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Amoebic gill disease (AGD) is caused by the marine amoeba Neoparamoeba perurans, a facultative parasite. Despite the significant impact this disease has on production of Atlantic salmon worldwide, the mechanisms involved in host-parasite interaction remains unknown. Excessive gill mucus secretion is reported as a host defence mechanism to prevent microbial colonization in the gill epithelium. Despite this response, N. perurans still attaches and proliferates. The present study aimed to investigate the interaction between N. perurans and mucin, the most abundant component in mucus. An in vitro adhesion assay using bovine submaxillary mucin (BSM) demonstrated that amoeba binding to mucin-coated substrate was significantly higher than to the BSA control. This binding interaction is likely glycan-mediated as pre-incubation with galactose, galactosamine, N-acetylgalactosamine and fucose reduced mucin adhesion to control levels. The ability of N. perurans to secrete proteases that target mucin was also investigated. Protease activity was detected in the amoeba culture media in the presence of BSM, but not when protease inhibitor was added. Mucin degradation was visually assessed on protein gels. This study provides preliminary evidence that N. perurans has developed mechanisms to interact with and evade mucus by binding to mucin glycan receptors and secreting proteases with mucolytic activity.

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naive Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.

Amoebic gill disease (AGD), caused byNeoparamoeba 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 degrees C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16 degrees 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. peruransDNA 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 (AGD) is the primary health concern for Atlantic salmon Salmo salar farmed in Tasmania, Australia. Neoparamoeba perurans is the aetiological agent of AGD; however, a diversity of other amoebae colonise the gills, and their role in AGD is unknown. Previous studies which document these accompanying amoebae on AGD-affected farmed Atlantic salmon relied on culture-based techniques which do not accurately determine the prevalence and abundance of these species nor whether they correlate with AGD pathology. Drawing on our previous culture-based study, here we develop and apply 5 new Taqman quantitative PCR (qPCR) assays to profile the prevalence of multiple amoeba species on the gills of AGD-affected Atlantic salmon held at 2 Tasmanian farm sites over a 1 yr period. The prevalence and abundance of N. perurans was also assessed using a previously established qPCR method. N. perurans was the dominant species, and its abundance positively correlated with the progression of gross gill pathology. Only a small number of sporadic detections of Pseudoparamoeba and Vannellida species were observed. Nolandella spp. was the notable exception, as it was the most prevalent amoeba (92 %) at 1 site at 1 sample time, during which no N. perurans were detected on gills but low levels of gross gill pathology were observed. N. perurans is the predominant species and primary pathogen of AGD; however, there were instances when they were not detected on diseased gills and Nolandella spp. were highly prevalent. The significance of Nolandella spp. in relation to AGD is not yet understood.

Disease caused by parasitic amoebae impacts a range of aquatic organisms including finfish, crustaceans, echinoderms and molluscs. Despite the significant economic impact caused in both aquaculture and fisheries, the aetiology of most aquatic amoebic diseases is uncertain, which then affects diagnosis, treatment and prevention. The main factors hampering research effort in this area are the confusion around amoeba taxonomy and the difficulty proving that a particular species causes specific lesions. These issues stem from morphological and genetic similarities between cryptic species and technical challenges such as establishing and maintaining pure amoeba cultures, scarcity of Amoebozoa sequence data, and the inability to trigger pathogenesis under experimental conditions. This review provides a critical analysis of how amoebae are commonly identified and defined as aetiological agents of disease in aquatic animals and highlights gaps in the available knowledge regarding determining pathogenic Amoebozoa.

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.

