobranchicola was detected in both farmed and wild salmonids, indicating that wild Atlantic salmon, sea trout, Salmo trutta, and Arctic char, Salvelinus alpinus, may be natural hosts of the parasite. Parvicapsula pseudobranchicola was found in samples from wild salmonids in the far south and the far north of Norway, displaying a wide geographic range of the parasite. Farmed salmonids showed P. pseudobranchicola infection levels many folds higher than that observed for wild sea trout, indicating that farmed Atlantic salmon are subjected to an elevated infection pressure compared with wild salmonids.

Patterns of Ichthyobodo necator sensu stricto infections on hatchery-reared Atlantic salmon Salmo salar in Norway.

Infection patterns with ectoparasitic flagellates belonging to the genus Ichthyobodo were studied in an Atlantic salmon Salmo salar (L.) hatchery in western Norway during an 11 mo period, from eyed eggs to smoltification. Since the earlier species designation Ichthyobodo necator (sensu lato, s.l.) has been shown to represent a complex of several species, the epizootiology of different Ichthyobodo spp. is poorly known. Therefore, we employed molecular methods to ascertain the specific identity of the parasites detected in our study. Only I. necator in the recently redefined and restricted sense occurred (I. necator sensu stricto, s.s.). We observed a 2-peak pattern of infection; the first peak occurred among fry in March and the second peak among fingerlings and pre-smolt in August and September. Skin lesions observed on fingerlings and pre-smolt were significantly associated with Ichthyobodo infections. Also, these infections were negatively correlated with both haematocrit values (Hct) and the condition factor (K) of the fish. The patterns of infection on the farmed salmon suggest that I. necator s.s. is an opportunistic parasite of salmon, flourishing in periods when susceptible hosts are present and the environment favours parasite proliferation. Our study is the first to detect and identify I. necator s.s. on wild-caught adult salmonids (brown trout S. trutta L.). Wild salmonids and sticklebacks Gasterosteus aculeatus (L.) caught in the lakes serving as a water supply to the hatchery were found infected with I. necator s.s., hence these are the likely sources of parasites entering the hatchery via the inlet water.

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.

Elevation of Francisella philomiragia subsp. noatunensis Mikalsen et al. (2007) to Francisella noatunensis comb. nov. [syn. Francisella piscicida Ottem et al. (2008) syn. nov.] and characterization of Francisella noatunensis subsp. orientalis subsp. nov., two important fish pathogens.

AIMS: This study was conducted to clarify the taxonomic status of Francisella sp. strain Ehime-1, a fish pathogen, in relation to the fish pathogens F. piscicida and F. philomiragia subsp. noatunensis and to F. philomiragia subsp. philomiragia. METHODS AND RESULTS: Francisella sp. Ehime-1 was compared to F. piscicida, F. philomiragia subsp. noatunensis and several F. philomiragia subsp. philomiragia isolates through sequencing of the 16S rRNA-gene and several house-keeping genes and determination of biochemical and phenotypic properties. Results show that F. piscicida is indistinguishable from F. philomiragia subsp. noatunensis by sequence and phenotypic traits. Francisella sp. Ehime-1 and F. philomiragia subsp. noatunensis are clearly separated from F. philomiragia. Francisella sp. Ehime-1 is biochemically, phenotypically and genetically different from F. philomiragia subsp. noatunensis (=F. piscicida), but DNA-DNA hybridization does not clearly support establishment as a separate species (level of relatedness 64% and 73.4%, mean 68.7%). CONCLUSIONS: We propose to elevate F. philomiragia subsp. noatunensis to species rank as F. noatunensis comb. nov., while F. piscicida is considered a heterotypic synonym of F. noatunensis comb. nov. Evidence suggests that Francisella sp. Ehime-1 represents a novel subspecies of F. noatunensis, for which the name F. noatunensis subsp. orientalis subsp. nov. is proposed (=DSM21254(T), = LMG24544(T)). SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the taxonomy and characteristics of fish-pathogenic Francisella spp.

Morphogenesis of salmonid gill poxvirus associated with proliferative gill disease in farmed Atlantic salmon (Salmo salar) in Norway.

