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.     

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.     

Presence of anti-Neoparamoeba sp. antibodies in Tasmanian cultured Atlantic salmon, Salmo salar L

Previous studies have indicated that when Atlantic salmon, Salmo salar L., are exposed to Neoparamoeba sp. the fish produce anti-Neoparamoeba sp. antibodies. It appears unlikely that these antibodies elicit any specific protection against amoebic gill disease (AGD) as fish with demonstrable activities have been affected by AGD. Experiments were conducted on Atlantic salmon cultured throughout Tasmania to assess the natural production of antibodies towards Neoparamoeba sp. Fish were sampled from areas where AGD was prevalent and from areas where there had been no reported cases. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-Neoparamoeba sp. antibody activities in serum. All fish from sea water had antibody activities greater than the negative control fish, including fish from areas with no reported cases of AGD. Time trial samples indicated that time after transfer to sea water did not appear to be a significant (P > 0.05) factor in antibody activity, however location was (P < 0.05). There was no agreement (corrected kappa value, 0.16) between the ELISA result and the isolation of Neoparamoeba sp. from the gills of the same fish. The results suggest that Atlantic salmon in seawater culture in Tasmania produce anti-Neoparamoeba sp. antibodies regardless of infection history, suggesting the presence of Neoparamoeba sp. in the environment.     

Environmental risk factors associated with amoebic gill disease in cultured salmon, Salmo salar L., smolts in Ireland

A 2-year study was carried out on amoebic gill disease (AGD) involving monthly samples of 1+ Atlantic salmon, Salmo salar L., smolts, histological assessment of the gills and analysis of environmental data. Gill pathology was seen before amoebae could be detected microscopically. These changes in gill integrity were associated with marine environmental conditions, particularly elevated ammonium, nitrite and chlorophyll levels. The results suggest that the environmental changes predispose salmon to colonization by amoebae and ciliates. High densities of histophagous scuticociliates were observed in the gills during periods of advanced gill pathology. A number of different amoebae were observed in close association with gill pathology. Neoparamoeba was not seen in high densities, nor was it associated with gill pathology, indicating that Neoparamoeba may not be the primary agent of the AGD in Irish salmonid culture.     

The effect of environmental factors on the distribution of Neoparamoeba pemaquidensis in Tasmania

Aquaculture in Tasmania is mostly carried out in estuaries. These estuarine habitats show a great variety and form unique environments in which Neoparamoeba pemaquidensis, the amoebic gill disease (AGD)-causing protozoan, may or may not survive. Tasmania is divided into two zones, one where AGD is present and one where AGD is absent, but any ecological data to rationalize this distribution is lacking. In in vitro trials N. pemaquidensis strains were exposed to different concentrations of ammonium sulphate, copper sulphate, copper sulphate and tannin, and different Neoparamoeba densities, salinities and temperatures. A trial using field water samples investigated the survival of N. pemaquidensis in waters sourced from AGD-free and AGD-positive zones, and water analysis was performed to determine any differences. Significantly decreased protozoan survival was found with exposure to increasing copper sulphate concentrations from 10 to 100,000 microM (P < 0.001), salinity of 15 per thousand (P < 0.001), low Neoparamoeba densities of 625 and 1,250 cells mL(-1) (P = 0.0005), and water sourced from Macquarie Harbour (P < 0.001). The water chemistry of this AGD-free zone showed significantly lower dissolved calcium and magnesium concentrations which may contribute to this area being AGD-free. Understanding of the ecology of N. pemaquidensis will enable better control and prevention strategies for Tasmanian salmon growers.     

Amoebic gill disease resistance is not related to the systemic antibody response of Atlantic salmon, Salmo salar L.

