Effect of hydrogen peroxide as treatment for amoebic gill disease in Atlantic salmon (Salmo salar L.) in different temperatures

Amoebic gill disease (AGD) is a pathogenic disease in salmonids caused by Neoparamoeba perurans. Treatment of AGD infection has been through freshwater bathing of the fish. However, as the availability of fresh water is often limited, hydrogen peroxide has been introduced as an alternative treatment. This study investigated the effect of hydrogen peroxide as treatment for AGD‐infected salmon (Salmo salar L.,) at different seawater temperatures and hydrogen peroxide dosages. In total, 600 fish were challenged with N. perurans and the severity of the AGD infection was measured using a gill score scale. After challenge and disease development, the fish were distributed into 12 tanks. The treatment was performed at different seawater temperatures (8°C, 12°C, 17°C) using different hydrogen peroxide doses. Each temperature included an untreated control group. Linear models were used to analyse gill score. A significant effect of treatment was found (?0.68 ± 0.05) regardless of dose and temperature, suggesting that hydrogen peroxide was effective in treating AGD. When the model included dose, a negative linear relationship between dose and gill score was found. The study proved that treatment of AGD with hydrogen peroxide was successful, as gills partially recovered following treatment and further disease development was delayed.     

Preliminary success using hydrogen peroxide to treat Atlantic salmon,Salmo salar L., affected with experimentally induced amoebic gill disease (AGD)

Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H₂O₂), commonly used throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H₂O₂ in sea water (500, 1000 and 1500 mg L^?1) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post‐exposure and after 24 h. A concentration/duration combination of 1000 mg L^?1 for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H₂O₂ at 12 and 18 °C for 15 min at 1250 mg L^?1 and their re‐infection rate was compared to freshwater‐treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post‐bath. However, the results were largely equivocal in terms of disease resolution over a 3‐week period following treatment. These data suggest that H₂O₂ treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.     

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single‐sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.     

Effects of temperature on amoebic gill disease development: Does it play a role?

A relationship between increasing water temperature and amoebic gill disease (AGD) prevalence in Atlantic salmon (Salmo salar) has been noted at fish farms in numerous countries. In Scotland (UK), temperatures above 12°C are considered to be an important risk factor for AGD outbreaks. Thus, the purpose of this study was to test for the presence of an association between temperature and variation in the severity of AGD in Atlantic salmon at 10 and 15°C. The results showed an association between temperature and variation in AGD severity in salmon from analysis of histopathology and Paramoeba perurans load, reflecting an earlier and stronger infection post‐amoebae exposure at the higher temperature. While no significant difference between the two temperature treatment groups was found in plasma cortisol levels, both glucose and lactate levels increased when gill pathology was evident at both temperatures. Expression analysis of immune‐ and stress‐related genes showed more modulation in gills than in head kidney, revealing an organ‐specific response and an interplay between temperature and infection. In conclusion, temperature may not only affect the host response, but perhaps also favour higher attachment/growth capacity of the amoebae as seen with the earlier and stronger P. perurans infection at 15°C.     

The induction of laboratory-based amoebic gill disease revisited

Previous work in our laboratory defined a method of inducing laboratory‐based amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L. Gills of AGD‐affected fish were scraped and the debris placed into fish‐holding systems, eliciting AGD in naïve Atlantic salmon. While this method is consistently successful in inducing AGD, variability in the kinetics and severity of infections has been observed. It is believed that the infections are influenced by inherently variable viability of post‐harvest amoeba trophozoites. Here, a new method of experimental induction of AGD is presented that redefines the infection model including the minimum infective dose. Amoebae were partially purified from the gills of AGD‐affected Atlantic salmon. Trophozoites were characterized by light microscopy and immunocytochemistry and designated Neoparamoeba sp., possibly Neoparamoeba pemaquidensis. Cells were placed into experimental infection systems ranging in concentration from 0 to 500 cells L^?1. AGD was detected by gross and histological examination in fish held in all systems inoculated with amoebae. The number of gross and histological AGD lesions per gill was proportional to the inoculating concentration of amoebae indicating that the severity of disease is a function of amoeba density in the water column. The implications of these observations are discussed in the context of the existing AGD literature base as well as Atlantic salmon farming in south‐eastern Tasmania.     

