Infectivity study of Streptococcus phocae to seven fish and mammalian cell lines by confocal microscopy

Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells.     

The susceptibility of salmonid fish to an atypical strain of Aeromonas salmonicida that infects goldfish, Carassius auratus (L.), in Australia

Abstract. An enzootic, Australian, atypical strain of Aeromonas salmonicida isolated from diseased goldfish, Carassius auratus (L.), was inoculated into Atlantic salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, S. gairdneri Richardson, and brook trout, Salvelinus fontinalis (Mitchill), fingerlings by intraperitoneal injection (i.p.) and by bath challenge, the latter with and without prior abrasion of skin. The 10-day LD₅₀ (i.p.) was estimated to be 7·4 × 10^-3 colony forming units (cfu) for Atlantic salmon, 3·0 × 10^-2 cfu for brown trout, 3·7 × 10² cfu for brook trout and 6·4 × 10³ cfu for rainbow trout. Brown, rainbow and brook trout succumbed to bath challenges with between 10⁵–10⁶ cfu/ml, developing ulcers of the skin and septicaemia. The organism was trasmitted from inoculated fish to five of 195 within-tank control fish via water and established a carrier state in one of 14 Atlantic salmon. It was concluded that the organism poses a significant threat to the salmonid farming industry and wild salmonid fisheries in Australia.     

Aeromonas salmonicida infection in wrasse (Labridae), used as cleaner fish, on an Atlantic salmon, Salmo salar L., farm

Abstract. Typical Aeromonas salmonicida with similar biochemical characteristics to A. salmonicida from Atlantic salmon, was isolated from wrasse, Ctenolabrus rupestris (L.) and Centrolabrus exoletus (L.), stocked as cleaner fish with these salmon. Although no external clinical signs were apparent, localized bacterial microcolonies were observed in muscle, gills, intestine, kidney and myocardial tissue. Mortalities attributed to A. salmonicida comprised 55% (n = 32) of total mortalities. No carriers of A. salmonicida were found in wild wrasse following stress testing. Although salmon post-smolts died when challenged with 1 × 10⁵ ml^-1 of a virulent strain, there were no mortalities in challenged wrasse. An oral route of infection is suggested rather than water-borne transfer as wrasse browsed on salmon mortalities. Wrasse were treated for A. salmonicida infection by injection with antibiotic and were also vaccinated, and in the latter case, elevation of antibody levels was noted.     

A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Abstract. A natural bath challenge method has been developed for furunculosis using Atlantic salmon, Salmo salar L., and rainbow trout, Salmo gairdneri Richardson. Fish were placed in an enclosed, continuously circulating tank system, supplemented with additional oxygen, the temperature raised gradually (overnight) to 15–16°C, a low dose of Aeromonas salmonicida (strain 184/186) introduced into the tank and the challenge maintained for 14 days. The challenge strain was characterized with respect to possible virulence factors and possessed an A-layer, ability to auto-agglutinate, haemagglutinate, adhere to Atlantic salmon cells and resist destruction by serum. No caseinase activity was detected. LD₅₀ for salmon using this method was 1.8×10³ cells per millilitre while trout had an LD₅₀ of 9.5×10⁴ cells per millilitre. Onset of the disease appeared to depend on fish size with larger trout (50 g) taking up to 10 days to show signs of the disease while mortalities in smaller trout (8.5g) commenced on day 1 post-challenge.     

Urea in feeds for sea water farmed Atlantic salmon: effect on growth, carcass quality and outbreaks of winter ulcer

The main objective of the present study was to evaluate whether dietary supplementation of urea might reduce the incidence of winter ulcer in sea water-farmed Atlantic salmon. Salmon destined to be S0 smolt were fed with a urea-supplemented diet (0 or 20 g kg⁻¹ urea) over an 8-week period prior to sea water transfer and were then fed supplemented diet (0, 5, 10 or 20 g kg⁻¹ urea) during the first and second winters in the sea. During the first winter positive relationships between dietary urea and plasma urea and between plasma urea and plasma osmolality were observed. Further, plasma osmolality displayed a negative relationship to mortality. Of the salmon that died during the first winter in the sea 90% had one or more skin ulcers. Both during the first and second winter there were fewer salmon with ulcer among fish fed with the diets supplemented with urea. Salmon fed with 20 g kg⁻¹ urea tended to have higher percentage water in the muscle. Mortality and incidence of salmon with ulcer seemed to relate to plasma osmolality amongst fish fed on diets that differed in levels of urea supplementation, suggesting that an osmotic imbalance may contribute to the development of winter ulcer in farmed salmon. Salmon fed with 20 g kg⁻¹ urea showed significantly greater body weight during the second winter in sea. Fish killed without prior starvation had significantly higher level of muscle urea in the 20 g kg⁻¹ urea group compared with fish fed with the unsupplemented diet. However, a 13-day starvation period reduced urea content in the muscle to the level of the control. No effects of dietary urea supplementation on the sensory quality of market size Atlantic salmon were observed.     

