Respiratory pathogenesis of amoebic gill disease (AGD) in experimentally infected Atlantic salmon Salmo salar

The aim of this study was to investigate the respiratory responses of Atlantic salmon, Salmo salar, experimentally affected with amoebic gill disease (AGD). In Series I, arterial blood samples were taken over a 96 h period following amoebae addition to examine potential respiratory effects associated with initial exposure. No major significant treatment effects were found between fish exposed to amoebae and control (non-exposed) fish. Arterial pH (pHa) was seen to be significantly elevated at 48 h in AGD fish relative to the 0 h time point. To investigate the long-term respiratory effects associated with infection, fish were similarly exposed to amoebae and sampled over a 16 d period. As for Series I, caudal blood pH was significantly elevated by Day 2 (48 h) compared to the pre (Day 0)-time point, suggesting that initial exposure to amoebae and/or amoebae attachment may have induced an initial respiratory alkalosis via increased ventilation frequency and/or amplitude. From Day 7 onwards, and coinciding with a significant increase in the percentage of affected gill filaments, blood pH decreased significantly, possibly indicating the onset of the characteristic respiratory acidosis that has previously been described for experimentally AGD-affected Atlantic salmon. Although fish in this study showed up to 90% AGD-affected filaments, the corresponding respiratory results do not reflect a major acid-base disturbance. Therefore, the findings from the present study support the contention that, although AGD only affects the gill, AGD-associated mortality in Atlantic salmon may not be primarily associated with respiratory failure.     

Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.     

Cardiovascular responses of three salmonid species affected with amoebic gill disease (AGD)

The cardiovascular effects of amoebic gill disease (AGD) were investigated immediately following surgery in three salmonid species; Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum). Fish, both naïve (control) and infected (AGD-affected) of each species, were fitted with dorsal aorta catheters and cardiac flow probes. Cardiac output and dorsal aortic pressures were then continuously measured over a 6-h period following surgery. Results showed that Atlantic salmon, brown trout and rainbow trout displayed similar dorsal aortic pressure, cardiac output, and systemic vascular resistance (mean dorsal aotic pressure divided by cardiac output) values. However, the only significant differences relating to disease status i.e. infected or control, were found in Atlantic salmon. Although no significant differences were seen in dorsal aortic pressure values, AGD-affected salmon displayed significantly elevated systemic vascular resistance at 4 and 6 h post surgery. Cardiac output was also approximately 35% lower in AGD-affected salmon compared to the non-affected control counterparts. These results comparatively examine cardiac function in response to AGD across three salmonid species and highlight species-specific cardiovascular responses that occur in association with disease. It is suggested that the apparent cardiac dysfunction seen in AGD-affected Atlantic salmon could, under stressful conditions, become exacerbated. Cardiac failure is therefore suggested to be a possible physiological mechanism by which AGD causes or contributes to mortality in Atlantic salmon.     

Metabolic effects of amoebic gill disease (AGD) and chloramine-T exposure in seawater-acclimated Atlantic salmon Salmo salar

Our aim was to determine possible metabolic effects amoebic gill disease (AGD) on Atlantic salmon Salmo salar. Standard (R(S)) and routine (R(ROU)) metabolic rates were evaluated by continually measuring oxygen consumption in 2 independent tanks of fish (18.69 +/- 1.01 kg m(-3), mean +/- SE). Active metabolic rate (R(ACT)) and metabolic scope (R(ACT) - R(S)) were assessed using a chasing protocol and determined at 3 time periods: (1) pre-infection, (2) 3 d post-infection, and (3) 2 d post-treatment. On Day 3 of the study, the fish were infected with amoebae isolated from the gills of AGD-affected salmon (2300 cells l(-1)). No significant elevations in R(ACT) or metabolic scope were detected 3 d post-infection and 2 d post-treatment; however, significant elevations in R(S) and R(ROU) were detected 3 d post-infection and 2 d post-treatment. Assessment of R(ROU) data, especially for the light period, also indicated a rise in oxygen consumption rate over the course of the experiment. Treatment of AGD-affected Atlantic salmon with chloramine-T (CL-T) appeared to briefly mitigate the rise in R(S), as there was a 30% drop (though non-significant) in R(S) following treatment. Despite this, R(S) continued the upward trend 1 d following treatment. These results suggest that over the course of AGD development, R(S) in Atlantic salmon increases. Therefore, considering the physical conditions which constrain R(ACT), we expect that metabolic scope would become compromised in fish more heavily affected with AGD. Treatment with CL-T shows promise for mitigating the respiratory effects of AGD and potentially minimising the loss of metabolic scope.     

