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.     

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.     

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 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.     

A review of infectious gill disease in marine salmonid fish

Infectious gill diseases of marine salmonid fish present a significant challenge in salmon-farming regions. Infectious syndromes or disease conditions affecting marine-farmed salmonids include amoebic gill disease (AGD), proliferative gill inflammation (PGI) and tenacibaculosis. Pathogens involved include parasites, such as Neoparamoeba perurans, bacteria, such as Piscichlamydia salmonis and Tenacibaculum maritimum, and viruses, such as the Atlantic salmon paramyxovirus (ASPV). The present level of understanding of these is reviewed with regard to risk factors, potential impacting factors, methods of best practice to mitigate infectious gill disease, as well as knowledge gaps and avenues for future research.     

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.     

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.     

Amoebic gill disease (AGD)-affected Atlantic salmon, Salmo salar L., are resistant to subsequent AGD challenge

There is inconsistent evidence of resistance of Atlantic salmon, Salmo salar L., to amoebic gill disease (AGD). Here, evidence is presented that demonstrates that Atlantic salmon exposed and subsequently challenged with AGD are more resistant than naïve control fish. Seventy‐three per cent of Atlantic salmon previously exposed to AGD survived to day 35 post‐challenge compared with 26% exposed to Neoparamoeba sp. for the first time, yet the gill pathology of surviving naïve control or previously exposed fish was not significantly different. Development of resistance to AGD is associated with anti‐Neoparamoeba sp. antibodies that were detectable in serum of 50% of surviving Atlantic salmon previously exposed to AGD. However, anti‐Neoparamoeba sp. antibodies were not detectable in cutaneous mucus of resistant fish. Increased resistance of Atlantic salmon after secondary Neoparamoeba sp. infection and detection of specific serum antibodies provides support for the development of a vaccine for AGD.     

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.     

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.     

A comparison of the use of different swab materials for optimal diagnosis of amoebic gill disease (AGD) in Atlantic salmon (Salmo salar L.)

Routine gill swabbing is a non-destructive sampling method used for the downstream qPCR detection and quantitation of the pathogen Neoparamoeba perurans, a causative agent of amoebic gill disease (AGD). Three commercially available swabs were compared aiming their application for timelier AGD diagnosis (Calgiswab^® (calcium alginate fibre-tipped), Isohelix^® DNA buccal and cotton wool-tipped). Calcium alginate is soluble in most sodium salts, which potentially allows the total recovery of biological material, hence a better extraction of target organisms’ DNA. Thus, this study consisted of (a) an in vitro assessment involving spiking of the swabs with known amounts of amoebae and additional assessment of retrieval efficiency of amoebae from agar plates; (b) in vivo testing by swabbing of gill arches (second, third and fourth) of AGD-infected fish. Both in vitro and in vivo experiments identified an enhanced amoeba retrieval with Calgiswab® and Isohelix® swabs in comparison with cotton swabs. Additionally, the third and fourth gill arches presented significantly higher amoebic loads compared to the second gill arch. Results suggest that limiting routine gill swabbing to one or two arches, instead of all, could likely lead to reduced stress-related effects incurred by handling and sampling and a timelier diagnosis of AGD.     

Changes in the innate immune response of Atlantic salmon, Salmo salar L., exposed to experimental infection with Neoparamoeba sp

An experiment was conducted to determine the effect of Neoparamoeba sp. infection on the innate immune responses of Atlantic salmon. Atlantic salmon were experimentally infected with Neoparamoeba sp. and serially sampled 0, 1, 4, 6, 8 and 11 days post-exposure (dpe). Histological analysis of infected fish gill arches identified the presence of characteristic amoebic gill disease lesions as early as 1 dpe with a steady increase in the number of affected gill filaments over time. Immune parameters investigated were anterior kidney phagocyte function (respiratory burst, chemotaxis and phagocytosis) and total plasma protein and lysozyme. In comparison with non-exposed control fish basal respiratory burst responses were suppressed at 8 and 11 dpe, while phorbol myristate acetate-stimulated activity was significantly suppressed at 11 dpe. Variable differences in phagocytic activity and phagocytic rate following infection were identified. There was an increase in the chemotactic response of anterior kidney macrophages isolated from exposed fish relative to control fish at 8 dpe. Total protein and lysozyme levels were not affected by Neoparamoeba sp. exposure.     

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.     

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.     

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.     

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.