Evaluation of sodium percarbonate as a bath treatment for amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a major health challenge for Atlantic salmon aquaculture globally. While freshwater bathing for 2 hr is effective in reducing infection severity, there is need for more rapid and lower cost alternatives. To this end, a combination of sodium percarbonate (SPC) in freshwater was examined for its treatment efficacy. Initial in vitro studies showed a reduction in amoeba viability when exposed for 30 min to freshwater containing >500 mg/L SPC. Subsequently, AGD‐affected salmon were bathed for 30 min in 16°C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16°C freshwater to mimic industry practice. Treatment at the highest SPC concentration caused extensive gill damage and substantial mortality. Neither occurred to a significant extent at lower SPC concentrations. Gill pathology of surviving fish 10 days post‐treatment (dpt) was comparable to or more severe than pre‐treatment, and significantly (p < .001) more severe than in 2 hr freshwater bathed fish. N. perurans DNA was confirmed by qPCR in all treatment groups at 10 dpt. The data indicate that a 30‐min exposure to SPC in freshwater is not a suitable alternative to existing freshwater treatment of AGD.     

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.     

Amoebic gill disease: sequential pathology in cultured Atlantic salmon, Salmo salar L

Amoebic gill disease (AGD) affects the marine culture phase of Atlantic salmon, Salmo salar L., in Tasmania. Here, we describe histopathological observations of AGD from smolts, sampled weekly, following transfer to estuarine/marine sites. AGD was initially detected histologically at week 13 post-transfer while gross signs were not observed for a further week post-transfer. Significant increases (P < 0.001) in the proportion of affected gill filaments occurred at weeks 18 and 19 post-transfer coinciding with the cessation of a halocline and increased water temperature at the cage sites. The progression of AGD histopathology, during the sampling period, was characterized by three phases. (1) Primary attachment/interaction associated with extremely localized host cellular alterations, juxtaposed to amoebae, including epithelial desquamation and oedema. (2) Innate immune response activation and initial focal hyperplasia of undifferentiated epithelial cells. (3) Finally, lesion expansion, squamation-stratification of epithelia at lesion surfaces and variable recruitment of mucous cells to these regions. A pattern of preferential colonization of amoebae at lesion margins was apparent during stage 3 of disease development. Together, these data suggest that AGD progression was linked to retraction of the estuarine halocline and increases in water temperature. The host response to gill infection with Neoparamoeba sp. is characterized by a focal fortification strategy concurrent with a migration of immunoregulatory cells to lesion-affected regions.     

Non-lethal loop-mediated isothermal amplification assay as a point-of-care diagnostics tool for Neoparamoeba perurans,the causative agent of amoebic gill disease

Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD). Two loop-mediated isothermal amplification (LAMP) assays targeting the parasite 18S rRNA and the Atlantic salmon EF1α, used as internal control, were designed. The N. perurans LAMP assay did not amplify close relatives N. pemaquidensis and N. branchiphila, or the host DNA. This assay detected 10⁶ copies of the parasite 18S rRNA gene under 13 min and 10³ copies under 35 min. Five “fast-and-dirty” DNA extraction methods were compared with a reference method and further validated by TaqMan™ qPCR. Of those, the QuickExtract buffer was selected for field tests. Seventy-one non-lethal gill swabs were analysed from AGD-clinically infected Atlantic salmon. The pathogen was detected under 23 min in fish of gill score >2 and under 39 min for lower gill scores. About 1.6% of the tests were invalid (no amplification of the internal control). 100% of positives were obtained from swabs taken from fish showing gill score ˃3, but only ~50% of positives for lower gill scores. The present LAMP assay could be implemented as a point-of-care test for the on-site identification of N. perurans; however, further work is required to improve its performance for lower scores.     

Effects of dissolved organic carbon and hardness in freshwater used to treat amoebic gill disease

Amoebic gill disease (AGD) is a significant disease of Atlantic salmon farmed in South East Tasmania. The commercial treatment for the disease is a freshwater bath for up to 4 h. Previous studies have shown that the chemical composition of the freshwater, in particular total water hardness, affects the efficacy of the treatment. The aim of this study was to determine if other water chemistry parameters, such as dissolved organic carbon (DOC), interact with total water hardness to affect treatment success. Firstly, the relative survival of isolated gill amoebae incubated for up to 3 h with hard or soft water (346.0 and 34.6 mg L^?1 CaCO₃ respectively) with low or high concentrations of humic or tannic acid (5 and 50 mg L^?1 respectively) was determined. Secondly, fish with AGD were bathed for 2.5 h in hard or soft water (249.3 and 35.3 mg L^?1 CaCO₃) containing either 5 or 20 mg L^?1 humic acid. The number of viable amoebae surviving on the gills and number of gill lesions were determined. It was found that the concentration of DOC used in this study that represents the levels commonly found around SE Tasmania is unlikely to have any commercial significance in the reduction in amoebae on the gills of Atlantic salmon. However, this study provided further support that freshwater selected for bathing AGD‐affected salmonids should be chosen primarily on its total water hardness.     

