Quantitation of immune response gene expression and cellular localisation of interleukin-1β mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

• Published in: Veterinary Immunology and Immunopathology 2006-11, Vol.114 (1), p.121-134
• Main Authors: Bridle, Andrew R., Morrison, Richard N., Cupit Cunningham, Pauline M., Nowak, Barbara F.
• Format: Article
• Language: English
• Subjects: AGD; amebiasis; amoebic gill disease; Amoebida; animal proteins; Atlantic salmon; fish diseases; Gene expression; gene expression regulation; genes; Gill; gills; immune response; In situ hybridisation; infection; interleukin-1; Interleukin-1β; kidneys; liver; Marine; messenger RNA; Neoparamoeba; nitric oxide synthase; nucleic acid hybridization; protein synthesis; quantitative analysis; Real-time PCR; reverse transcriptase polymerase chain reaction; Salmo salar; salmon; serum amyloid A; serum amyloid P-like pentraxin
• ISSN: 0165-2427; 1873-2534; 1365-2567
• DOI: 10.1016/j.vetimm.2006.08.002

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-1β (IL-1β), 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, β-actin. Interleukin-1β (IL-1β) 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-1β mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1β mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1β-specific biotinylated cRNA probe. Expression of IL-1β 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-1β at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1β is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1β mRNA expression during infection.