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Amoebic gill disease, caused by the protozoan ectoparasite Neoparamoeba perurans, remains a significant threat to commercial Atlantic salmon aquaculture operations worldwide, despite partial control afforded by selective breeding and therapeutic intervention. Anecdotal reports from commercial producers suggest that historically, smaller Atlantic salmon smolts are more susceptible to AGD than larger smolts. Here, large (350 g) and small (200 g) commercially sourced, AGD-naïve Atlantic salmon cohorts were experimentally exposed to 50 N. perurans trophozoites L

The interbranchial lymphoid tissue (ILT) was recently described in the gills of salmonids. This study examined changes in the ILT during a parasitic infection in marine environment, using amoebic gill disease (AGD) as a model. Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans were sampled at 0, 3, 7, 14 and 28 days post challenge. Transversal sections of three areas of the gills (dorsal, medial and ventral) were histologically assessed for morphological and cellular changes. AGD induced morphological changes and a cellular response in the ILT of affected fish. These changes included a significant increase in the ILT surface area in fish 28 days after AGD challenge, compared to control fish at the same time point. The length of the ILT increased significantly 28 days post exposure in the dorsal area of the gill arch in the fish affected by AGD. The lymphocyte density of the ILT increased after AGD challenge, peaking at 7 days post exposure; however, by 28 days post exposure, a reduction of lymphocyte density to values close to pre-infection levels was observed. PCNA immunostaining revealed that epithelial hyperplasia was the most likely factor contributing to the ILT enlargement in the affected fish.

Amoebic gill disease can be experimentally induced by the exposure of salmonids to Neoparamoeba spp. freshly isolated from infected fish, while cultured amoebae are non-infective. Results from our previous work suggested that one key difference between infectious and non-infectious Neoparamoeba were the highly glycosylated molecules in the glycocalyx. To characterise these surface glycans or glycoproteins we used a monoclonal antibody (mAb 44C12) specific to a surface molecule unique to infective parasites. This mAb recognised a carbohydrate epitope on a high molecular weight antigen (HMWA) that make up 15-19% of the total protein in a soluble extract of infectious parasites. The HMWA consisted of at least four glycoprotein subunits of molecular weight (MW) greater than 150 kDa that form disulfide-linked complexes of MW greater than 600 kDa. Chemical deglycosylation yielded at least four protein bands of approximate MW 46, 34, 28 and 18 kDA. While a similar HMWA complex was present in non-infective parasites, the glycoprotein subunits were of lower MW and exhibited differences in glycosylation. The four glycoproteins subunits recognised by mAb 44C12 were resistant to degradation by PNGase F, PNGase A, O-glycosidase plus β-1, 4-galactosidase, β-N-acetylglucosaminidase and neuraminidase. The major monosaccharides in the HMWA from infectious parasites were rhamnose, fucose, galactose, and mannose while sialic acids were absent. The carbohydrate portion constituted more than 90% of the total weight of the HMWA from infectious Neoparamoeba spp. Preliminary results indicate that immunisation of salmon with HMWA does not lead to protection against challenge infection; rather it may even have an immunosuppressive effect. © 2010 Elsevier Ltd.

Tumour necrosis factor-alpha (TNF-alpha) is a key mediator of inflammation during amoebiasis of humans and mice. Atlantic salmon (Salmo salar L.) are also susceptible to infection by amoebae (Neoparamoeba spp.), inflicting a condition known as amoebic gill disease (AGD). Here, the role of TNF-alpha in AGD-pathogenesis was examined. Two Atlantic salmon TNF-alpha transcripts designated TNF-alpha1 and TNF-alpha2 together with their respective genes were cloned and sequenced. TNF-alpha1 is 1379 bp and consists of a 738 bp open reading frame (ORF) translating into a predicted protein of 246 amino acids. TNF-alpha2 is 1412 bp containing an ORF and translated protein the same lengths as TNF-alpha1. An anti-rainbow trout TNF-alpha polyclonal antibody that bound recombinant Atlantic salmon TNF-alpha1 and TNF-alpha2 was used to detect constitutive and inducible expression of TNF-alpha in various tissues. The anti-TNF-alpha antibody bound to a TNF-like protein approximately 60 kDa that was constitutively expressed in a number of tissues in healthy Atlantic salmon. However, this protein was not detected in lysates from mitogen-stimulated head kidney leucocytes, despite up-regulation of TNF-alpha mRNAs under the same conditions. During the early onset of AGD in Atlantic salmon, there were no demonstrable differences in the gill tissue expression of TNF-alpha1, TNF-alpha2 nor the interleukin-1 beta (IL-1beta), inducible nitric oxide synthase (iNOS) and interferon gamma (IFN-gamma) mRNAs compared to tissue from healthy fish. In Atlantic salmon with advanced AGD, IL-1beta but not TNF-alpha1 or TNF-alpha2 mRNAs was up-regulated and was lesion-restricted. Given that Neoparamoeba spp. modulated both TNF-alpha2 and IL-1beta in head kidney leucocytes in vitro, it appears that rather than being recalcitrant to Neoparamoeba spp.-mediated TNF-alpha expression, either the parasite can influence the cytokine response during infection, there is ineffective signalling for TNF-alpha expression, or there are too few cells at the site of infection with the capacity to produce TNF-alpha. These data support our previous observation that IL-1beta mRNA expression is up-regulated in AGD-affected tissue and that TNF-alpha is not intrinsic in AGD-pathogenesis.