A. salmonicida subsp. salmonicida strain A449 [11] was grown in broth (in vitro) and in implants (in vivo) for this study because differences in the growth and characteristics of the bacteria, e.g. capsule formation and changes in superoxide dismutase activity [2] and [7] have been shown to result from these different growth milieus. For broth-grown cultures, bacteria were cultured to mid-logarithmic growth in tryptic soy broth at 17 °C and were resuspended to approximately 1.5×107 colony forming units (cfu)/ml in sterile Hank's balanced salt solution (HBSS). For implant-grown cultures, the same strain was cultured to mid-logarithmic phase as above, and placed in 12 cm lengths of dialysis tubing (SpectraPor 2 MWC 12–14,000 Da) that had been presoaked in HBSS and autoclaved [3]. The dialysis tubing was closed at one end by tying with suture, filled with 2×108 cells of A. salmonicida and closed at the other end by twisting and tying with suture. Dialysis bags were washed 6 times in 200 ml of HBSS and kept on ice until implanted into the peritoneal cavities of six salmon. Three days post-surgery, the dialysis bags were removed and the bacterial cells were collected and quantified. The concentrations of bacteria used for infection of macrophages were estimated by absorbance at 600 nm and calculated exactly by plate counts on tryptic soy agar. Head kidney tissue was dissociated by repeated passage through a 3 ml syringe and centrifuged at 500g for 10 min at 4 °C. The cell pellet was resuspended in 10 ml of L-15/2% FBS, centrifuged as before and the cell pellet finally resuspended in 4 ml of L-15/2% FBS. The cell suspension was layered on a Percoll gradient consisting of 4 ml of 51% Percoll and 4 ml of 34% Percoll and centrifuged at 400g for 20 min at 4 °C [12]. The macrophage-enriched cell preparation was collected from the 34%/51% interphase, the volume adjusted to 10 ml with L-15/2% FBS and cells pelleted by centrifugation at 500g for 10 min at 4 °C. The cell pellet was resuspended again in L-15/2% FBS, centrifuged as above and resuspended in 5 ml L-15/2% FBS. Viable cells were enumerated using Trypan blue and then resuspended in L-15/0.1% FBS at a concentration of 1×107 cells/ml. Macrophage-enriched cells (100 μl; 1×106 cells) isolated from each of the 24 fish were plated in individual wells of 96-well flat-bottom polystyrene tissue culture plates (Costar) and incubated in L-15 medium for 40 h at 17 °C. Half of the plate was used for the control treatment and half for exposure to bacteria. Additional 96-well plates were seeded at the same density for respiratory burst assay using phorbol myristate (Sigma) and assayed as described above. The cells were allowed to rest for 1 day prior to treatment. Medium was removed, the adherent cells gently washed once in L-15 medium and incubated in L-15/5% FBS until the following day. A. salmonicida (10 μl) grown in broth (1×105 cells) or in implants (2×105 cells) were warmed to 17 °C, added to the macrophages and incubated for 0.5, 1.0 or 2.0 h. Control wells received 10 μl of HBSS.
Growth protocol
Saint John River stock Atlantic salmon (Salmo salar L.) were maintained in single-pass, heated, dechlorinated fresh water at 12 °C at the Aquatron at Dalhousie University, Halifax, NS, Canada. Immediately before sampling, fish (100 g) were transferred to a bucket containing an overdose of TMS-Aqua MS-222 (Syndel). Using sterile technique, head kidneys were removed and placed in ice-cold L-15 media (Sigma-Aldrich, Canada) containing 2% FBS, 10 U/ml sodium heparin (Sigma-Aldrich, Canada) and 100 U/ml penicillin–streptomycin (Sigma-Aldrich, Canada) that had been passed through a 0.22 μm filter unit.
Extracted molecule
polyA RNA
Extraction protocol
Medium was removed from the macrophages and replaced with 100 μl Trizol. Plates were placed at 4 °C. Cell lysates from macrophages from individual fish were pooled and stored at −80 °C until use. Since all fish were responders as judged by a respiratory burst assay, lysates from the three replicates were pooled for each time point and for each condition (infected versus control) and total RNA was isolated according to the manufacturer's recommendations. The mRNA was isolated using the Micro FastTrack 2.0 kit (Invitrogen) and stored at −80 °C until use.