One of the first critical steps in the pathogenesis of amoebic gill disease (AGD) of farmed salmon is the adhesion of the causative amoeba to the host. The current study aimed to investigate the potential involvement of glycan-binding proteins expressed on the extracellular surface of Neoparamoeba perurans in gill tissue recognition and binding. The glycan-binding properties of the surface membrane of N. perurans and the carbohydrate binding profile of Atlantic salmon gill-derived epithelial cells were identified through the use of glycan and lectin microarrays, respectively. The occurrence of specific carbohydrate-mediated binding was then further assessed by in vitro attachment assays using microtitre plates pre-coated with the main glycan candidates. Adhesion assays were also performed in the presence of exogenous saccharides with the aim of blocking glycan-specific binding activity. Comparative analysis of the results from both lectin and glycan arrays showed significant overlap, as some glycans to which binding by the amoeba was seen were reflected as being present on the gill epithelial cells. The two main candidates proposed to be involved in amoeba attachment to the gills are mannobiose and N-acetylgalactosamine (GalNAc). Adhesion of amoebae significantly increased by 33.5 and 23% when cells were added to α1,3-Mannobiose-BSA and GalNAc-BSA coated plates. The observed increased in attachment was significantly reduced when the amoebae were incubated with exogenous glycans, further demonstrating the presence of mannobiose- and GalNAc-binding sites on the surfaces of the cells. We believe this study provides the first evidence for the presence of a highly specific carbohydrate recognition and binding system in N. perurans. These preliminary findings could be of extreme importance given that AGD is an external parasitic infestation and much of the current research on the development of alternative treatment strategies relies on either instant amoeba detachment or blocking parasite attachment.

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

RNA interference (RNAi) mediated by double stranded RNA (dsRNA) has emerged as one of the most promising techniques to study gene function of non-model protozoan parasites. We have previously demonstrated that bacterially expressed dsRNA delivered by immersion elicited successful knockdown in Neoparamoeba pemaquidensis, the non-infective species closely related to the causative agent of salmonid amoebic gill disease (AGD). However, considering that amoebae naturally feeds on microorganisms, direct ingestion of bacteria designed to express dsRNA could allow rapid and low-cost analysis of gene function on a large-scale. Therefore, the main objective of this study was to investigate whether ingestion of bacteria-expressing dsRNAs would also induce suppression of N. pemaquidensis β-actin and EF1α. Despite effective bacterial uptake, no significant variation in EF1α relative copy number was triggered by dsRNA ingestion. β-actin, on the other hand, presented similar silencing efficiency to what was observed in our previous soaking study. However, the observed RNAi response was delayed by at least 72 h. The present work provides evidence that delivery of bacterially expressed dsRNA through feeding can be successfully achieved in N. pemaquidensis, albeit not as efficiently as by soaking. Therefore, further investigation is required to develop more efficient and specific RNAi delivery systems in Neoparamoeba species. To our knowledge, this is the first time that RNAi-mediated knockdown through ingestion was attempted to manipulate gene function of a marine amoeba.

RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using β-actin and EF1-α as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 μg/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, β-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.

Aquatic animal diseases are one of the most significant constraints to the development and management of aquaculture worldwide. As a result, measures to combat diseases of fish and shellfish have assumed a high priority in many aquaculture-producing countries. RNA interference (RNAi), a natural mechanism for post-transcriptional silencing of homologous genes by double-stranded RNA (dsRNA), has emerged as a powerful tool not only to investigate the function of specific genes, but also to suppress infection or replication of many pathogens that cause severe economic losses in aquaculture. However, despite the enormous potential as a novel therapeutical approach, many obstacles must still be overcome before RNAi therapy finds practical application in aquaculture, largely due to the potential for off-target effects and the difficulties in providing safe and effective delivery of RNAi molecules in vivo. In the present review, we discuss the current knowledge of RNAi as an experimental tool, as well as the concerns and challenges ahead for the application of such technology to combat infectious disease of farmed aquatic animals.

After the publication of the work entitled "Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfate)β-1→3GalNAc(4-Sulfate)β-1→] motifs in dermatan sulfate on heparin cofactor II activity", by Kozlowski et al., BMC Biochemistry 2011, 12:29, we found that the legends to Figures 2 to 5 contain serious mistakes that compromise the comprehension of the work. This correction article contains the correct text of the legends to Figures 2 to 5.

Background Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Results Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units. Conclusions Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.