Amoebic gill disease (AGD) is a proliferative gill tissue response caused by Neoparamoeba perurans and is the main disease affecting Australian marine farmed Atlantic salmon. We have previously proposed that macroscopic gill health ('gill score') trajectories and challenge survival provide evidence of a change in the nature of resistance to AGD. In order to examine whether the apparent development of resistance was because of an adaptive response, serum was sequentially sampled from the same individuals over the first three rounds of natural AGD infection and from survivors of a subsequent non-intervention AGD survival challenge. The systemic immune reaction to 'wildtype' Neoparamoeba sp. was characterized by Western blot analysis and differentiated to putative carbohydrate or peptide epitopes by periodate oxidation reactions. The proportion of seropositive fish increased from 46% to 77% with each AGD round. Antibody response to carbohydrate epitope(s) was immunodominant, occurring in 43–64% of samples. Antibodies that bound peptide epitope were identified in 16% of the challenge survivors. A 1:50 (single-dilution) enzyme-linked immunosorbent assay confirmed a measurable immune titre in 13% of the survivors. There was no evidence that antibodies recognizing wildtype Neoparamoeba provided significant protection against AGD.     

Wild fish are not a significant reservoir for Neoparamoeba pemaquidensis (Page, 1987)

Amoebic gill disease (AGD), caused by the protozoan Neoparamoeba pemaquidensis (Page, 1987) is the most important disease affecting salmon farms in Tasmania. Reservoirs for this protozoan parasite are largely unknown. This study investigated wild fish as a potential reservoir of N. pemaquidensis . A total of 325 wild fish, comprising 12 different fish species, were caught from and around salmon farms and examined for the presence of AGD. None of the wild fish were infected with AGD. In a laboratory trial, seahorse, Hippocampus abdominalis , greenback flounder, Rhombosolea tapirina, and Atlantic salmon, Salmo salar, were challenged with N. pemaquidensis . Neoparamoeba pemaquidensis was detected on the gills on 10 of 15 (66.7%) flounder, nine of 24 (37.5%) seahorses, and six of six (100%) Atlantic salmon. However, paramoebae positive flounder and seahorse lacked the characteristic AGD gill pathology. It is concluded that AGD does not appear in wild fish and wild fish do not seem to be a reservoir of the pathogen.     

High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.     

Gill disease of marine fish caused by infection with Neoparamoeba pemaquidensis

Amoebic gill disease (AGD) of maricultured salmonids, turbot, Scophthalmus maximus (L.), European seabass, Dicentrarchus labrax (L.), and sharpsnout seabream, Diplodus puntazzo (Cetti), caused by Neoparamoeba pemaquidensis has been reported from Australia (Tasmania), Ireland, France, Chile, North America (Washington State and California) and Spain. Of the salmonids, Atlantic salmon, Salmo salar L., appears to be the most susceptible with rainbow trout, Oncorhynchus mykiss (Walbaum), also suffering significant disease. Only minor outbreaks have been reported in coho, O. kisutch (Walbaum), and chinook salmon, O. tshawytscha (Walbaum). The disease now accounts for 10–20% of production costs of Atlantic salmon in Tasmania and has lead to temporary abandonment of culture of this species in parts of Spain. It is of lesser, but still significant, importance in other countries. Much is known about the pathology of AGD but the pathophysiology of the disease is poorly understood. There is evidence that non-specific immunity is involved in fish acquiring resistance to AGD, but no unequivocal evidence exists for protection as a result of specific immune responses. To date, for salmonids, the only effective treatment for AGD is a freshwater bath. Control procedures based on modification of management strategies have been minimal and virtually unresearched.     

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.     

Gross pathology and its relationship with histopathology of amoebic gill disease (AGD) in farmed Atlantic salmon, Salmo salar L

Gross pathological assessment of amoebic gill disease (AGD) is the only non-destructive, financially viable method for rapid and broad-scale disease management of farmed Atlantic salmon, Salmo salar L., in Tasmania. However, given the presumptive nature of this diagnosis, the technique has been considered questionable. This study investigated the degree of conformity between clinical signs and histological lesions observed in a commercial setting. Three groups of Atlantic salmon (n = 42, 100 and 100, respectively) were collected from various farm sites in southern Tasmania between December 2001 and April 2003. Micro-stereoscopic analysis showed that grossly affected tissue regions correspond to areas of hyperplastic lamellar fusion, generally in association with attached Neoparamoeba sp. Agreement between gross signs of AGD and histopathological diagnosis was compared. Kappa analysis indicated moderate to good agreement between methods (kappa = 0.52-0.74). Individual cases of disagreement were further scrutinized and several factors were found to influence the level of agreement between the two methods. Stage of disease development, lesions derived from other pathogens, assessor interpretation/experience, sampling methods, histological technique and/or experience were potential confounding factors. It was concluded that clinical diagnosis is acceptable as a farm-monitoring tool only. Removal of grossly affected tissue and subsequent histological examination is recommended to improve diagnostic accuracy.     

