Microarray analysis of the response of Atlantic salmon (Salmo salar) primary head kidney macrophage cultures to infection with Piscirickettsia salmonis

Piscirickettsia salmonis (PSAL) is an intracellular bacterial pathogen and the etiological agent of salmonid rickettsial septicemia (SRS). The organism appears to be ubiquitous and in Chile, infections cause high mortality and are responsible for economic losses in salmon aquaculture. The bacterium infects, survives and replicates in salmonid macrophages/monocytes suggesting that early interactions between the bacterium and macrophages/monocytes play a key role in the progression of SRS. The goal of this work was to investigate the transcriptomic response of primary culture of Atlantic salmon head kidney macrophage/monocytes following infection with PSAL. Leucocytes were isolated from head kidney homogenates of 8 individual fish and cultured in 6-well (10⁶ cells per well) plates. For each individual fish, 2 6-well plates were seeded (non-adherent cells were removed after 24 hours). In every 6-well plate, 4 wells were infected with 100 µL of PSAL culture (4.5 x10⁵ TCID₅₀) and 2 wells received 100 µL of MEM from a clean CHSE-214 culture. Three wells for each individual (1 non-infected and 2 infected) were sampled at 2, 6, 24 and 72 hours post-infection (hpi). One infected well and one non-infected well were used for RNA extractions and the second infected well was sampled for electron microscopy. Transcriptomic data were obtained at 2 and 24 hpi using a 44K Atlantic salmon microarray. Differentially expressed genes were identified in R. Interleukin-1 beta, NF-kappa B inhibitor alpha, CD83 antigen precursor Cholesterol-25-hydrolase and tumor necrosis factor were differentially expressed at 2 hpi and the differential expression was validated by using QPCR. In addition, transcripts encoding amebocyte aggregation factor, C-type lectin

and tumor necrosis factor precursor 2 were confirmed as molecular biomarkers of

salmon macrophage response to PSAL infection. Lastly, PSAL cells were identified within macrophages by transmission electron microscopy as early as 2 hpi and in all other time-points except 72 hours. These findings are consistent with the early up-regulation of cholesterol-25-hydrolase, which is in known to inhibit membrane fusion between viruses and host cells in mammals. Taken together, these results shed light on the early interaction between macrophages and PSAL and highlight the role of pro-inflammatory cytokines in the response to PSAL infection.

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