http://eprints.utas.edu.au/4195/ Molecular cloning and expression analysis of tumour necrosis factor-alpha in amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.) Morrison, RN Zou, J Secombes, CJ Scapigliati, G Adams, MB Nowak, BF 300703 Aquaculture Tumour necrosis factor-alpha (TNF-a) 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-a in AGD-pathogenesis was examined. Two Atlantic salmon TNF-a transcripts designated TNF-a1 and TNF-a2 together with their respective genes were cloned and sequenced. TNF-a1 is 1379 bp and consists of a 738 bp open reading frame (ORF) translating into a predicted protein of 246 amino acids. TNF-a2 is 1412 bp containing an ORF and translated protein the same lengths as TNF-a1. An anti-rainbow trout TNF-a polyclonal antibody that bound recombinant Atlantic salmon TNF-a1 and TNF-a2 was used to detect constitutive and inducible expression of TNF-a in various tissues. The anti-TNF-a antibody bound to a TNF-like protein z60 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-a 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-a1, TNF-a2 nor the interleukin-1 beta (IL-1b), inducible nitric oxide synthase (iNOS) and interferon gamma (IFN-g) mRNAs compared to tissue from healthy fish. In Atlantic salmon with advanced AGD, IL-1b but not TNF-a1 or TNF-a2 mRNAs was up-regulated and was lesion-restricted. Given that Neoparamoeba spp. modulated both TNF-a2 and IL-1b in head kidney leucocytes in vitro, it appears that rather than being recalcitrant to Neoparamoeba spp.-mediated TNF-a expression, either the parasite can influence the cytokine response during infection, there is ineffective signalling for TNF-a expression, or there are too few cells at the site of infection with the capacity to produce TNF-a. These data support our previous observation that IL-1b mRNA expression is up-regulated in AGD-affected tissue and that TNF-a is not intrinsic in AGD-pathogenesis. Academic Press 2007 Article PeerReviewed application/pdf http://eprints.utas.edu.au/4195/1/4195.pdf http://dx.doi.org/10.1016/j.fsi.2007.04.003 Morrison, RN and Zou, J and Secombes, CJ and Scapigliati, G and Adams, MB and Nowak, BF (2007) Molecular cloning and expression analysis of tumour necrosis factor-alpha in amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.). Fish and Shellfish Immunology, 23 (5). pp. 1015-1031. ISSN 1050-4648 http://eprints.utas.edu.au/4195/http://eprints.utas.edu.au/4195/1/