Growth and digestibility effects of soya lecithin and choline chloride on juvenile Atlantic salmon

Atlantic salmon, Salmo salar L., fingerlings with a mean weight of 1.5 g were fed one of four casein-gelatine-based purified diets supplemented with soya lecithin (LC) and choline chloride (CH) for 84 days. The diets were supplemented with either: 0 g kg⁻¹ CH and 0 g kg⁻¹ LC, 0 g kg⁻¹ CH and 30 g kg⁻¹ LC, or 5 g kg⁻¹ CH and 0 g kg⁻¹ LC, or 5 g kg⁻¹ CH and 30 g kg⁻¹ LC. The same diets were also fed to 100-g salmon to assess the effects of LC and CH supplementation on digestibility. Fingerlings fed the diet with neither LC nor CH (0 g kg⁻¹ LC and 0 g kg⁻¹ CH) grew at a significantly slower rate than fish fed the supplemented diets. There were significant effects on growth of supplementation of both LC and CH. The results indicate that the choline requirement of Atlantic salmon fingerlings is satisfied by 4 g kg⁻¹ inclusion in a gelatine-casein-based diet, and that dietary soya lecithin can fully replace choline chloride. The digestibility study with the larger fish indicated a beneficial effect of lecithin on the digestibility of both protein and energy.     

Dietary phosphorus requirement of juvenile Atlantic salmon, Salmo salar L.

The objective of this study was to determine the dietary phosphorus (P) requirement of juvenile Atlantic salmon, Salmon salar L. Triplicate groups of fish (mean initial weight 1.4 g) were fed semipurified, casein-gelatine-based diets containing one of five levels of P (4, 8, 10, 15 and 25 g kg⁻¹) from Ca(H₂PO₄)₂·H₂O, or a commercial feed (17 g kg⁻¹ P) for 9 weeks. Weight gains did not differ significantly among treatment groups fed the experimental diets but were slightly less than gains in fish fed the commercial feed. Feed efficiency (wet weight gain/dry feed consumed) was similar in all groups, averaging 1.45. Availability of dietary P, estimated from apparent retention and apparent digestibility, was 86%. Whole-body P concentrations declined in fish fed diets containing less than 10 g kg⁻¹ P. Fitting a logistic curve to dietary P vs. whole-body P concentrations indicated that a minimum of 11 g kg⁻¹ dietary P (9 g kg⁻¹ digestible P) was required by juvenile Atlantic salmon to maintain whole-body P concentrations at initial levels. Calculation of a dietary requirement using a simple factorial model which incorporated measurements of P availability, feed efficiency and normal whole-body P concentration indicated that the dietary requirement was approximately 10 g kg⁻¹. The dietary requirement established in this study (10–11 g kg⁻¹) is higher than previously reported for Atlantic salmon or other fishes. Possible reasons for the wide range of reported dietary P requirements in fishes are discussed.     

First description of atypical furunculosis in freshwater farmed Atlantic salmon,Salmo salar L., in Chile

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that the atypical isolates belong to two genetic groups that were associated with the geographical origin.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

Three different levels of dietary vitamin D3 fed to first-feeding fry of Atlantic salmon (Salmo salar L.): effect on growth, mortality, calcium content and bone formation

Atlantic salmon ( Salmo salar L.) fry were reared on a fishmeal-based diet with three different levels of vitamin D₃ (0.2, 5 and 57 mg vitamin D₃ kg^–1 feed, ww) from first-feeding for 14 weeks. No significant differences were recorded in weight, length, specific growth rate, mortality, or kidney calcium concentration between the different dietary groups. No skeletal malformations or histopathological changes were recorded in any of the dietary groups. These results suggest Atlantic salmon fry to be highly tolerant of megadoses of vitamin D₃ over a limited period of time.     

Infectious pancreatic necrosis virus: experimental infection of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae)

The use of wrasse as cleaner fish in farming of Atlantic salmon, Salmo salar L., is now widespread in Scotland, Ireland and Norway, but little is known about the susceptibility of these fish to the common pathogens of cage-cultured salmon. The susceptibility of goldsinny wrasse, Ctenolabrus rupestris L., to infectious pancreatic necrosis virus (IPNV) was investigated. Captive-bred C. rupestris were infected with IPNV-Sp (Shetland strain) using a bath challenge method. Two infection doses were used, low (4.1 × 10⁵ pfu mL⁻⁻¹) and high (2.4 × 10⁶ pfu mL⁻⁻¹), with two replicates for each experiment. Bathing times were 1 h for the low dose and 5 h for the high dose. A maximum prevalence of infection (30%) was seen 2 weeks post-infection in replicate 1 of the low dose experiment and was accompanied by low tissue titres. The tissue titres dropped to an undetectable level by 4 weeks post-infection. In the high dose experiment, a high prevalence of infection was seen along with moderate titres in tissues as well as a marked pathological response. Both prevalence and intensity declined rapidly and the fish recovered. In the high dose experiment, faecal titres followed the same pattern as those in the tissues. The implications for the use of wrasse in the farming of Atlantic salmon are discussed.     

