Molecular cloning and expression analysis of tumour necrosis factor-alpha in amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.).

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.