Accelerated recovery of Atlantic salmon (Salmo salar) from effects of crowding by swimming

The effects of post-crowding swimming velocity (0, 0.35, and 0.70 m/s) and recovery time (1.5, 6, and 12 h) on physiological recovery and processing quality parameters of adult Atlantic salmon (Salmo salar) were determined. Atlantic salmon crowded to a density similar to that of a commercial slaughter process (>200 kg/m(3), 40 min) were transferred to a swimming chamber for recovery treatment. Osmolality and concentrations of cortisol, glucose and lactate in blood plasma were used as physiological stress indicators, whereas image analyses of extent and duration of rigor contraction, and fillet gaping were used as measures of processing quality. Crowded salmon had a 5.8-fold higher plasma cortisol concentration than control salmon (P<0.05). The elevated plasma cortisol concentration was reduced by increasing the swimming velocity, and had returned to control levels after 6 h recovery at high water velocity. Similar effects of swimming velocity were observed for plasma osmolality and lactate concentration. A lower plasma glucose concentration was present in crowded than in control fish (P<0.05), although a typical post-stress elevation in plasma glucose was observed after the recovery treatments. Lower muscle pH was found in crowded compared with control salmon (P<0.05), but muscle pH returned to control levels after 6 h recovery at intermediate and high swimming velocities and after 12 h in the low velocity group. Crowding caused an early onset of rigor mortis contraction. However, subjecting crowded salmon to active swimming for 6 h before slaughter delayed the onset of rigor mortis contraction from 2.5 to 7.5 h post mortem. The extent of rigor mortis contraction was also affected by crowding and post-stress swimming activity (P<0.05), and the largest degree of contraction was found in crowded salmon. In conclusion, active swimming accelerated the return of plasma cortisol, hydromineral balance, and the energy metabolism of adult Atlantic salmon to pre-stress levels. Moreover, an active swimming period delayed the onset of rigor mortis contraction, which has a positive technological implication for the salmon processing industry.     

Concentration effects of Winogradskyella sp. on the incidence and severity of amoebic gill disease

To study the concentration effects of the bacterium Winogradskyella sp. on amoebic gill disease (AGD), Atlantic salmon Salmo salar were pre-exposed to 2 different doses (10(8) or 10(10) cells 1(-1)) of Winogradskyella sp. before being challenged with Neoparamoeba spp. Exposure of fish to Winogradskyella sp. caused a significant increase in the percentage of AGD-affected filaments compared with controls challenged with Neoparamoeba only; however, these percentages did not increase significantly with an increase in bacterial concentration. The results show that the presence of Winogradskyella sp. on salmonid gills can increase the severity of AGD.     

The effects of an acute temperature change on the metabolic recovery from exhaustive exercise in luvenile Atlantic salmon (Salmo salar)

This research examined the influence of acute changes of water temperature on the recovery processes following exhaustive exercise in juvenile Atlantic salmon (Salmo salar). White muscle phosphocreatine (PCr), ATP, lactate, glycogen, glucose, pyruvate, plasma lactate, and plasma osmolality were measured during rest and at 0, 1, 2, and 4 h following exhaustive exercise in fish acclimated and exercised at 12 degrees C and acutely exposed to either 6 degrees C or 18 degrees C water during recovery. An acute exposure to 6 degrees C water during the recovery period resulted in a severe reduction of metabolic recovery in salmon. However, metabolites such as muscle PCr and ATP and plasma lactate recovered very quickly (2-4 h) in fish acutely exposed to 18 degrees C during recovery. Overall, differences exist when postexercise metabolite levels are compared between acclimated fish and those fish acutely exposed to different water temperatures (either higher or lower). Taken together, the findings of the acute experiments suggest that at some point following exercise fish may seek warmer environments to speed the recovery process. However, the relationship between behavioural thermoregulation and recovery following exhaustive exercise in fish is not well understood.     

In vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD)

The objective of the present study was to evaluate the in vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD). The current treatment for AGD-affected Atlantic salmon involves bathing sea-caged fish in freshwater for a minimum of 3 h, a labour-intensive and costly exercise. Previous attempts to identify alternative treatments have suggested bithionol as an alternate therapeutic, but extensive in vitro efficacy testing has not yet been done. In vitro toxicity to Neoparamoeba spp. was examined using amoebae isolated from the gill of AGD-affected Atlantic salmon and exposing the parasites to freshwater, alumina (10 mg l(-1)), seawater, bithionol or bithionol sulphoxide at nominal concentrations of 0.1, 0.5, 1, 5 and 10 mg l(-1) in seawater. The numbers of viable amoebae were counted using the trypan blue exclusion method at 0, 24, 48 and 72 h. Both bithionol and bithionol sulphoxide demonstrated in vitro toxicity to Neoparamoeba spp. at all concentrations examined (0.1 to 10 mg l(-1) over 72 h), with a comparable toxicity to freshwater observed for both chemicals at concentrations > 5 mg l(-1) following a 72 h treatment. Freshwater remained the most effective treatment, with only 6% viable amoebae seen after 24 h and no viable amoebae observed after 48 h.     

Comparative ionic flux and gill mucous cell histochemistry: effects of salinity and disease status in Atlantic salmon (Salmo salar L.)

Two experiments were conducted to assess the physiological effects of freshwater exposure and amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.). The first experiment monitored marine salmon during a 3 h freshwater exposure, the standard treatment for AGD in Tasmania. The second experiment described the gill mucous cell histochemistry for freshwater adapted and seawater acclimated fish (AGD affected and unaffected) for possible correlations to ionoregulation. When exposed to freshwater, marine Atlantic salmon experienced a minor ionoregulatory dysfunction represented by a net efflux of Cl(-) ions at 3 h. AGD affected fish experienced the net efflux of Cl(-) ions 1 h sooner, and had a significantly greater net efflux of total ammonia. Changes to gill mucous cell populations corresponded to differing salinity and the presence of AGD. In AGD affected fish, these populations significantly differed between lesion and non-lesion associated areas of the gill filament. Our results have shown changes in the ionoregulatory capacity of Atlantic salmon due to freshwater exposure and AGD. Gill mucous cell histochemistry indicates the potential importance of the mucous layer in ionoregulation and disease. In comparison to previous studies on rainbow trout, these results suggest that Atlantic salmon have a greater short-term ionoregulatory capacity.     

Detection of serum anti-Neoparamoeba spp. antibodies in amoebic gill disease-affected Atlantic salmon Salmo salar

Amoebic gill disease (AGD)-affected Atlantic salmon Salmo salar sometimes developed a serum-antibody response to wild-type Neoparamoeba spp. Five of 103 AGD-affected S. salar sampled possessed detectable antibodies that bound wild-type Neoparamoeba spp. western blotting revealed two distinctly different binding profiles.     

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD)

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.     

Migration of hatchery‐reared Atlantic salmon and wild anadromous brown trout post‐smolts in a Norwegian fjord system

Hatchery‐reared Atlantic salmon Salmo salar ( n  = 25) and wild anadromous brown trout (sea trout) Salmo trutta ( n  = 15) smolts were tagged with coded acoustic transmitters and released at the mouth of the River Eira on the west coast of Norway. Data logging receivers recorded the fish during their outward migration at 9, 32, 48 and 77 km from the release site. Seventeen Atlantic salmon (68%) and eight sea trout (53%) were recorded after release. Mean migratory speeds between different receiver sites ranged from 0·49 to 1·82 body lengths (total length) per second (bl s⁻¹) for Atlantic salmon and 0·11–2·60 bl s⁻¹ for sea trout. Atlantic salmon were recorded 9, 48 and 77 km from the river mouth on average 28, 65 and 83 h after release, respectively. Sea trout were recorded 9 km from the release site 438 h after release. Only four (23%) sea trout were detected in the outer part of the fjord system, while the rest of the fish seemed to stay in the inner fjord system. The Atlantic salmon stayed for a longer time in the inner part than in the outer parts of the fjord system, but distinct from sea trout, migrated through the whole fjord system into the ocean.     

