pathogen: Enteromyxum leei pathogen exposure: EXPOSED, NON-INFECTED fish description: FISH 3 - 195g, 19.5cm - Tank 23-6
Biomaterial provider
Jaume Pérez-Sánchez, Ariadna Sitjà-Bobadilla, Josep Calduch: GinerInstituto de Acuicultura de Torre de la Sal (CSIC) 12595 Ribera de Cabanes, Castellón, Spain
Treatment protocol
One fish group was exposed to E. leei-contaminated effluent (recipient group = R). R fish (n= 66, average weight = 134 g) were placed in two replicated 200L fibre-glass tanks which were set to receive exclusively the effluent water from another tank containing 24 infected (donors = D; average weight = 127.3 g; prevalence of infection = 54%) gilthead sea bream. The D to R fish ratio was 0.8. Other 66 naïve fish were allocated in two replicated tanks (control group = CTRL) under the same conditions, but without receiving contaminated effluent. Over the course of the study, day length followed natural changes and water was heated in order to keep temperature always above 18 oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight. Disease signs and daily mortalities were recorded throughout the experiments. The parasitic status of dead fish was checked by microscopic examination of fresh intestinal scrapings. Fish were sampled after 113 days post exposure. Feeding was stopped one day prior to the sampling to ensure that the digestive tract was empty. Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80 oC until RNA extraction and analysis.
Growth protocol
Gilthead sea bream were checked for the absence of the parasite and acclimated to the experimental conditions two weeks before the beginning of the experiment. Day length followed natural changes and water was heated in order to keep temperature always above 18oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy Maxi combination protocol with Qiazol reagent
Label
Cy5
Label protocol
Ten micrograms of total RNA per individual fish (control/experimental) and ten micrograms of reference RNA per slide were indirectly labelled following an "in house" protocol with the red fluorescent dye ester cyanine 5 (Cy5) and green fluorescent dye ester Cyanine 3 (Cy3) both from GE Healthcare (UK). In brief, cDNA was synthesised using Superscript III MMLV reverse transcriptase (Invitrogen) incorporating aminoallyl dUTP (Sigma Aldrich). Synthesis took place at 42 oC for 2 hours. RNA was hydrolysed with 10M NaOH/0.5M EDTA for 15 minutes and the reaction was then neutralised with 1M HEPES. Reverse transcription reactions were purified using Microcon 30 columns (Millipore, UK) according to manufacturer~s instructions. Purified cDNAs were dried using a vacuum concentrator (Eppendorf) and were then resuspended in 0.1M sodium bicarbonate buffer. Cy dyes were resuspended in the same buffer and coupling to the Cy dye ester took place in the dark for 2 hours at room temperature. The removal of unincorporated dye was performed using the Illustra CyScribe GFX purification kit (GE Healthcare). Labelled cDNA samples were tested for successful dye incorporation using a Nanodrop spectrophotometer (Nanodrop technologies).
Channel 2
Source name
PATHOGEN EXPOSURE REFERENCE POOL OF ALL RNA (EXCEPT HKI1,HKI2,HKI3,HKC7,HKN2, HKN3, HKN4,HKN5,HKN7,INTN1, INTN5, INTN8, HKC7, HKC8, HKI1, HKI2, HKI3, HKI6, INTC3, INTC5, INTC6)
Jaume Pérez-Sánchez, Ariadna Sitjà-Bobadilla, Josep Calduch: GinerInstituto de Acuicultura de Torre de la Sal (CSIC) 12595 Ribera de Cabanes, Castellón, Spain
Treatment protocol
One fish group was exposed to E. leei-contaminated effluent (recipient group = R). R fish (n= 66, average weight = 134 g) were placed in two replicated 200L fibre-glass tanks which were set to receive exclusively the effluent water from another tank containing 24 infected (donors = D; average weight = 127.3 g; prevalence of infection = 54%) gilthead sea bream. The D to R fish ratio was 0.8. Other 66 naïve fish were allocated in two replicated tanks (control group = CTRL) under the same conditions, but without receiving contaminated effluent. Over the course of the study, day length followed natural changes and water was heated in order to keep temperature always above 18 oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight. Disease signs and daily mortalities were recorded throughout the experiments. The parasitic status of dead fish was checked by microscopic examination of fresh intestinal scrapings. Fish were sampled after 113 days post exposure. Feeding was stopped one day prior to the sampling to ensure that the digestive tract was empty. Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80 oC until RNA extraction and analysis.