Label
Cy3
Label protocol
Synthesis and amplification of cDNA was carried out as previously described [30] on pooled mRNA samples from infected (cohab) and from uninfected control fish, with the following modifications: 100 ng of salmon polyA+ RNA and 80 pg of Arabidopsis thaliana chlorophyll synthetase G4 mRNA (Accession U19382) were reverse-transcribed using the SuperSMARTTM PCR cDNA synthesis kit (Clontech) and the RNase Inhibitor used previously was excluded. All of column-purified single-stranded cDNA (85 ul) was combined with 40 ul PCR mixture and 75 ul PCR-grade dH2O, mixed well and split to yield two 100 ul reaction mixtures. Amplification was performed in a Model 9700 thermocycler (Perkin Elmer) using the following conditions: 95 degC for 1 minute; 15-24 cycles of 95 degC for 5 s, 65 degC for 5 s and 68 degC for 3 min. After 15 cycles, 70 ul of the reaction were removed and stored on ice, while the remaining 30 ul were returned to the thermocycler for further cycles. The optimal number of cycles was determined by the removal of 5 ul aliquots after 15, 18, 21 and 24 cycles and assessment by agarose gel electrophoresis. The original 70 ul reaction mix was returned to the thermocycler for the required number of additional cycles. The amplified cDNA was purified using QIAquick columns (Qiagen) and labelled in two 60 ul reactions containing 1X Advantage 2 PCR buffer, 0.2 mM d(A/T/G)TPs, 0.04 mM dCTP, 120 pmol anchor dT primer and 3 nmol Cy3-dCTP or Cy5-dCTP.
A. salmonicida subsp. salmonicida strain A449 [11] was grown in broth (in vitro) and in implants (in vivo) for this study because differences in the growth and characteristics of the bacteria, e.g. capsule formation and changes in superoxide dismutase activity [2] and [7] have been shown to result from these different growth milieus. For broth-grown cultures, bacteria were cultured to mid-logarithmic growth in tryptic soy broth at 17 °C and were resuspended to approximately 1.5×107 colony forming units (cfu)/ml in sterile Hank's balanced salt solution (HBSS). For implant-grown cultures, the same strain was cultured to mid-logarithmic phase as above, and placed in 12 cm lengths of dialysis tubing (SpectraPor 2 MWC 12–14,000 Da) that had been presoaked in HBSS and autoclaved [3]. The dialysis tubing was closed at one end by tying with suture, filled with 2×108 cells of A. salmonicida and closed at the other end by twisting and tying with suture. Dialysis bags were washed 6 times in 200 ml of HBSS and kept on ice until implanted into the peritoneal cavities of six salmon. Three days post-surgery, the dialysis bags were removed and the bacterial cells were collected and quantified. The concentrations of bacteria used for infection of macrophages were estimated by absorbance at 600 nm and calculated exactly by plate counts on tryptic soy agar. Head kidney tissue was dissociated by repeated passage through a 3 ml syringe and centrifuged at 500g for 10 min at 4 °C. The cell pellet was resuspended in 10 ml of L-15/2% FBS, centrifuged as before and the cell pellet finally resuspended in 4 ml of L-15/2% FBS. The cell suspension was layered on a Percoll gradient consisting of 4 ml of 51% Percoll and 4 ml of 34% Percoll and centrifuged at 400g for 20 min at 4 °C [12]. The macrophage-enriched cell preparation was collected from the 34%/51% interphase, the volume adjusted to 10 ml with L-15/2% FBS and cells pelleted by centrifugation at 500g for 10 min at 4 °C. The cell pellet was resuspended again in L-15/2% FBS, centrifuged as above and resuspended in 5 ml L-15/2% FBS. Viable cells were enumerated using Trypan blue and then resuspended in L-15/0.1% FBS at a concentration of 1×107 cells/ml. Macrophage-enriched cells (100 μl; 1×106 cells) isolated from each of the 24 fish were plated in individual wells of 96-well flat-bottom polystyrene tissue culture plates (Costar) and incubated in L-15 medium for 40 h at 17 °C. Half of the plate was used for the control treatment and half for exposure to bacteria. Additional 96-well plates were seeded at the same density for respiratory burst assay using phorbol myristate (Sigma) and assayed as described above. The cells were allowed to rest for 1 day prior to treatment. Medium was removed, the adherent cells gently washed once in L-15 medium and incubated in L-15/5% FBS until the following day. A. salmonicida (10 μl) grown in broth (1×105 cells) or in implants (2×105 cells) were warmed to 17 °C, added to the macrophages and incubated for 0.5, 1.0 or 2.0 h. Control wells received 10 μl of HBSS.
