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.     

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.     

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.     

Piscine reovirus (PRV) in wild Atlantic salmon,Salmo salar L., and sea‐trout,Salmo trutta L., in Norway

This is the first comprehensive study on the occurrence and distribution of piscine reovirus (PRV) in Atlantic salmon, Salmo salar L., caught in Norwegian rivers. PRV is a newly discovered reovirus associated with heart and skeletal muscle inflammation (HSMI), a serious and commercially important disease affecting farmed Atlantic salmon in Norway. A cross‐sectional survey based on real‐time RT‐PCR screening of head kidney samples from wild, cultivated and escaped farmed Atlantic salmon caught from 2007 to 2009 in Norwegian rivers has been conducted. In addition, anadromous trout (sea‐trout), Salmo trutta L., caught from 2007 to 2010, and anadromous Arctic char, Salvelinus alpinus (L.), caught from 2007 to 2009, were tested. PRV was detected in Atlantic salmon from all counties included in the study and in 31 of 36 examined rivers. PRV was also detected in sea‐trout but not in anadromous Arctic char. In this study, the mean proportion of PRV positives was 13.4% in wild Atlantic salmon, 24.0% in salmon released for stock enhancement purposes and 55.2% in escaped farmed salmon. Histopathological examination of hearts from 21 PRV‐positive wild and one cultivated salmon (Ct values ranging from 17.0 to 39.8) revealed no HSMI‐related lesions. Thus, it seems that PRV is widespread in Atlantic salmon returning to Norwegian rivers, and that the virus can be present in high titres without causing lesions traditionally associated with HSMI.     

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.     

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.     

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter,Scortum barcoo (McCulloch & Waite)

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus‐specific PCR. The full‐length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV‐like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.     

Isolation,characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.     

Edwardsiella tarda infection in Korean catfish,Silurus asotus,in a Korean fish farm

Mass mortality of Korean catfish, Silurus asotus, occurred in a culture farm situated in Jeollabukdo Province, Korea. The cumulative mortality rates reached up to 5% of the total fish in the farm per day. In clinical signs, the affected fish showed abdominal distension, vent protrusion, enteritis, liver congestion and abscess‐like lesions in enlarged spleen and kidney. Histopathologically, in the liver, hepatocytes lost fat and underwent atrophy or necrosis. The spleen showed necrotized splenocytes and a haemorrhagic pulp. In the kidney, glomerular destruction, degeneration of renal tubular epithelial cell and haemorrhage were observed. However, necrotic muscular lesions were not observed. A pure bacterial isolate was obtained from the liver, spleen and kidney lesions of affected fish. Experimental infection of normal catfish with the isolate resulted in the development of clinical signs similar to those seen on the farm. The isolates were identified as Edwardsiella tarda through biochemical tests (99.4%) and analysis of bacterial genes (16S rDNA) sequences (98%). The bacteria possessed two virulent genes: sodB and katB genes. These results suggest that E. tarda can act as a pathogen of farmed catfish. This is the first report showing that E. tarda caused mortality in cultured Korean catfish.     

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.     

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.     

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.     

DNA fragmentation in blue mussel (Mytilus edulis) sperm: aquaculture and fisheries implications

The objective of this study was to assess sperm DNA longevity in blue mussels (Mytilus edulis) using a dynamic assessment of sperm DNA fragmentation (SDF) after sperm activation. Mature blue mussels (n = 57) in Vigo (Galicia, Spain) were obtained, specifically rope farmed blue mussels (n = 38) and wild blue mussels (n = 19). After the sperm collection, a subsample was assessed for SDF (0 h), while the rest of the sample was incubated for 6, 9, 12, 24 and 48 h at 15°C, assessing each time point using the Sperm‐Halomax kit (Halotech DNA, Madrid, Spain). The Kaplan–Meier estimator, log‐rank (Mantel–Cox) test and Mann–Whitney U‐test were used for statistical analyses (spss v. 16.0), α = 0.05. The rate of SDF (r‐SDF) between rope farmed and wild blue mussels over 0–6 h incubation was not significantly different (P = 0.278), but was for 6–24 h (P = 0.004). Differences in r‐SDF were observed when comparing the means between the two groups (P < 0.0001). Individual differences in r‐SDF existed among the rope farmed (P < 0.0001) and wild blue mussels (P < 0.0001). Wild blue mussels presented a higher DNA longevity than the farmed blue mussels. Selection of blue mussel males with a low level of sperm DNA damage and greater sperm DNA longevity may result in better fertilization and seed production.     

A survey on Tetracapsuloides bryosalmonae infections in Slovene fresh waters

Slovenia has no history of health problems related to proliferative kidney disease (PKD) either in farmed or in wild fish. However, due to the past molecular evidence for the presence of Tetracapsuloides bryosalmonae DNA in tissues of some fish from open waters, a survey was conducted on wild salmonids that were primarily sampled for other purposes. In winter 2010–2011, specimens from a total of 244 rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., from 30 bodies of fresh water were examined for T. bryosalmonae using a PCR method. The sampled fish showed no clinical signs or gross pathological lesions characteristic of PKD. Nineteen (7.8%) fish from seven (23.3%) fresh waters were positive for T. bryosalmonae. The identity of PCR amplicons was confirmed by sequencing. With one exception, all the positive fish were found in waters from the regions where the average yearly temperatures and the environmental pollution are higher. This preliminary countrywide survey provided the first insight into the situation regarding T. bryosalmonae infection of wild salmonids in Slovenia.     

A parasite survey of farmed Southern bluefin tuna, Thunnus maccoyii (Castelnau)

Farmed Southern bluefin tuna (SBT) were examined for parasites. Samples of harvest fish, mortalities and some fish showing clinical signs of disease were investigated. Targeted screening was conducted for a scuticociliate, Uronema nigricans, the myxosporean Kudoa sp. and a sanguinicolid digenean, Cardicola forsteri. General parasitological investigation revealed a diverse parasite community of didymozoid digeneans, two species of copepods, a polyopisthocotylean monogenean and larval cestodes. Targeted screening for U. nigricans exposed a low prevalence, most probably due to a lack of sensitivity in the test method. Few of the parasites examined pose a risk to the health of farmed SBT.     

Detection of tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria

Tilapia lake virus disease (TiLVD) has emerged to be an important viral disease of farmed Nile tilapia (Oreochromis niloticus) having the potential to impede expansion of aquaculture production. There is a need for rapid diagnostic tools to identify infected fish to limit the spread in individual farms. We report the first detection of TiLV infection by PCR in farmed and wild Nile tilapia from Lake Victoria. There was no difference in prevalence between farmed and wild fish samples (p = .65), and of the 442 samples examined from 191 fish, 28 were positive for TiLV by PCR. In terms of tissue distribution, the head kidney (7.69%, N = 65) and spleen (10.99%, N = 191), samples had the highest prevalence (p < .0028) followed by heart samples (3.45%, N = 29). Conversely, the prevalence was low in the liver (0.71%, N = 140) and absent in brain samples (0.0%, N = 17), which have previously been shown to be target organs during acute infections. Phylogenetic analysis showed homology between our sequences and those from recent outbreaks in Israel and Thailand. Given that these findings were based on nucleic acid detection by PCR, future studies should seek to isolate the virus from fish in Lake Victoria and show its ability to cause disease and virulence in susceptible fish.     

Sequential pathology after initial freshwater bath treatment for amoebic gill disease in cultured Atlantic salmon, Salmo salar L

Freshwater bathing is essential for control of amoebic gill disease (AGD) during the marine phase of the Tasmanian Atlantic salmon production cycle, a practice that is costly, production limiting and increasing in frequency. Although the pathogenesis of gill infection with Neoparamoeba sp. in naïve Atlantic salmon, Salmo salar, is now understood, the progression of re‐infection (post‐treatment) required elucidation. Here, we describe the weekly histopathological progression of AGD from first to second freshwater bath. Halocline cessation and increased water temperature appeared to drive the rapid onset of initial infection prior to bathing. Freshwater bathing cleared lesions of attached trophozoites and associated cellular debris. Subsequent gill re‐infection with Neoparamoeba sp. was evident at 2 weeks post‐bath and had significantly increased (P < 0.001), in severity by 4 weeks post‐bath. No significant difference in gross pathology was observed until 4 weeks post‐bath (P < 0.05). The re‐infective progression of AGD was characterized by localized host tissue responses juxtaposed to adhered trophozoites (epithelial oedema, hypertrophy and hyperplasia), non‐specific inflammatory cell infiltration (macrophages, neutrophils and eosinophilic granule cells) and finally advanced hyperplasia with epithelial fortification. During the post‐bath period, non‐AGD lesions including haemorrhage, necrosis and regenerative hyperplasia were occasionally observed, although no evidence of secondary colonization of these lesions by Neoparamoeba sp. was noted. We conclude that pathogenesis during the inter‐bath period was identical to initial infection although the source of re‐infection remains to be established.     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

Pathology of gas bubble disease in two air-breathing catfishes (Clarias batrachus Linn. and Heteropneustes fossilis Bloch.)

Gross pathology in Clarias batrachus and Heteropneustes fossilis, and histopathology in the liver, intestine and kidney and abnormalities in erythrocyte count/mm³ and haemoglobin g% of C. batrachus afflicted with gas bubble disease were studied over 96 h. Histopathology and blood abnormalities in H. fossilis could not be observed due to heavy mortality. A tilt in the swimming posture and an upward bend in the tail region were the first signs of the disease. Exophthalmia was noted and blebs containing gas bubbles appeared on the fins and other parts of the body. The blebs burst producing open lesions after 72 h, and the fins became necrotic.Histopathologic changes included, in the liver, vacuolation of cells, necrosis and cordal disarray; in the intestine, lesions in the villi, degeneration of the submucosa, cellular exudate production in the lumen and air bubbles in the villi; and in the kidney, degeneration of tubules and glomeruli and necrosis of haemopoietic tissue. Erythrocyte count and haemoglobin content showed a consistent and significant fall from 12.87 × 10⁶ to 6.91 × 10⁶/mm³ and from 9.33 to 7.00 g%, respectively, up to 72 h. Empty blood vessels were noted in the liver, intestine and kidney. Fish of both species regained normal swimming orientation, and erythrocyte count and haemoglobin content also showed recovery from the disease.