Growth protocol
Gilthead sea bream were checked for the absence of the parasite and acclimated to the experimental conditions two weeks before the beginning of the experiment. Day length followed natural changes and water was heated in order to keep temperature always above 18oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy Maxi combination protocol with Qiazol reagent
Label
Cy3
Label protocol
Ten micrograms of total RNA per individual fish (control/experimental) and ten micrograms of reference RNA per slide were indirectly labelled following an "in house" protocol with the red fluorescent dye ester cyanine 5 (Cy5) and green fluorescent dye ester Cyanine 3 (Cy3) both from GE Healthcare (UK). In brief, cDNA was synthesised using Superscript III MMLV reverse transcriptase (Invitrogen) incorporating aminoallyl dUTP (Sigma Aldrich). Synthesis took place at 42 oC for 2 hours. RNA was hydrolysed with 10M NaOH/0.5M EDTA for 15 minutes and the reaction was then neutralised with 1M HEPES. Reverse transcription reactions were purified using Microcon 30 columns (Millipore, UK) according to manufacturer~s instructions. Purified cDNAs were dried using a vacuum concentrator (Eppendorf) and were then resuspended in 0.1M sodium bicarbonate buffer. Cy dyes were resuspended in the same buffer and coupling to the Cy dye ester took place in the dark for 2 hours at room temperature. The removal of unincorporated dye was performed using the Illustra CyScribe GFX purification kit (GE Healthcare). Labelled cDNA samples were tested for successful dye incorporation using a Nanodrop spectrophotometer (Nanodrop technologies).
Hybridization protocol
The two cDNA samples per slide (e.g. reference cDNA Cy3 labelled and experimental cDNA Cy5 labelled) were mixed and appropriate blockers were added (sheared salmon sperm genomic DNA, poly dA). The cDNA mixture was concentrated by precipitation with 3M sodium acetate and absolute ethanol at -20oC for 30 min. followed by centrifugation at 12500 x g for 30 min. Following removal of supernatant, the cDNA pellet was resuspended in 70 µl slide hyb buffer #1 (Ambion) and was then denatured at 95oC for 2 min. Slides were placed in Pre-Hyb solution (3xSSC, 0.1 % BSA, 0.1 % SDS) in 50 ml Sarsted tubes pre-warmed @ 42 °C and pre-hybed @ 42 °C for 30 min. Slides were rinsed 3 times in water and spun dry for 3 min @ 1600rpm. The hybridisation mixture was then added to the pre-hybed microarray slide using a coverslip (Erie Scientific). Hybridisation was performed in a Genetix hybidisation chamber (Genetix, UK) at 42 °C for 16 h. Following hybridisation the slides were washed (2 x 5 min. washes using 0.2XSSC/0.1% SDS and 2 x 5 min. washes using 0.2XSSC) using an Advawash automatic washing station (Advalytix).
Scan protocol
Microarray slides were scanned in two channels (543 & 633nm) at 5 µM resolution using a confocal laser scanner (ScanArray Express, Perkin Elmer)
Description
Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80 oC until RNA extraction and analysis.
Data processing
TIFF image files were imported into Genepix Pro (version 5.0.1.24) (Axon instruments) for feature finding and alignment using a batch alignment process. Features were flagged as present, absent or bad by this software program and pixel intensities for feature and background were quantified. Output Genepix results (GPR) files were imported into the Genespring GX 7.3 software program (Agilent technologies). To account for dye swap, the signal channel and control channel measurements for appropriate samples were reversed. Values below 0.01 were set to 0.01. A Lowess curve was fit to the log-intensity versus log-ratio plot using 20.0% of the data to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. VALUE column represents Log2 Ratio (CONTROL/REFERENCE) or Log2 Ratio (EXPERIMENTAL/REFERENCE).