Oral L-cysteine ethyl ester (LCEE) reduces amoebic gill disease (AGD) in Atlantic salmon Salmo salar

There is a need for the development of alternative therapeutic treatments for amoebic gill disease (AGD) in Atlantic salmon Salmo salar L. to maintain the sustainability of the Tasmanian Atlantic salmon aquaculture industry. This study aimed to assess the effects of the mucolytic drug L-cysteine ethyl ester (LCEE) on marine Atlantic salmon mucus and whether or not it may have a therapeutic advantage for the alleviation of AGD when administered orally. We also aimed to document any physiological consequences of LCEE. Results showed that LCEE significantly decreased the viscosity of marine Atlantic salmon mucus both in vitro, where LCEE concentration showed a negative relationship to mucus viscosity (R2 = 0.95 at 11.5 s(-1)), and in vivo. Oral administration of LCEE at 52.7 mg LCEE kg(-1) fish d(-1) over 2 wk significantly delayed the progression of AGD-associated pathology during an aggressive, cohabitation induced, laboratory infection. Medicated fish had approximately 50% less gill filaments affected by AGD than control fed fish at 3 d post-infection when assessed using histology. Palatability of medicated feed was shown to be approximately 65% of control feed. No osmoregulatory disturbance was seen in medicated fish, although blood and whole body flux data indicated a slight acidosis coinciding with an increased plasma total ammonia concentration. However, both variables were within a tolerable physiological range and returned to control levels 3 d post-cessation of medicated feed. LCEE holds potential as an in-feed additive when administered over 2 wk prior to infection to delay the progression of AGD associated pathology. From the parameters measured, LCEE seems to have minimal physiological consequences after 2 wk of administration.     

Metabolic recovery in Atlantic salmon fry and parr following forced activity

Atlantic salmon Salmo salar fry and parr were subjected to 5 min of forced activity and the subsequent changes in oxygen consumption and ammonia excretion rates were evaluated over a 24 h period. In a second experiment, individual Atlantic salmon fry and parr were freeze‐clamped in liquid nitrogen, before, immediately following a 5 min activity period, or after periods of recovery up to 2 h. Samples were analysed for whole body phosphocreatine (PCr), ATP and lactate. Five minutes of forced activity resulted in significant increases in both oxygen consumption and ammonia excretion rates. Changes in the oxygen consumption rates were greater in the parr compared with the fry. In contrast, the post‐exercise ammonia excretion rates were nearly twice as high for the fry compared with the parr. Exercise also caused a marked decrease in PCr levels (c. 47 and 65% in fry and parr, respectively), no change in ATP levels and a significant increase in lactate levels in Atlantic salmon fry and parr. Recovery of PCr occurred quickly (between 15 and 30 min) in fry and parr. Although the post‐activity levels of lactate were lower in fry (c. 3 μmol g^?1) compared with parr (c. 14 μmol g^?1), lactate levels returned to control levels within 60 min in fry, but it took >2 h for this metabolite to recover in parr. Compared with parr, these findings show that Atlantic salmon fry possess a reduced anaerobic capacity, and these results are consistent with the theoretical and experimental evidence that smaller fish support burst swimming through aerobic processes.     

Reproductive ecology of cultured fish in the wild

Domestication has been shown to have an effect on morphology and behaviour of Atlantic salmon ( Salmo salar ). We compared swimming costs of three groups of juvenile Atlantic salmon subject to different levels of domestication: (1) wild fish; (2) first generation farmed fish origination from wild genitors; and (2) seventh generation farmed fish originating from Norwegian aquaculture stocks. We assessed swimming costs under two types of turbulent flow (one mean flow velocity of 23 cm s⁻¹ and two standard deviations of flow velocity of 5 and 8 cm s⁻¹). Respirometry experiments were conducted with fish in a mass range of 5–15 g wet at a water temperature of 15° C. Our results confirm (1) that net swimming costs are affected by different levels of turbulence such that, for a given mean flow velocity, fish spent 1·5‐times more energy as turbulence increased, (2) that domesticated fish differed in their morphology (having deeper bodies and smaller fins) and in their net swimming costs (being up to 30·3% higher than for wild fish) and (3) that swimming cost models developed for farmed fish may be also be applied to wild fish in turbulent environments.     

Coho salmon haematological, metabolic and acid‐base changes during exercise and recovery in sea water

Cannulated, seawater‐acclimated coho salmon Oncorhynchus kisutch were swum to exhaustion in a seawater tunnel (10° C, mean U _crit 50 cm s⁻¹), resulting in metabolic acidosis and increased plasma electrolyte and cortisol concentrations, which were corrected during a 4 h recovery. Because the swimming and physiological performance data were similar to those of other salmonids, it was concluded that life‐history limitations, besides their exercise capabilities in upwelling zones, probably explain declining coho salmon populations.