Growth protocol
Saint John River stock Atlantic salmon (Salmo salar L.) were maintained in single-pass, heated, dechlorinated fresh water at 12 °C at the Aquatron at Dalhousie University, Halifax, NS, Canada. Immediately before sampling, fish (100 g) were transferred to a bucket containing an overdose of TMS-Aqua MS-222 (Syndel). Using sterile technique, head kidneys were removed and placed in ice-cold L-15 media (Sigma-Aldrich, Canada) containing 2% FBS, 10 U/ml sodium heparin (Sigma-Aldrich, Canada) and 100 U/ml penicillin–streptomycin (Sigma-Aldrich, Canada) that had been passed through a 0.22 μm filter unit.
Extracted molecule
polyA RNA
Extraction protocol
Medium was removed from the macrophages and replaced with 100 μl Trizol. Plates were placed at 4 °C. Cell lysates from macrophages from individual fish were pooled and stored at −80 °C until use. Since all fish were responders as judged by a respiratory burst assay, lysates from the three replicates were pooled for each time point and for each condition (infected versus control) and total RNA was isolated according to the manufacturer's recommendations. The mRNA was isolated using the Micro FastTrack 2.0 kit (Invitrogen) and stored at −80 °C until use.
Label
Cy5
Label protocol
Synthesis and amplification of cDNA was carried out as previously described [30] on pooled mRNA samples from infected (cohab) and from uninfected control fish, with the following modifications: 100 ng of salmon polyA+ RNA and 80 pg of Arabidopsis thaliana chlorophyll synthetase G4 mRNA (Accession U19382) were reverse-transcribed using the SuperSMARTTM PCR cDNA synthesis kit (Clontech) and the RNase Inhibitor used previously was excluded. All of column-purified single-stranded cDNA (85 ul) was combined with 40 ul PCR mixture and 75 ul PCR-grade dH2O, mixed well and split to yield two 100 ul reaction mixtures. Amplification was performed in a Model 9700 thermocycler (Perkin Elmer) using the following conditions: 95 degC for 1 minute; 15-24 cycles of 95 degC for 5 s, 65 degC for 5 s and 68 degC for 3 min. After 15 cycles, 70 ul of the reaction were removed and stored on ice, while the remaining 30 ul were returned to the thermocycler for further cycles. The optimal number of cycles was determined by the removal of 5 ul aliquots after 15, 18, 21 and 24 cycles and assessment by agarose gel electrophoresis. The original 70 ul reaction mix was returned to the thermocycler for the required number of additional cycles. The amplified cDNA was purified using QIAquick columns (Qiagen) and labelled in two 60 ul reactions containing 1X Advantage 2 PCR buffer, 0.2 mM d(A/T/G)TPs, 0.04 mM dCTP, 120 pmol anchor dT primer and 3 nmol Cy3-dCTP or Cy5-dCTP.
Hybridization protocol
Arrays were prehybridized at 42 °C for 30 min–3 h with 5× sodium chloride–sodium citrate (SSC), 0.1% SDS and 1% BSA (Sigma-Aldrich, Canada) to block non-specific hybridizations. They were washed twice in sterile dH2O and once in filtered isopropanol and then dried in a plate centrifuge at 500 rpm for 5 min. The labelled cDNAs were resuspended in hybridization solution containing 20 μg poly(dA), 50% formamide and 25% hybridization buffer (GE Healthcare Life Sciences) and were incubated at 42 °C for 10 min. The warmed hybridization mixture was added to the prehybridized array under a glass coverslip, placed in a hybridization chamber (Die-Tech) and incubated at 42 °C in a water bath for 18 h. Arrays were washed once for 10 min at 42 °C in 1× SSC with 0.2% SDS, twice for 10 min each at 42 °C in 0.1× SSC with 0.2% SDS and once for 5 min at 25 °C in 0.1× SSC. Arrays were dried by centrifugation for 5 min at 500 rpm before scanning.
Scan protocol
Scanned on Perkin Elmer ScanArray 5000XL, ScanArray Express software v3.0.1
Description
pooled replicate for 0.5h
Data processing
Spot intensities were quantified using the QuantArray microarray analysis software from the same supplier, and normalization and data analysis were carried out using the GeneTraffic software (Iobion Informatics). Background fluorescence was subtracted from the spot intensities and spots were flagged and removed from the data set if the signal to background ratio was less than 1, the signal to average background ratio was less than 1, or the signal intensity was less than 250. Data were normalized on a global basis by the Lowess method [15]. Normalization values were generated from the ratio of the total (combined) intensity of valid Cy3 spots to the total intensity of valid Cy5 spots on each array subgrid.
