Exophiala angulospora infection in hatchery‐reared lumpfish (Cyclopterus lumpus) broodstock

Samples from moribund lumpfish were collected in a marine hatchery in Scotland in 2015. Black nodules were noted on the skin, and gills and fungal hyphae were extensively distributed in musculature and internal organs. Multifocal chronic inflammatory lesions displaced structures in all affected organs. Mortalities commenced on completion of spawning in May and were evenly distributed over the second year in the temperature range 11–15°C. The main systemic infection causing agent was initially identified based on morphological characteristics as an Exophiala species. Ribosomal DNA (rDNA) ITS regions of the isolates were subsequently sequenced confirming the isolates belonged to Exophiala genus. All isolates fell in a single phylogenetic cluster, which is represented by Exophiala angulospora. Fish were treated with either formalin or Bronopol or a combination of both, but there was no effect on the pattern or numbers of mortalities. Isolates were also tested against three different concentrations of Latrunculin A, Amphotericin B and Itraconazole with no success. It is of utmost importance to increase the knowledge on pathogen–host interactions to successfully develop sustainable control methods.     

Piscirickettsia salmonis infection in cultured lumpfish (Cyclopterus lumpus L.)

A Piscirickettsia salmonis infection was diagnosed in lumpfish (Cyclopterus lumpus L.) juveniles held in a marine research facility on the west coast of Ireland. The main clinical signs and pathology included marked ascites, severe multifocal liver necrosis and severe diffuse inflammation and necrosis of the exocrine pancreas and peri‐pancreatic adipose tissue. Numerous Piscirickettsia‐like organisms were observed by histopathology in the affected organs, and the bacterial species was characterized by molecular analysis. Sequencing of the partial 16S rDNA gene and internal transcribed spacer region showed the lumpfish sequences to be closely related to previously identified Atlantic salmon (Salmo salar L.) sequences from Ireland. To the authors’ knowledge, this is the first detection of P. salmonis in lumpfish worldwide. The infection is considered potentially significant in terms of lumpfish health and biosecurity.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Pathogenicity of Pasteurella sp. in lumpsuckers (Cyclopterus lumpus L.)

The incidence of disease caused by Pasteurella sp. in farmed lumpsuckers in Norway has been steadily increasing in recent years, causing significant economic losses and fish welfare issues. The disease affects all life stages, both in hatcheries and after release into salmon cages. Therefore, it is important to establish robust challenge models, to be used for vaccine development. Exposure experiments via intramuscular and intraperitoneal injection underlined the high virulence of the bacteria, whereas the cohabitation and bath models allowed the chronic symptoms of the disease to be studied more accurately. Skin lesions and haemorrhage at the base of fins were observed in the more acute cases of the disease. Symptoms including white spots over the skin, especially around the eyes, characterized the chronic cases. The latter were most prominent from the bath challenge model. Histopathology indicated a systemic pattern of disease, whereas qPCR analysis from head kidney showed that bacteria may be present in survivor fish at the end of the challenges. In all the challenge models investigated, Pasteurella sp. was re‐isolated from the fish, thus fulfilling Koch's postulates. These findings highlight the importance of screening of lumpsuckers prior to transfer to minimize the risks of carrying over asymptomatic carriers.     

Feeding behaviour and growth of lumpfish (Cyclopterus lumpus L.) fed with feed blocks

The aim of this project was to investigate if lumpfish can be fed using specially designed feed blocks instead of regular fish feed pellets. Two studies were performed. In Part I different designs of feed blocks were introduced and fish observed with underwater cameras to record feeding behaviour. Results indicate that lumpfish require feed blocks with grooves in order to graze from them and that the acclimation period is relatively short (2–4 hr) before the fish will use them as a feed source. In the second part of the project two duplicate groups of lumpfish with an initial mean (±SD) weight of 125.4 ± 45.7 g were individually weighed and randomly distributed into six 3.5 m³ circular flow‐through tanks with 45 fish in each tank. Fish in three tanks were fed using feed blocks with grooves and fish in three tanks were fed using a regular commercially available lumpfish extruded feed. Both groups received a daily feeding rate of 2% body/weight. From day 14 onwards, fish fed with marine pelleted feed had a significantly higher mean weight compared to fish fed with feed blocks. Although not significant, the condition factor was higher in the feed block group during the study period. Results from this study show that lumpfish will readily graze from feed blocks if they are presented in a way that allows them to do. In addition, the acclimation period required before the fish will utilize them appears to be short thus potentially allowing for their use in commercial salmon cages.     