Further development of bithionol therapy as a treatment for amoebic gill disease in Atlantic salmon, Salmo salar L.

This study examined the efficacy of bithionol as a prophylactic or therapeutic oral treatment for Atlantic salmon (AS), Salmo salar , affected by amoebic gill disease (AGD). Furthermore, it explored the interaction of bithionol oral therapy with the current standard treatment (a freshwater bath for at least 3 h). The efficacy of three medicated feeds was determined in the trial by feeding AGD-affected AS at 1% body weight (BW) day⁻¹ either oil coated commercial feed (control) or prophylactic and therapeutic bithionol at 25 mg kg⁻¹ feed. Feeding commenced 2 weeks prior to exposure to Neoparamoeba spp. at 300 cells L⁻¹ and continued for 49 days post-exposure (PE). Bithionol when fed as a 2-week prophylactic or therapeutic treatment at 25 mg kg⁻¹ feed delayed the onset of AGD pathology and reduced the percentage of gill filaments with lesions. Administration of a 3-h freshwater bath at 28 days PE significantly reduced amoeba numbers to a similar level across all treatments; in contrast, gross gill score and percent lesioned filaments were reduced to different extents, the control having a significantly higher score than both bithionol treatments. Following the freshwater bath, clinical signs of AGD increased at a similar level across all treatments, albeit controls were significantly higher than the bithionol treatments immediately following freshwater treatment. This study demonstrated that bithionol at 25 mg kg⁻¹ feed, when fed as a 2-week prophylactic or a therapeutic treatment, delayed and reduced the intensity of AGD pathology and warrants further investigation as a treatment for AGD-affected AS.     

Efficacy of bithionol as an oral treatment for amoebic gill disease in Atlantic salmon Salmo salar (L.)

This study examined the efficacy of bithionol as an oral treatment for Atlantic salmon Salmo salar affected by amoebic gill disease (AGD). The current commercial management strategy of AGD is a costly 3 h freshwater bath. It is labour intensive and the number of baths needed appears to be increasing; hence, there is an effort to identify alternative treatments. Efficacy was examined by feeding AGD-affected Atlantic salmon twice daily to satiation with bithionol, an antiprotozoal, at 25 mg kg^− 1 feed. Three seawater (35‰, 17 °C) re-circulation systems were used each consisting of three tanks containing 32 Atlantic salmon smolts with an average (± SEM) mass of 90.4 g (± 5.2). Three feeds were examined in the trial including bithionol, plain commercial control and oil coated commercial control. Feeding commenced 2 weeks prior to exposure to Neoparamoeba spp. at 300 cells L^− 1 and continued for 28 days post-exposure. Efficacy was determined by examining gross gill score and identifying percent lesioned gill filaments twice weekly for 4 weeks post-exposure. Bithionol when fed as a two-week prophylactic treatment at 25 mg kg^− 1 feed delayed the onset of AGD pathology and reduced the percent lesioned gill filaments by 53% and halved the gill score from 2 to 1 when compared with both the plain and oil controls during an experimental challenge. There were no palatability problems observed with mean feed intake of bithionol over the trial duration with fish consuming higher levels of the bithionol diet compared to both the oil and plain controls. This study demonstrated that bithionol at 25 mg kg^− 1 feed, when fed as a two-week prophylactic treatment for Neoparamoeba spp. exposure, delayed and reduced the intensity of AGD pathology and warrants further investigation as an alternative to the current freshwater bath treatment for AGD-affected Atlantic salmon.     

Hydrogen peroxide oxygenation and disinfection capacity in recirculating aquaculture systems

