title: Molecular cloning and expression analysis of tumour necrosis factor-alpha in amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.).
abstract: 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.
authors.affiliation: Aquafin CRC, School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia. rmorriso@utas.edu.au
authors.forename: R N
authors.initials: RN
authors.lastname: Morrison
authors.forename: J
authors.initials: J
authors.lastname: Zou
authors.forename: C J
authors.initials: CJ
authors.lastname: Secombes
authors.forename: G
authors.initials: G
authors.lastname: Scapigliati
authors.forename: M B
authors.initials: MB
authors.lastname: Adams
authors.forename: B F
authors.initials: BF
authors.lastname: Nowak
mesh_terms: D000562:Amebiasis / Q000378:metabolism / Q000662:veterinary*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005393:Fish Diseases / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005880:Gills / Q000469:parasitology; D053582:Interleukin-1alpha / Q000235:genetics; D053583:Interleukin-1beta / Q000235:genetics; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D020317:Salmo salar / Q000378:metabolism*; D016415:Sequence Alignment; D014409:Tumor Necrosis Factor-alpha / Q000097:blood / Q000235:genetics*; D015854:Up-Regulation
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha