Experimental challenge of Atlantic salmon Salmo salar using clones of Paramoeba perurans, P. pemaquidensis and Tetramitus sp.

Salmon gill disease in Norway is in most cases associated with a range of different pathogens, stress and environmental factors. Paramoeba perurans and other amoebae have been isolated during such disease outbreaks. Other amoebae isolated from salmon with gill disease in Norway include P. pemaquidensis, Tetramitus sp. and Vannella sp. Here we tested the pathogenicity of the first 2 species in challenge experiments. We found that even when clonal cultures of P. pemaquidensis established an infection on the gills of salmon, it failed to cause gill disease, while Tetramitus sp. appeared to be unable to establish a lasting infection on the gills of healthy salmon. The result of the challenge with P. pemaquidensis confirms the results of similar studies performed in the USA and in Australia. Tetramitus sp. is probably a common amoeba in the marine environment, and its presence on the gills of farmed salmon may just be accidental. Based on this study, we conclude that P. perurans is the only known amoeba in marine salmon farming associated with amoebic gill disease in Norway.publishedVersio

Growth characteristics and morphology of Paramoeba perurans from Atlantic salmon Salmo salar L. and ballan wrasse Labrus bergylta in Norway

Background Paramoeba perurans is the causative agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar L. and many other farmed marine fish species worldwide. The first cases of AGD in Norway were reported in 2006, and it has subsequently become established as a significant gill disease that affects the country’s salmonid aquaculture industry. Despite several decades of research on AGD, there is still a lack of knowledge of the biology of P. perurans and its interactions with its hosts and the environment. Methods The growth and morphology of 10 clonal isolates of P. perurans were studied. The isolates were from farmed Atlantic salmon and ballan wrasse that had been obtained from different sites along the Norwegian coast between 2013 and 2015. The morphology and population growth patterns of these clonal amoeba isolates were examined in vitro using light microscopy and real-time reverse transcription polymerase chain reaction under a range of temperatures (4, 12, 15 and 21 °C) and salinities (20, 25, 30 and 34 ‰). Results We found distinct morphological differences between both locomotive and floating forms of the amoeba isolates. The locomotive amoebae of the clonal isolates varied in size (area) from 453 µm2 to 802 µm2. There were differences in the growth patterns of the clonal amoeba isolates under similar conditions, and in their responses to variations in temperature and salinity. While most of the isolates grew well at salinities of 25–34 ‰, a significant reduction in growth was seen at 20 ‰. Most of the amoeba isolates grew well at 12 °C and 15 °C. At 4 °C, amoebae grew slower and, in contrast to the other temperatures, no extended pseudopodia could be seen in their floating form. The isolates seemed to reach a plateau phase faster at 21 °C, with a higher number of smaller, rounded amoebae. Conclusions The differences observed here between clonal isolates of P. perurans should be further examined in experimental in vivo challenge studies, as they may be of relevance to the virulence and proliferation potential of this amoeba on gills. Potential differences in virulence within P. perurans could have implications for management strategies for AGD.publishedVersio

Method for cryopreservation of Paramoeba perurans

Paramoeba perurans causes amoebic gill disease (AGD), which is a major problem in aquaculture worldwide. The parasite can be cultured in vitro, but to this date, no method for long-term storage of the clones exists. In this study, we describe a method for cryopreservation of Paramoeba perurans. The method was successfully employed on four out the five clones we tested. The thawing success rate, that is the percentage of successfully thawed vials relative to the total number of vials that were thawed, differed for the clones and ranged from 25% to 100%. The age of the clones seemed to have a negative impact on the ability to survive cryopreservation.publishedVersio

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.publishedVersio

A new aquareovirus causing high mortality in farmed Atlantic halibut fry in Norway

A new aquareovirus was isolated from cultured Atlantic halibut (Hippoglossus hippoglossus) fry at a facility where massive mortalities had occurred during the start-feeding phase. The same virus was also detected in juveniles (about 10 grams) of the 2013 generation at two other production sites, but not in larger fish from generations 2007–2012. The virus replicated in BF-2 and CHSE-214 cell cultures and produced syncytia and plaque-like cytopathic effects. This Atlantic halibut reovirus (AHRV) was associated with necrosis of the liver and pancreas, syncytium formation in these tissues, and distinct viroplasm areas within the syncytium in halibut fry. Transmission electron microscopy revealed that the viroplasm contained virions, non-enveloped, icosahedral particles approximately 70 nm in diameter with a double capsid layer, amorphous material, and tubular structures. The RNA-dependent RNA polymerase (RdRp) gene from the AHRV isolates showed the highest amino acid sequence identity (80 %) to an isolate belonging to the species Aquareovirus A, Atlantic salmon reovirus TS (ASRV-TS). A partial sequence from the putative fusion-associated small transmembrane (FAST) protein of AHRV was obtained, and this sequence showed the highest amino acid sequence identity (46.8 %) to Green River Chinook virus which is an unassigned member of the genus Aquareovirus, while a comparison with isolates belonging to the species Aquareovirus A showed <33 % identity. A proper assessment of the relationship of AHRV to all members of the genus Aquareovirus, however, is hampered by the absence of genetic data from members of several Aquareovirus species. AHRV is the first aquareovirus isolated from a marine coldwater fish species and the second reovirus detected in farmed fish in Norway. A similar disease of halibut fry, as described in this paper, has also been described in halibut production facilities in Canada and Scotland