Proliferative gill disease (PGD) is an emerging problem in Norwegian culture of Atlantic salmon (Salmo salar). Parasites (Ichthyobodo spp.) and bacteria (Flexibacter/Flavobacterium) may cause PGD, but for most cases of PGD in farmed salmon in Norway, no specific pathogen has been identified as the causative agent. However, Neoparamoeba sp. and several bacteria and viruses have been associated with this disease. In the spring of 2006, a new poxvirus, salmon gill poxvirus (SGPV), was discovered on the gills of salmon suffering from PGD in fresh water in northern Norway. Later the same year, this virus was also found on gills of salmon at two marine sites in western Norway. All farms suffered high losses associated with the presence of this virus. In this study, we describe the entry and morphogenesis of the SGP virus in epithelial gill cells from Atlantic salmon. Intracellular mature virions (IMVs) are the only infective particles that seem to be produced. These are spread by cell lysis and by "budding" of virus packages, containing more that 100 IMVs, from the apical surface of infected cells. Entry of the IMVs appears to occur by attachment to microridges on the cell surface and fusion of the viral and cell membranes, delivering the cores into the cytoplasm. The morphogenesis starts with the emergence of crescents in viroplasm foci in perinuclear areas of infected cells. These crescents consist of two tightly apposed unit membranes (each 5 nm thick) that seem to be derived from membranes of the endoplasmic reticulum. The crescents develop into spheres, immature virions (IVs), that are 350 nm in diameter and surrounded by two unit membranes. The maturation of the IVs occurs by condensation of the core material and a change from spherical to boat-shaped particles, intracellular mature virions (IMVs), that are about 300 nm long. Hence, the IMVs from the SGP virus have a different morphology compared to other vertebrate poxviruses that are members of the subfamily Chordopoxvirinae, and they are more similar to members of subfamily Entomopoxvirinae, genus Alphaentomopoxvirus. However, it is premature to make a taxonomic assignment until the genome of the SGP virus has been sequenced, but morphogenesis clearly shows that this virus is a member of family Poxviridae.

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.

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.

Parvicapsula pseudobranchicola (Myxosporea) in farmed Atlantic salmon Salmo salar. Tissue distribution, diagnosis and phylogeny.

Parvicapsula pseudobranchicola infections in farmed Atlantic salmon in Norway are associated with low-grade to significant mortalities. The parasite is found as mature spores in pseudobranchs, but has also been detected in the gills, liver and kidney. Diagnosis has relied on the detection of Parvicapsula spores, with the pseudobranch being the preferred organ. A better understanding of the epizootiology of this myxosporean is a prerequisite for appropriate management and control. Hence, early detection of infections and life cycle studies are needed. We sequenced the small subunit (ssu) rDNA (18S) from P. pseudobranchicola and developed a sensitive diagnostic PCR protocol. This allowed us to (1) identify appropriate tissues for diagnostic assays, (2) examine the intraspecific variation in ssu rDNA in the parasite's Norwegian range, (3) examine annelid potential primary hosts and (4) obtain additional ssu rDNA sequences of marine Parvicapsula species to perform a phylogenetic study. Primers were constructed targeting the ssu rDNA from P. minibicornis. With these we obtained a partial ssu sequence of the P. pseudobranchicola type isolate. A new set of primers (PCF3/PCR3) was constructed for diagnostic purposes. These were tested against DNA from the host and several myxozoan species infecting Norwegian salmon. The primers give a positive product of 203 bp and pick out P. pseudobranchicola in salmnonids. They also amplify the congeners P. unicornis and P. asymmetrica infecting unrelated fish. The PCR protocol developed showed a greater sensitivity than light microscopy. The pseudobranchs were always positive and are the recommended organ for PCR diagnostics. There was no sequence variation between geographic isolates from farmed salmon. Preliminary examinations of marine polychaetes and oligochaetes collected from farm sites with parvicapsulose problems were negative. A comparison of the sequence of the ssu rDNA from P. pseudobranchicola with that of other myxozoans shows that it groups closely together with P. unicornis and P. asymmetrica. The closest relative to this group is P. minibicornis.

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 host's tissues can cause extensive damage to the delicate herring larvae.

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.