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.     

The viscosity and glycoprotein biochemistry of salmonid mucus varies with species,salinity and the presence of amoebic gill disease

Fish mucus has previously been reported to change in appearance and composition among species and in response to changes in salinity and disease status. This study reports on the mucus viscosity and glycoprotein biochemistry of Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum) in freshwater and seawater, both naïve to and affected by amoebic gill disease (AGD). Cutaneous mucus viscosity was measured over a range of shear rates (11.5, 23, 46 and 115 s^–1), and non-Newtonian behaviour was demonstrated for all three species. Mucus viscosity was significantly greater in seawater than in freshwater for all species, and significantly lower in AGD-affected Atlantic salmon and brown trout. Mucus glucose, total protein and osmolality data indicated that differences in viscosity due to salinity were mostly attributed to changes in mucus hydration, while differences due to disease were mostly attributed to changes in mucus composition. Trends in gill mucus cell histochemistry included shifts in glycoproteins from neutral mucins in freshwater to acidic mucins in seawater, and shifts towards neutral mucins, with an increase in mucus cell numbers, in response to AGD. Results suggested that Atlantic salmon and brown trout are more similar to one another in their mucus profile than to rainbow trout. Atlantic salmon and brown trout both exhibited a whole-body mucus response to AGD, whereas rainbow trout exhibited only a local gill response. Findings hold implications for fish physiology and pathology, and indicate that future fish-disease management strategies should be species and condition specific.Communicated by I.D. HumeThe word mucus has been used in its noun form throughout the paper for clarity

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An erratum to this article can be found at .     

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.     

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.     

In vitro cultured Neoparamoeba perurans causes amoebic gill disease in Atlantic salmon and fulfils Koch's postulates

Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Koch's postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Koch's postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature.     

Cloning and functional characterisation of a peroxiredoxin 1 (NKEF A) cDNA from Atlantic salmon (Salmo salar) and its expression in fish infected with Neoparamoeba perurans

Peroxiredoxin 1 (Prx 1), also known as natural killer enhancing factor A (NKEF A), has been implicated in the immune response of both mammals and fish. Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a significant problem for the Atlantic salmon (Salmo salar L.) aquaculture industry based in Tasmania, Australia. Here we have cloned and functionally characterized a Prx 1 open reading frame (ORF) from Atlantic salmon liver and shown that Prx 1 gene expression was down-regulated in the gills of Atlantic salmon displaying symptoms of AGD. The Prx 1 ORF encoded all of the residues and motifs characteristic of typical 2-Cys Prx proteins from eukaryotes and the recombinant protein expressed in Escherichia coli catalyzed thioredoxin (Trx)-dependent reduction of H(2)O(2), cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH) with K(m) values of 122, 77 and 91 μM, respectively, confirming that it was a genuine 2-Cys Prx. The recombinant protein also displayed a double displacement reaction mechanism and a catalytic efficiency (k(cat)/K(m)) with H(2)O(2) of 1.5 × 10(5) M(-1) s(-1) which was consistent with previous reports for the 2-Cys Prx family of proteins. This is the first time that a Prx 1 protein has been functionally characterized from any fish species and it paves the way for further investigation of this important 2-Cys Prx family member in fish.     

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.     

Randomized clinical trial to investigate the effectiveness of teflubenzuron for treating sea lice on Atlantic salmon

A double-blind, randomized control clinical trial was performed to investigate the effectiveness of teflubenzuron in controlling sea lice Lepeophtheirus salmonis on farmed Atlantic salmon Salmo salar. A total of 40 sea cages from 3 commercial cage sites in Atlantic Canada were used in this Good Clinical Practice (GCP) trial. The teflubenzuron was administered in the feed at a dosage of 10 mg kg(-1) biomass d(-1) for 7 d. Medicated and control cages were matched by site, cage size, and pre-treatment mean lice counts using cages as the unit of concern. Post-treatment lice counts and staging of developmental stages were performed at 1 and 2 wk after the end of treatment. Chalimus stages in medicated cages were significantly lower than in control cages at 1 wk (79% reduction in mean lice counts, p < 0.001), and at 2 wk (53% reduction, p < 0.001). Mobile (pre-adult and adult) stages were also significantly reduced in medicated cages at 1 wk (69% reduction, p < 0.01), and at 2 wk (40% reduction, p < 0.01) post-treatment, respectively. Teflubenzuron was proven effective for reducing lice burdens on salmon despite the low parasite levels experienced during the trial and the recruitment of lice from the untreated cages. The use of cage as the unit of concern was an important design component of this trial.     