Hydrogen peroxide (H₂O₂) treatment is an alternative for disinfection in aquaculture, which may be advantageous as it dissociates and disinfects while increasing water oxygen concentration. Yet, accurate dosing remains undeveloped in Recirculating Aquaculture Systems (RAS). Dosage requirements can depend on organic burden, stocking density, feeding frequency, salinity, temperature and biofilter performance. The present case study investigated the dual effect of H₂O₂ application for oxygen enrichment and disinfection when continuously applied to a RAS rearing European seabass. H₂O₂ addition equivalent to 2.4 and 15.8 H₂O₂ mg L⁻¹ were applied for 4 h per day in three 5-days experiments. H₂O₂ was injected at the inlet of protein skimmer and/or the rearing tanks in or without combination with traditional disinfection methods. Water microbial load and oxygen saturation were determined, along with stress markers glucose and cortisol in blood plasma of fish. Doses of 15.8 mg L⁻¹ H₂O₂ steadily increased oxygen levels in holding tank water from ∼50 % to over 100 % saturation while reducing microbial load (from 604.4 CFU ml⁻¹ in the rearing tanks before dosing to 159.8 CFU ml⁻¹ after application), achieving suitable conditions for commercial fish densities in RAS. The doses used had negligible impact on biofilter performance and did not affect the fish in terms of stress. Overall results indicate H₂O₂ is effective for disinfection and oxygenation of RAS systems when applied at appropriate dosage and we recommend the protein skimmer as the safest position in order to protect the bacterial community of the biofilters and the reared fish.     

Experimental amoebic gill disease of Atlantic salmon, Salmo salar L.: further evidence for the primary pathogenic role of Neoparamoeba sp. (Page, 1987)

Amoebic gill disease (AGD) has been attributed to infection by Neoparamoeba sp. The causal mechanisms for AGD lesion development and the primary pathogenic role of Neoparamoeba sp. require elucidation. Three groups of Atlantic salmon were exposed to viable gill isolated amoebae, to sonicated amoebae, or to sea water containing viable amoebae without direct contact with gill epithelia. Fish were removed 8 days post-exposure and the gills assessed histologically for AGD. AGD occurred only when fish were exposed to viable trophozoites. Consequently, in an accompanying experiment, infection was evaluated histologically at 12, 24 and 48 h post-exposure in three groups of salmon, one group being mechanically injured 12 h prior to exposure. A progressive host response and significant increase (P < 0.001) in the numbers of attached amoebae was apparent over the 48-h duration in undamaged hemibranchs in both treatment groups. There were no significant differences to mucous cell populations. Attachment of Neoparamoeba sp. to damaged gill filaments was significantly reduced (P < 0.05) by 48 h post-exposure. These data further confirm and describe the primary pathogenic role of Neoparamoeba sp. and the early host response in AGD. Preliminary evidence suggests that lesions resulting from physical gill damage are not preferentially colonized by Neoparamoeba sp.     

Detection of Neoparamoeba perurans by duplex quantitative Taqman real-time PCR in formalin-fixed, paraffin-embedded Atlantic salmonid gill tissues

The development and the application of a quantitative duplex real‐time PCR for the detection of Neoparamoeba perurans and the elongation factor α 1 gene (ELF) of Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), are described. A set of primers and probe was designed to amplify a 139‐bp fragment specific to the N. perurans 18S rRNA gene. The test was shown to be very sensitive, being able to detect as little as 13.4 DNA copies per μL corresponding to 0.15 fg of template DNA. In addition, the reaction that detected N. perurans was found to have a high degree of repeatability and reproducibility, to have a linear dynamic range (R² = 0.999) extending over 5 log₁₀ dilutions and to have a high efficiency (104%). The assay was applied to DNA samples extracted from 48 formalin‐fixed, paraffin‐embedded (FFPE) salmon gill tissues showing varying degrees of gill histopathology and amoebic gill disease (AGD)‐type histopathology ranging from absent to severe (each scored 0–3). Neoparamoeba perurans DNA was detected in all the blocks where AGD‐type histopathology was diagnosed microscopically and in 43.6% of the blocks showing signs of gill pathology. The association between parasitic load and gill histopathology and AGD‐type histopathology severity was also investigated. This study also describes the development and the application of a second real‐time PCR for the generic detection of Neoparamoeba spp., Page, 1987. A set of primers and probe conserved among the Neoparamoeba spp. was designed to amplify a 150‐bp fragment within the 18S rRNA gene. Applied to N. perurans‐negative gill tissues, the method was used to exclude the presence of other Neoparamoeba spp. in those blocks where gill pathology was observed microscopically.     