Effect of dietary lipid level on muscle composition in Atlantic salmon Salmo salar

This study was conducted to determine the effects of feeding increasing lipid concentrations (310, 380 and 470 g kg^–1 lipid on dry weight) in diets based mainly on herring byproducts to Atlantic salmon Salmo salar . The diets were isonitrogenous, varying in dietary lipid content at the expense dietary starch. Average fish weight increased from 1.2 kg in April to 2.2–2.7 kg at the end of the feeding trial in September. Significantly greater growth was found in fish fed either the 380 g kg⁻¹ or the 470 g kg⁻¹ lipid diets compared with the 310 g kg⁻¹ lipid diet. Muscle lipid content increased in all dietary groups on a wet weight basis from 7.7 ± 1.4% to 12 ± 3% in salmon fed the 310 g kg⁻¹ lipid diet, and to 16 ± 2% in salmon fed the 380 g kg⁻¹ and 470 g kg⁻¹ lipid diets. In fish of similar weight there was a positive correlation between dietary lipid and muscle lipid concentrations. Low concentrations of muscle glycogen were detected in fish fed each of the diets, while muscle vitamin E concentrations slowly decreased as muscle lipid increased. Muscle fatty acid composition reflected dietary fatty acid profiles, containing similar percentages of total saturated, monoenic and n-3 fatty acids (20:5n-3 and 22:6n-3) in fish from all dietary treatment groups. However, a higher ratio of n-3/n-6 was found in muscle from fish fed the 470 g kg⁻¹ lipid diet compared with the other two groups. Blood chemistry values varied somewhat, but all values were within normal ranges for Atlantic salmon of these sizes.     

Distended, water-filled stomach in seawater farmed rainbow trout, Oncorhynchus mykiss (Walbaum), provoked experimentally by osmoregulatory stress

The present study evaluates if abdominal dis-tension caused by a water-filled stomach in seawater farmed rainbow trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon, Salmo salar L., could be provoked experimentally by live chilling in sea water (0.5 °C). Fifty rainbow trout and 50 salmon were visually classified, either as normal or suffering from the condition. Prior to chilling, no rainbow trout or salmon suffered from a water-filled stomach. During chilling, 25% of the rainbow trout and 2.5% of the salmon developed water-filled stomach. Affected rainbow trout had significantly higher blood plasma osmolality and significantly lower body weight than normal trout. The frequency of trout with a water-filled stomach increased significantly with increasing chilling time and increasing plasma osmolality. The regression coefficients revealed that the proportion of affected rainbow trout in-creased by 0.82% h⁻¹ of chilling and by 0.46% per unit increase in mosmol L^−1. The present study revealed that abdominal distension in seawater farmed rainbow trout was far more severe than in Atlantic salmon and could be provoked experimentally by osmoregulatory stress. This is consistent with the observation that water-filled stomach appear only occasionally in seawater farmed Atlantic salmon.     

Swimming performance of wild and F1-hatchery-reared Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) smolts

Abstract –  The swimming performance of wild and hatchery-reared smolts of two salmonid species was investigated. Wild Atlantic salmon smolts (WS) and brown trout smolts (WT) of equal size were caught in fish traps during migration. Hatchery-reared smolts of both species (HS and HT for salmon and trout respectively) were first generation offspring from wild broodstock. The swimming performance of individual smolts from the four groups (WS, HS, WT, HT) was tested three consecutive times using a swimming flume with water flowing at a start rate of 0.16 m·s⁻¹ and a constant acceleration rate of 0.167 cm·s⁻² (10 cm·s⁻¹·min⁻¹). Wild caught smolts of both species performed significantly better than those reared in hatchery conditions. The WS group were observed to maintain an average swimming speed ( U _burst) that was 30% faster than the HS group, whereas the wild trout smolts were superior to HT by approximately 25%. Repeated measures revealed species-specific exhaustion patterns. Brown trout smolts maintained consecutive U _burst indicating significant stamina compared with Atlantic salmon smolts that were found to be exhausted by the initial trial.     