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.     

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.     

Salmonid gill bacteria and their relationship to amoebic gill disease

16S ribosomal RNA gene analysis was used to assess the bacterial community associated with Atlantic salmon, Salmo salar L., gills which were either affected by amoebic gill disease (AGD) or were AGD-negative, in order to determine the role that bacteria may play in the development of AGD. AGD-positive specimens were either infected in the laboratory with Neoparamoeba pemaquidensis, the causative agent of AGD, or were obtained from commercial salmon cages. Samples from laboratory fish maintained in sea water possessed a marine-type community while field samples which had been treated by a series of freshwater baths possessed a more diverse community which included variable proportions of different bacterial ecotypes, including groups typically associated with soil, skin surfaces and faeces. Samples from fish infected with AGD in the laboratory and a sample from one of two salmon cage fish specimens were dominated by a phylotype belonging to the strictly marine bacterial genus Psychroserpens (family Flavobacteriaceae, phylum Bacteroidetes). The phylotype was not detected in any of the AGD-negative samples or in one of two AGD-positive samples obtained from fish subjected to temporary freshwater immersion. The possibility of certain Psychroserpens species as potential opportunistic pathogens associated with salmonid AGD is proposed.     

Microfauna associated with amoebic gill disease in sea-farmed Atlantic salmon, Salmo salar L., smolts

A study of microfauna, associated with pathological changes in the gills of Atlantic salmon, Salmo salar L., was conducted over 2001-2002. Monthly samples of 1(+) salmon smolts were taken, protozoan populations were quantified and gill health was assessed histologically. Protozoan densities were correlated with pathological changes, in order to determine their possible role in lesions in the gills. The most severe gill tissue changes were observed in summer/autumn and the least in spring. A diverse polyphyletic protozoan community was observed colonizing the gills, including Neoparamoeba sp., other amoebae, scuticociliates, Ichthyobodo-like flagellates, trichodinid ciliates and prostomatean ciliates. The earlier gill tissue changes in the gill were not always associated with the presence of these microorganisms, whereas amoebae (other than Neoparamoeba sp.), Ichthyobodo-like flagellates and trichodinid ciliates correlated with augmenting gill lesions. Neoparamoeba sp. was present, but its abundance did not correlate with the disease. This study suggests that a diversity of protozoans including Ichthyobodo-like flagellates, trichodinid ciliates and amoebae other than Neoparamoeba sp. are involved in the aetiology of amoebic gill disease in the Irish situation.     

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.     

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.     

Cardiac morphology in relation to amoebic gill disease history in Atlantic salmon, Salmo salar L.

Fish from cages with histories of heavy and light amoebic gill disease (AGD) outbreaks were harvested and the morphology, histology and activities of lactate dehydrogenase determined. Although fish with a history of heavy AGD were smaller, their heart somatic indices were similar to those of fish with a history of light AGD. However, morphometrically the ratios of ventricle axis length and width and axis length and height were significantly higher, and there was an overall thickening of the muscularis compactum in the ventricle of fish with heavy AGD history. There was no difference in the lactate dehydrogenase activity of the ventricle muscle in the two fish groups. These results suggest that the change in ventricle shape associated with AGD was a possible compensation for an increased afterload where the lengthening of the ventricle was compensated for by an increase in muscle thickness, but without any overall ventricular hypertrophy or gain in ventricular mass. This suggests that AGD may be associated with cardiovascular compromise in affected fish.     

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.