Effect of hydrogen peroxide and/or Flavobacterium psychrophilum on the gills of rainbow trout,Oncorhynchus mykiss (Walbaum)

The immune response and morphological changes in the gills of rainbow trout fry after immersion in hydrogen peroxide (H₂O₂), Flavobacterium psychrophilum or combined exposure were examined. The gills were sampled 4, 48, 125 and 192 h after exposure, and the regulation of expression of the following genes was investigated using qPCR: IgT, IgM, CD8, CD4, MHC I, MHC II, IL-4/13A, TcR-β, IL-10, IL-1β, IL-17, SAA and FoxP3. Bacteria were not observed in haematoxylin-and-eosin-stained gill tissue, but the presence of F. psychrophilum 16S rRNA was detected using qPCR. The 16S rRNA levels were correlated with gene expression. Although pretreatment with H₂O₂ before immersion in F. psychrophilum did not significantly alter the amount of bacteria found in the gill, the immune response was influenced: exposure to F. psychrophilum resulted in a negative correlation with expression of IL-17c1, MHC I and MHC II, while pretreatment with H₂O₂ resulted in a positive correlation with IL-4/13A and IgM. Exposure to either H₂O₂ or F. psychrophilum influenced the regulation of gene expression and damaged tissue. Exposure to both combined altered the immune response to infection and postponed healing of gill tissue.     

Evaluation of the immune response in rainbow trout fry,Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion‐based challenge with or without prior exposure to hydrogen peroxide (H₂O₂). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post‐infection. Separately, both H₂O₂ and F. psychrophilum influenced gene expression, and pre‐treatment with H₂O₂ influenced the response to infection with F. psychrophilum. Pre‐treatment with H₂O₂ also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H₂O₂‐treated fish in the early phase of infection was indicated, but H₂O₂ exposure did not affect antibody levels 50 days post‐infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre‐treated with H₂O₂ relative to the F. psychrophilum group. The results show that a single pre‐treatment with H₂O₂ impairs the response against F. psychrophilum and may intensify infection.     

Short communication: Acute toxicity of hydrogen peroxide in juvenile white shrimp Litopenaeus vannamei reared in biofloc technology systems

Biofloc technology (BFT) has been used to rear white shrimp, Litopenaeus vannamei. In this culturing system, the absence of aeration causes a rapid drop in dissolved oxygen levels, and hydrogen peroxide (H₂O₂) can be used as an emergency source of oxygen. This study aimed to determine the lethal concentration and safe level of H₂O₂ applied as a source of oxygen for juvenile white shrimp L. vannamei in a BFT system. Juveniles (1.39 ± 0.37 g) were exposed for 2 h to different concentrations of H₂O₂ [29 (100), 58 (200), 116 (400), 174 (600), 232 (800), 290 (1,000) and 348 (1,200) μL H₂O₂/L (ppm H₂O₂-29 %/L)] in addition to a control group without addition of H₂O₂, and the survival rates were monitored for 96 h. The LC₅₀ values and 95 % confidence intervals at 24, 48, 72 and 96 h were 235.5 (207–268), 199.1 (172–229), 171.1 (146–198) and 143.3 (120–170) μL H₂O₂/L, respectively. The safe level was 14.3 μL H₂O₂/L, and the highest concentration with survival rates similar to the control group (NOAEC) was 29 μL H₂O₂/L. In these concentrations, H₂O₂ can be used as a safe source of oxygen for L. vannamei reared in BFT systems.     

Reduced total hardness of fresh water enhances the efficacy of bathing as a treatment for amoebic gill disease in Atlantic salmon, Salmo salar L

The current treatment for amoebic gill disease (AGD)-affected Atlantic salmon involves bathing sea-caged fish in fresh water, often sourced from local dams, for 3-4 h. In both a small-scale laboratory and an on-farm field experiment, the effects of water hardness on the efficacy of freshwater bathing were assessed. Results showed that soft fresh water (19.3-37.4 mg L(-1) CaCO3), whether it be naturally soft city mains water or artificially softened dam water, was more efficacious at alleviating AGD in affected fish than hard fresh water (173-236.3 mg L(-1) CaCO3). Soft freshwater bathing significantly reduced viable gill amoebae numbers (from 73.9 to 40.9% of total count) and significantly alleviated gill pathology, both gross and histological. Following bathing, gross gill pathological scores of soft freshwater bathed fish lagged 2 weeks behind hard freshwater bathed fish. Significant gill lesion fragmentation, and shedding of lesion-associated hyperplastic tissue, was accompanied by a significant reduction in AGD-affected gill filaments in soft freshwater bathed fish. Furthermore, soft freshwater bathing alleviated the blood plasma electrolyte imbalance seen in control (sea water) and hard freshwater bathed fish. This study showed that the use of soft fresh water for bathing AGD-affected Atlantic salmon could be an improvement to the current method of treatment. Not only does it reduce gill amoeba numbers, but also, it is of a therapeutic advantage with the potential to reduce bathing frequency.