Experimental challenge of Atlantic salmon Salmo salar using clones of Paramoeba perurans, P. pemaquidensis and Tetramitus sp.

Salmon gill disease in Norway is in most cases associated with a range of different pathogens, stress and environmental factors. Paramoeba perurans and other amoebae have been isolated during such disease outbreaks. Other amoebae isolated from salmon with gill disease in Norway include P. pemaquidensis, Tetramitus sp. and Vannella sp. Here we tested the pathogenicity of the first 2 species in challenge experiments. We found that even when clonal cultures of P. pemaquidensis established an infection on the gills of salmon, it failed to cause gill disease, while Tetramitus sp. appeared to be unable to establish a lasting infection on the gills of healthy salmon. The result of the challenge with P. pemaquidensis confirms the results of similar studies performed in the USA and in Australia. Tetramitus sp. is probably a common amoeba in the marine environment, and its presence on the gills of farmed salmon may just be accidental. Based on this study, we conclude that P. perurans is the only known amoeba in marine salmon farming associated with amoebic gill disease in Norway

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

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

Nutrition as a mean to reduce sea lice infestation in Atlantic salmon

Aller Aqua Norway AS ble i 2017 gjennom en åpen utlysning tildelt midler fra FHF (prosjekt nr 901458) for å dokumentere effekten av mineralingrediensen Biofeed Forte (BF) mot lusesmitte på laks. Forsøk på regnbueørret ved Aller Aqua sin FoU-konsesjon Floteneset (Seamatech AS) har i 2 produksjoner 2015-2018 indikert effekter av BF-fôr på lusetall av hunnlus. Det er utført tilsvarende forsøk på laks ved lokaliteten Leland (Fylkesnes Fisk AS) i 2017, men effekter av BF-fôret på lusetall kunne ikke påvises i dette forsøket. Formålet med prosjektet var derfor å gjøre endringer på ingrediensen slik at den gav bedre virkning mot lus på laks. Aller Aqua Norway AS har vært prosjektansvarlig, og stått for avgjørelser i prosjektet. Dette gjelder valg av mineralingredienser til uttesting i forsøk, godkjenning av fôrresepter og forsøksdesign. Nofima har vært FoU-ansvarlig og stått for planlegging av fôringsforsøk med laks ved Ilab og småmerdforsøk ved Seamatech sitt anlegg ved Vadheim. Til disse forsøkene ble det brukt en mineralingrediens valgt av Aller Aqua ut fra in vitro forsøk ved Ilab, tilsatt etter anbefalinger 4 % i forsøksfôret produsert ved Nofimas Fôrteknologisenter. Nofima-produsert BF-fôr viste seg høst 2018 å inneholde verdier av jod (1200 mg/kg) over øvre tillatte grenseverdi (20 mg/kg) grunnet en blandingsfeil av mineralingrediensen Biofeed Forte gjort av produsenten, Pharmatech AS. Nofima-produsert kontrollfôr uten mineralingrediensen hadde et jodinnhold på 7 mg/kg. Effekter observert i karforsøk ved Ilab og småmerdforsøk Vadheim 2018 er derfor ikke kommersielt relevant, da de observerte effektene ikke kan tilskrives jodinnholdet eller øvrige komponenter i mineralingrediensen. Til tross for dette ble det observert interessante funn på fisk fôret med BF-fôr på lusetall og skinnets morfologi/fysiologi som diskuteres i rapporten. Disse funnene danner grunnlag for videre studier for å forstå effekter av fôringredienser som kan påvirke skinnhelse og redusere lusetall hos laks og regnbueørret.Forebyggende ernæring mot lus på laks - forsøk og dokumentasjon på betydning av samvirkning mellom aktive tilsetninger og grunnfôr. Faglig delrapportNutrition as a mean to reduce sea lice infestation in Atlantic salmonpublishedVersio