Phenotypic and genotypic characterization of Flavobacterium psychrophilum from Finnish fish farms

Finnish isolates ( n =37) of Flavobacterium psychrophilum isolated from farmed salmonids were studied using phenotypic and genotypic characteristics. The characteristics of isolates were compared with the characteristics of Swedish and Estonian F. psychrophilum isolates and the type strain, F. psychrophilum NCIMB 1947^T. Multiple isolates from eight disease outbreaks were examined to determine differences between isolates from a single outbreak. The F. psychrophilum isolates represented a biochemically homogeneous group. However, some minor differences in biochemical and physiological characteristics were observed. Seven different antigenic patterns among Finnish isolates were detected and the results suggest a new serotype of F. psychrophilum . Using Cla Ι, Hae ΙΙΙ and Pvu ΙΙ restriction enzymes in ribotyping analyses 13 different genotypes were demonstrated and a possible relationship between serotype Fd and genotype F1 was determined. There were no significant differences between the isolates from Finland, Estonia, Sweden and the type strain of F. psychrophilum .     

Effect of sudden increase in discharge in a large river on newly emerged Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) fry

Abstract– Downstrcam displacement of salmonid fry due to flow increase, from 12–15 m³/_s to >100 m³_s, was documented in the river Suldalslåen, Western Norway. In May only fry of brown trout ( Sulmo trutta ) occurred in the drift, while from the beginning of June only newly emerged Atlantic salmon ( Sulmo sular ) were found. The maximum number of Atlantic salmon fry drifting during a single day was estimated to be 17 000 individual. Their density in the drift was higher during the night than during the day, and their appearance in the drift coincided with the predicted period of emergence. Total brown trout numbers in the drift were estimated to vary between 4000 and 16 000 per day. Fry displaced downstream from the lowermost part of the river were lost from the population. The total losses were estimated to be between 75 000 and 100 000 Atlantic salmon fry which represents between 5.6 and 11.1%) of juvenile mortality during the first year of life.     

Dietary menadione nicotinamide bisulphite (vitamin K3) does not affect growth or bone health in first-feeding fry of Atlantic salmon (Salmo salar L.)

The aim of the present study was to elucidate if increasing levels of added vitamin K in the feed influenced fish growth, health or the incidences of bone deformities in Atlantic salmon ( Salmo salar L.) from onset of start feeding to ∼100 g in freshwater. Duplicate groups of Atlantic salmon fry (∼0.20 g) were fed eight levels (0, 2.5, 5, 7.5, 10, 15, 20 and 50 mg menadione kg⁻¹) of the vitamin K derivate menadione nicotinamide bisulphite (MNB) in a regression design for 28 weeks. All fish maintained high growth rates throughout the experiment, and showed no significant differences in specific growth rate, condition factor, whole body proximate analysis, blood coagulation time, vertebra morphology or mechanical properties of vertebrae. We found a dose-response between whole body vitamin K concentration and the dietary MNB supplementation level. Analysis of liver γ-glutamylcarboxylase activity revealed significant dose-dependent differences between groups given the 0, 10 and 50 mg MNB kg⁻¹ diets. In conclusion, Atlantic salmon seems to require low levels of dietary vitamin K, and the amount of vitamin K found naturally in the presently used feed ingredients may be enough to maintain optimal growth, health and bone strength in Atlantic salmon fry from start feeding.     

Supplementing copper to a fish meal based diet fed to Atlantic salmon parr affects liver copper and selenium concentrations

The present experiment was conducted to study growth and tissue responses in Atlantic salmon, Salmo salar L., fed a fish meal based diet supplemented with copper (Cu). The findings of the experiment were used to evaluate the need for dietary Cu supplementation. Atlantic salmon parr, initially weighing ≊ 7.5 g, were randomly distributed among 10 tanks, with 300 fish in each tank. Duplicate groups of fish were fed a fish meal based diet containing 3.5 mg Cu kg⁻¹, or this diet supplemented with 5, 10, 50 or 100 mg Cu kg⁻¹ (as CuSO₄*5 H₂O) for 12 weeks. Growth was recorded and blood haemoglobin measured. The Cu concentrations in whole body, liver, serum and selected muscle samples were measured, as was liver selenium (Se) concentration.
There were no difference in growth among the dietary treatments. There were, however, significant differences among the dietary groups in liver Cu and Se concentrations. The fish fed the diet supplemented with 5 mg Cu kg⁻¹ had increased liver Cu concentration compared with the other groups. Similar trends were found for serum Cu concentration and whole-body Cu concentration, but these effects were not significant. Liver Cu and Se concentrations were positively correlated and liver Se concentrations were inversely correlated to dietary Cu concentration, confirming an interaction between these two elements in salmon.
There may be a positive effect of a modest Cu supplementation level, and we suggest that a small amount of Cu (5–10 mg kg⁻¹) should be added to fish meal based diets.