Transcriptome profiling of Atlantic salmon (Salmo salar) parr with higher and lower pathogen loads following Piscirickettsia salmonis infection

Piscirickettsiosis or salmonid rickettsial septicemia (SRS) caused by intracellular Gram-negative bacterial pathogen, Piscirickettsia salmonis, constitutes one of the main infectious diseases in salmonid aquaculture. In the present study, we aimed to explore the transcriptomic responses in the Atlantic salmon kidney following a low dose of EM-90-like P. salmonis infection using the consortium for Genomic Research on All Salmonids Project (cGRASP)-designed Agilent 44K salmonid oligonucleotide microarray. Two infection level phenotypes [low (L-SRS) and high (H-SRS) infection level] were revealed in infected individuals at 21 days post-injection (DPI) based on multivariate analyses of expression of four antibacterial biomarker transcripts (campb, hampa, il8a, tlr5a) and pathogen level measured by qPCR. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling. We identified 1636 and 3076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups, respectively (FDR = 0.01). Atlantic salmon parr were anesthetized using Benzocaine (150 µl L-1 BZ-20®, Veterquímica S.A., Maipú, Santiago, Chile) and intraperitoneally (IP) injected with 0.1 mL of a low dose bacterial inoculum with a titer of 10^0.83 TCID50/mL (50% Tissue Culture Infective Dose) or 0.1 mL of a medium that was used to prepare the bacterial inoculum (i.e. minimum essential medium, MEM). Ten fish per tank [3 tanks from the challenge group (i.e. P. salmonis-injected) and 4 tanks from the control group (CON)] were euthanized using an overdose of Benzocaine (300 µl L-1 BZ-20®) and dissected on the day before injection (PRE) of the remaining fish, as well as 2, 7, 13, 21 and 42 days post-injection (DPI). Head kidney samples were collected and stored in RNAlater (Ambion/Life Technologies, Austin, TX, USA) for 48 h at 4°C and transferred to -80°C until RNA extraction. Two infection level phenotypes [low (L-SRS) and high (H-SRS) infection level] were revealed in infected individuals at 21 days post-injection (DPI) based on multivariate analyses of expression of four antibacterial biomarker transcripts (campb, hampa, il8a, tlr5a) and pathogen level measured by qPCR. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling with a common reference design. Test samples were labeled with Cy5, whereas the common reference (i.e. a RNA pool of all 15 samples included in microarray experiment) was labeled with Cy3. Each individual test sample was hybridized together with the common reference sample on an array (i.e. 15 arrays in total).