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)

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-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.