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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.

Neoparamoeba spp. are amphizoic amoebae with the capacity to colonize the gills of some marine fish, causing AGD. Here, the gill tissue transcriptome response of Atlantic salmon ( Salmo salar L.) to AGD is described. Tanks housing Atlantic salmon were inoculated with Neoparamoeba spp. and fish sampled at time points up to 8 days postinoculation (pi.). Gill tissues were taken from AGD-affected fish, and a DNA microarray was used to compare global gene expression against tissues from AGD-unaffected fish. A total of 206 genes, representing 190 unique transcripts, were reproducibly identified as up- or downregulated in response to Neoparamoeba spp. infection. Informative transcripts having GO biological process identifiers were grouped according to function. Although a number of genes were placed into each category, no distinct patterns were observed. One Atlantic salmon cDNA that was upregulated in infected gill relative to noninfected gill at 114 and 189 h pi. showed significant identity with the Xenopus, mouse, and human anterior gradient-2 (AG-2) homologs. Two Atlantic salmon AG-2 mRNA transcripts, designated asAG-2/1 and asAG-2/2, were cloned, sequenced, and shown to be predominantly expressed in the gill, intestine, and brain of a healthy fish. In AGD-affected fish, differential asAG-2 expression was confirmed in samples used for microarray analyses as well as in AGD-affected gill tissue taken from fish in an independent experiment. The asAG-2 upregulation was restricted to AGD lesions relative to unaffected tissue from the same gill arch, while p53 tumor suppressor protein mRNA was concurrently downregulated in AGD lesions. Differential expression of p53-regulated transcripts, proliferating cell nuclear antigen and growth arrest and DNA damage-inducible gene-45β (GADD45β) in AGD lesions, suggests a role for p53 in AGD pathogenesis. Thus AGD may represent a novel model for comparative analysis of p53 and p53-regulated pathways.

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry. KEY WORDS: Amoebic gill disease · Neoparamoeba perurans · In situ hybridisation · Aquaculture Resale or republication not permitted without written consent of the publisherDis Aquat Org 78: [217][218][219][220][221][222][223] 2008 cases of AGD reported elsewhere, it is not known what role N. perurans, N. pemaquidensis and/or N. branchiphila play. Therefore, our objective was to use species-specific molecular probes to determine the aetiological agent or agents of AGD in 4 host species in 6 countries. MATERIALS AND METHODSParaffin-embedded gill tissues were obtained from 4 fish species predominantly during or following epizootics at commercial fish farming operations in 6 countries (Table 1). An epizootic was not reported from Chinook salmon Oncorhynchus tshawytscha cultured in New Zealand; however, the smallest fish (runts) within the healthy population were observed to have gill lesions that corresponded with AGD and these fish were used in this study. Gill tissues were sectioned (3 to 7 μm), stained with haematoxylin and eosin (H&E) and examined with light microscopy. Alternatively, sections (7 μm) of gill tissues were placed onto Polysine glass slides (Menzel-Gläser) and dried overnight at 37°C. Sections were hybridised with a digoxigenin (DIG)-labelled 'universal' 18S rRNA oligonucleotide probe to verify the integrity of rRNA as previously described (Young et al. 2007). All gill tissues with suitable host and amoeba rRNA were serially sectioned, placed onto Polysine glass slides and incubated with Neoparamoeba perurans, N. branchiphila and N. pemaquidensis DIG-labelled oligonucleotide probes as previously described (Young et al. 2007). Positive and negative (no probe) controls were run in parallel with each in situ hybridisation experiment by hybridising each probe with a section containing representative strains of each Neoparamoeba species termed an 'amoebae array' as previously described (Young et al. 2007). Tissue sections were incubated for up to 1 h with in a premixed solution of 5-b...

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The recent description of Neoparamoeba perurans as an aetiological agent of amoebic gill disease (AGD) advanced our understanding of the condition and has forced a re-evaluation of methods used for the diagnosis of AGD. Currently, there are no tools available that are both specific for N. perurans and suitable for a routine diagnostic procedure. Therefore, in this study we describe an assay to detect N. perurans. The assay, which utilizes PCR to amplify the N. perurans 18S rRNA gene, was shown to be specific and highly sensitive. Neoparamoeba perurans was detected in both gill samples and primary isolates of non-cultured gill-derived amoebae obtained during necropsy or biopsy from AGD-affected Atlantic salmon, Salmo salar L. The PCR-based assay provides a simple, flexible tool that will be a useful addition to the diagnostic repertoire for AGD. It may also be used for the genotypic screening of trophozoites during culture and could facilitate further epidemiological and ecological studies of AGD.

Previous studies have demonstrated that beta-glucans stimulate Atlantic salmon, Salmo salar L., head kidney macrophages both in vitro and in vivo and increase protection against various pathogens. Based on our previous work that showed potent immunostimulatory CpG motif-containing oligodeoxynucleotides increased resistance to amoebic gill disease (AGD), the present study investigated the immunostimulatory effects of three commercial beta-glucan-containing feeds and their ability to increase resistance to AGD. All three commercial beta-glucans were able to stimulate the respiratory burst activity of Atlantic salmon head kidney macrophages in vitro, albeit at different times and concentrations. However, dietary incorporation of the beta-glucans was unable to stimulate the in vivo respiratory burst activity of head kidney macrophages, or serum lysozyme production, and did not increase resistance against AGD. However, this trial showed for the first time that a small subpopulation of Atlantic salmon subjected to a severe AGD infection was able to resist becoming heavily infected and furthermore survive the challenge.