title: Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD).
abstract: The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.
authors.affiliation: School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute and Aquafin CRC, University of Tasmania, Locked Bag 1370, Launceston 7250, Tasmania, Australia. Andrew.Bridle@utas.edu.au
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bridle
authors.forename: Richard N
authors.initials: RN
authors.lastname: Morrison
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Cupit Cunningham
authors.forename: Barbara F
authors.initials: BF
authors.lastname: Nowak
mesh_terms: D000562:Amebiasis / Q000235:genetics / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*; D000656:Amoeba / Q000276:immunology*; D000818:Animals; D005393:Fish Diseases / Q000235:genetics / Q000276:immunology / Q000469:parasitology*; D005786:Gene Expression Regulation / Q000276:immunology; D005880:Gills / Q000276:immunology / Q000469:parasitology; D017403:In Situ Hybridization / Q000662:veterinary; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D007668:Kidney / Q000276:immunology / Q000469:parasitology; D008099:Liver / Q000276:immunology / Q000469:parasitology; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D020317:Salmo salar / Q000235:genetics / Q000276:immunology*; D000685:Serum Amyloid A Protein / Q000235:genetics / Q000276:immunology; D000683:Serum Amyloid P-Component / Q000235:genetics / Q000276:immunology
chemical_list: D053583:Interleukin-1beta; D012333:RNA, Messenger; D000685:Serum Amyloid A Protein; D000683:Serum Amyloid P-Component; D052247:Nitric Oxide Synthase Type II