Tolerance of lumpfish,Cyclopterus lumpus,to freshwater bath treatment for amoebic gill disease,Neoparamoeba perurans,infection and efficacy of different treatment regimens

The tolerance of lumpfish to freshwater treatment and efficacy over a range of salinities and treatment durations is examined. No structural and cellular changes were noted in organs, including gills and gut, sampled 48 hr posttreatment because of freshwater baths for periods of up to 5 hr exposure at 0 ppt or continuous treatment in 15 ppt water for 10 days. Behavioral indicators such as activity and ventilation frequency were also not significantly different between treatments, with the exception of the first trial where lumpfish were inactive during treatment in 0 ppt water compared with active fish in 32 ppt seawater. Amoeba numbers declined from an initial 21.5/gill arch to 0.33 after 3 hr in freshwater and to zero after 5 hr, and to five amoebas/gill arch after 3 hr in brackish water treatment (15 ppt). When fish were treated continuously for 15 days in 15 ppt water, amoeba numbers declined from 231 to 3.7/gill arch after 3 days and a mean of 1.75 amoebae after 10 days. In the last trial, no amoebas were present per gill arch after 3 hr treatment in 0 and 3 ppt bath.     

Vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) at a low temperature leads to a low antibody response against Aeromonas salmonicida

We present a study on the effect of water temperature on immunization of Atlantic lumpfish. In total, 360 fish were vaccinated with either 50 μl of an oil‐based injection vaccine (VAX), with Aeromonas salmonicida and Vibrio salmonicida antigens, or PBS. Fish were vaccinated at three different water temperatures, 5°C, 10°C and 15°C, and sorted into six groups (N = 60). Lumpfish were weighed every 3 weeks after vaccination, sampled at 3, 6, 9 and 18 weeks post‐immunization (wpi) and evaluated by modified Speilberg score, ELISA and immunoblotting. Vaccinated fish showed low antibody response against V. salmonicida. Fish vaccinated at 5°C showed significantly lower antibody response against A. salmonicida throughout the study. At higher temperatures, vaccinated fish showed significantly increased antibody responses, at 18 wpi for 10°C and at 6 and 18 wpi for 15°C. Immunoblotting demonstrated specific response against the LPS antigen of A. salmonicida in the 10°C and 15°C VAX groups. Mean body weight increased in all groups throughout the study. Vaccinated fish had low Speilberg scores with no melanization of abdominal tissue. Our results show that vaccinating lumpfish at a lower water temperature may lead to a low antibody response against A. salmonicida.     

Lumpfish (Cyclopterus lumpus,Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail‐biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV‐infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.     

Effects of three commercial diets on growth,cataract development and histopathology of lumpfish (Cyclopterus lumpus L.)

Three duplicate groups of individually tagged lumpfish (mean initial weight: 20.0 ± 4.0 g) were fed three commercially available diets (A, B and C) for a period of 147 days. There were significant differences in growth rates between the groups with fish fed diet A having the highest growth rates whilst fish fed diet C achieved the lowest growth. Lumpfish fed diets A and B had lower prevalence and severity of cataracts compared with fish fed diet C, suggesting a possible dietary effect on cataract, alone or in combination with other environmental factors. There was a significant difference in the degree of liver vacuolization between the three dietary treatments at the end of the study period with fish fed diets A and B showing moderate to severe vacuolization compared with liver tissue from fish fed diet C, which had milder vacuolizations. There was little or no inflammation in pyloric caeca, midgut and hindgut and similarly for epithelial necrosis or vacuolization for all three dietary treatments at the end of the study. Fish fed diet A had the longest intestinal folds whilst fish fed diet C had the shortest.     

Ocular tissue changes associated with anterior segment opacity in lumpfish (Cyclopterus lumpus L) eye

Lumpfish use their vision to hunt prey or, in the case of aquaculture, to see and eat pelleted diets. A common anterior ocular opacity abnormality designated in the literature as “cataract” described in both farmed and wild lumpfish has not yet been characterized in detail at the pathobiological level. We describe here lens tissue changes associated with cataract in cultured and domesticated lumpfish. Methodology included gross observations, ophthalmoscopy and histology. Young adult cultured animals approaching 400 days post‐hatch presented a range of anterior segment opacities associated with lenticular abnormalities observable at a histological level. Wild aged domesticated animals also displayed cataracts characterized mainly by abnormalities of the lens observed by both ophthalmoscopy and histology. Dysplastic lesions of the lens in one aged domesticated lumpfish were accompanied with both retinal and optic nerve degeneration. These novel naturally occurring anatomical changes in lumpfish present both commonalities and unique features associated with cataract in young adult cultured animals versus aged domesticated broodstock animals.     