Feed intake and tissue distribution of florfenicol in cod (Gadus morhua) administered feed with different fat contents

This study was performed to examine the appetite and the corresponding plasma and tissue distribution of florfenicol when administered to healthy groups of cod using medicated and non‐medicated salmonid and marine feeds. Marine feed contains approximately 18% fat whereas salmonid feed contains approx. 30% fat. Two groups of fish were medicated with florfenicol at a dosage of 10 mg kg^?1 day^?1 for 10 consecutive days when the drug was administered either via marine or salmonid pellets. Two groups of fish also received either non‐medicated marine or salmonid pellets. Twenty‐four hours after giving the medicated marine feed, 14 out of 20 fish contained detectable concentrations of florfenicol with mean values (n = 14) of 4.67 ± 4.02 μg ml^?1 in plasma, 2.29 ± 2.11 μg g^?1 in muscle and 0.79 ± 0.69 μg g^?1 in the liver. In the fish given medicated salmonid feed, 18 of 20 fish contained detectable concentrations of florfenicol with mean values (n = 18) of 1.77 ± 1.84 μg ml^?1 in plasma, 0.75 ± 0.66 μg g^?1 in muscle and 0.30 ± 0.25 μg g^?1 in the liver. Decreased feed intake of the salmonid feed, both medicated and non‐medicated, was noted when compared to medicated and non‐medicated marine feed. No difference in feed consumption was registered between medicated and non‐medicated marine feed, however a difference was noted between the medicated and non‐medicated salmonid feed.     

Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.     

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.     

18S ribosomal DNA-based PCR identification of Neoparamoeba pemaquidensis, the agent of amoebic gill disease in sea-farmed salmonids

Neoparamoeba pemaquidensis is a parasomal amoeboid protozoan identified as the agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar reared in sea-pens in Tasmania, Australia, and coho salmon Oncorhynchus kisutch farmed on the west coast of the USA. Outbreaks of AGD caused by immunologically cross-reactive paramoebae have also been reported in sea-farmed salmonids in several other countries. Complete 18S rDNA sequences were determined for respective paramoebae isolated from infected gills of salmon from Tasmania and Ireland, and N. pemaquidensis isolates from the USA and UK, including representative free-living isolates. Alignments over 2110 bp revealed 98.1 to 99.0% sequence similarities among isolates, confirming that paramoebae implicated in AGD in geographically distant countries were homologous and belonged to the same species, N. pemaquidensis. The results supported previous findings that N. pemaquidensis exists as a widely distributed, amphizoic marine protozoan. Partial 18S rDNA sequences were obtained for the ultrastructurally similar species, N. aestuarina, and for the morphologically similar but non-parasomal amoeba Pseudoparamoeba pagei. N. aestuarina had 95.3 to 95.7% sequence similarities with N. pemaquidensis strains, which distinguished 2 closely related but separate species. Neoparamoeba spp. were not analogous to P. pagei or to other marine Gymnamoebia. We designed 4 oligonucleotide primers based on elucidated 18S rDNA sequences and applied them to single-step and nested 2-step PCR protocols developed to identify N. pemaquidensis to the exclusion of apparently closely related and non-related protistan taxa. Nested PCR was able to detect the AGD parasite from non-purified, culture-enriched net microfouling samples from Atlantic salmon sea-pens in Tasmania, and confirmed that N. pemaquidensis was also responsible for AGD in chinook salmon O. tshawytscha in New Zealand. Our sequence and PCR analyses have now shown that AGD affecting 3 different salmonid species farmed in 4 countries are associated with N. pemaquidensis. A species-specific diagnostic PCR provides for the first time, a highly specific detection and identification assay for N. pemaquidensis that will facilitate future ecological and epidemiological studies of AGD.     

Acid–base and respiratory effects of confinement in Atlantic salmon affected with amoebic gill disease

Amoebic gill disease (AGD) in cultured salmonids causes severe multifocal hyperplastic lesions in the gills with the potential to influence respiratory and acid–base physiology. Atlantic salmon Salmo salar affected with AGD were surgically implanted with dorsal aortic catheters and, following recovery, were confined for 5 min ( n  = 16) or left undisturbed ( n  = 8). Confinement caused an acute extracellular acidosis that was corrected in 6 h amongst surviving fish. There was a gradual increase in plasma lactate concentrations that peaked at 1 h post-confinement then declined by 9 h recovery. In a second experiment, AGD-affected fish were confined then recovered either in a tank of static water ( n  = 9) or while being forced to swim at 1·5 body lengths s⁻¹ ( n  = 6). There was no significant difference between fish recovered by swimming and those in static water in terms of recovery of the acute extracellular acidosis and lactate accumulations coincident with exhaustive exercise. Confinement severely compromised the survival of AGD-affected Atlantic salmon, although survivors appeared to recover similarly to other studies. Forced swimming of AGD-affected Atlantic salmon following confinement did not facilitate recovery and is unlikely to be a useful strategy for mitigating the effects of stressful episodes such as crowding and fish movement and commercial handling.     

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.     

Susceptibility of rainbow trout Oncorhynchus mykiss,Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.