Comparison of diet composition,feeding, growth and health of lumpfish (Cyclopterus lumpus L.) fed either feed blocks or pelleted commercial feed

Two duplicate groups of individually tagged lumpfish (mean initial weight: 21.5 ± 3.2 g) were fed either a commercially available lumpfish feed or feed blocks for a period of 123 days. The aim was to evaluate and compare the effects of these feed types on growth, cataract development and histopathology in lumpfish. There were significant differences in growth rates between the groups with fish fed pelleted feed having the highest growth rates. The development of cataracts was significantly different with fish fed pelleted feed having a cataract prevalence of 87% at the end of the study period whilst fish fed with feed blocks had 10% prevalence. The results of the histological examination undertaken in this study showed overall small differences between the two dietary treatments. In some individuals in groups receiving both diets, there was mild to moderate expansion of the lamina propria with tissue most likely to represent fibrous tissue with scattered leucocytes. Overall, the findings of the present study show that lumpfish will readily graze from feed blocks and although growth is lower the prevalence of cataract is greatly reduced using feed blocks.     

Intramuscular vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) induces inflammatory reactions and local immunoglobulin M production at the vaccine administration site

Atlantic lumpfish were vaccinated by intramuscular (im) or intraperitoneal (ip) injection with a multivalent oil‐based vaccine, while control fish were injected with phosphate‐buffered saline. Four lumpfish per group were sampled for skin/muscle and head kidney tissue at 0, 2, 7, 21 and 42 days post‐immunization (dpi) for histopathology and immunohistochemistry (IHC). Gene expressions of secretory IgM, membrane‐bound IgM, IgD, TCRα, CD3ε and MHC class IIβ were studied in tissues by using qPCR. Im. vaccinated fish showed vaccine‐induced inflammation with formation of granulomas and increasing number of eosinophilic granulocyte‐like cells over time. On IHC sections, we observed diffuse intercellular staining of secretory IgM at the injection site at 2 dpi, while IgM + cells appeared in small numbers at 21 and 42 dpi. Skin/muscle samples from im. vaccinated fish demonstrated an increase in gene expression of IgM mRNA (secretory and membrane‐bound) at 21 and 42 dpi and small changes for other genes. Our results indicated that im. vaccination of lumpfish induced local IgM production at the vaccine injection site, with no apparent proliferation of IgM + cells. Eosinophilic granulocyte‐like cells appeared shortly after im. injection and increased in numbers as the inflammation progressed.     

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land‐based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real‐time RT‐PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re‐isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real‐time RT‐PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.     

Studies on the antibody response and side effects after intramuscular and intraperitoneal injection of Atlantic lumpfish (Cyclopterus lumpus L.) with different oil‐based vaccines

Atlantic lumpfish (Cyclopterus lumpus L.) is used as a biological delousing agent for sea lice (Lepeophtheirus salmonis K.) infestations in Norwegian aquaculture. Here, we present a study on the antibody response and vaccine side effects after intramuscular and intraperitoneal injection of lumpfish with two vaccines. Both vaccines contained bacterial antigens from atypical Aeromonas salmonicida A‐layer types V and VI, Vibrio anguillarum serotype O1 and Moritella viscosa sp., but one vaccine contained a vegetable oil‐based adjuvant, while the other contained a mineral oil‐based adjuvant. Intramuscular injection of the mineral oil‐based vaccine caused a high acute mortality of fish within 48 hr after immunization. Intraperitoneal injection of the mineral oil‐based vaccine resulted in a lower severity of intra‐abdominal side effects than the vegetable oil‐based vaccine. Intramuscular injection of the mineral oil‐based vaccine resulted in a significantly higher antibody response against A. salmonicida when compared to controls and the vegetable oil‐based vaccine group. The antibody response was poor against V. anguillarum and M. viscosa for all groups. Our results indicate that intramuscular injection of oil‐based vaccines might be feasible for providing immunological protection for Atlantic lumpfish against bacterial diseases, especially atypical A. salmonicida, but more work is required to identity optimal adjuvants.     

An epidemiological study of cataracts in wild and farmed lumpfish (Cyclopterus lumpus L.) and the relation to nutrition

Twelve groups of farmed lumpfish and one of wild lumpfish were screened for cataract and sampled for fish muscle tissue, whole heart and both eye lenses to investigate possible relations between cataract and tissue free amino acid concentrations. Cataract prevalence ranged from 20% to 100%, with the highest average score of 7.3 (max 8) and incidences of severe cataract (>5) in all groups. Cataract could not be explained by suboptimal histidine concentrations in the feed. Neither muscle nor cardiac tissues had concentrations of free histidine compounds. The lumpfish lens contained N‐acetylhistidine (NAH), of which low concentrations were strongly related to cataract severity. However, no correlation between lens NAH and cataract severity was found in the present sample set. Wild lumpfish had higher levels compared to farmed lumpfish, suggesting that the farmed lumpfish may have been deficient in histidine or have a higher utilization of NAH due to osmotic problems. Thus, cataract in farmed lumpfish may be related to primary or secondary disturbed nutrient metabolism or malnutrition, shown by the high levels of specific amino acids in different tissues, which may cause osmotic imbalance and cataract development. This nutritional or environmental‐related welfare problem deserves further research.