Pigs were randomly allocated to either a control group or an infection group and kept in separate rooms. The pigs in the infection group were inoculated intra-tracheally with a 3 ml suspension of the pathogenic H. parasuis serovar 5, strain 29755, in phosphate buffered saline (PBS) containing 5 x 10^8 colony forming units (cfu) per ml. Control pigs were inoculated in the same manner with PBS alone. Of the 200 animals that started the experiment, 80.5 % of the animals were still alive at the time of challenge. This meant that 133 animals were inoculated with H. parasuis while 29 animals were designated ‘Uninfected control’ animals. The time-course of the experiment was 72 hours, containing 3 time-points at 24, 48 and 72 hours. At each time-point, 1 control animal and 4-5 challenged animals were euthanized. Pigs were monitored daily for clinical signs of disease and approximately equal numbers of pigs were euthanized at 24, 48, and 72 hours post-infection and scored semi-quantitatively for the presence and severity of lesions associated with Glasser's disease and the presence of H. parasuis by culture and PCR. The four clinical signs measured were: 1) demeanour. 2) dysponea. 3) lameness. 4) tremor. The 9 lesions assessed were: 1) pleuritis. 2) hydrothorax. 3) pericarditis. 4) hydropericardium. 5) pleuritis. 6) ascites. 7) meningitis. 8) arthritis. 9) penumonia. The six sites tested by culture and PCR were: 1) pleura. 2) pericardium. 3) peritoneum. 4) carpal joint. 5) foot joint. 6) meninges. The following tissue samples were also taken for each animal and stored in RNA Later (Ambion): lung, pericardium, carpal joint, foot joint, and meninges. The animals were classified into 4 susceptibility groups based on the extent of disease: 1) Fully Resistant. 2) Less Resistant. 3) Less Susceptible. 4) Fully Susceptible. The aim of the microarray experiment was to compare the extremes of susceptibility at early (24 hours) and late (72 hours) infection time-points. It was decided to use lung tissue to study the infection due to the intra-tracheal nature of the challenge. Consequently, 6 'Fully Resistant' animals were chosen for the experiment, and were matched by sire and time-point to 'Susceptible' animals (4 Fully Susceptible animals and 2 Less Susceptible animals). The Fully Resistant animals were negative for the presence of bacteria by culture and PCR at each of the 6 sites tested, and by RT-PCR in lung. They had clinical signs or lesion scores of no more than 1 in no more than 2 sign/lesion categories or a total clinical sign or lesion score of no more than 3. The Less susceptible animals were positive for H parasuis at 4 or fewer sites tested by culture or PCR and had a total score for clinical signs and lesions of 5 or less. The Fully Susceptible animals were positive for bacteria at 5 or more sites by culture or PCR and had total clinical sign and lesion scores of 3 or more. The scoring system is described in detail in the following paper: Blanco, I., Canals, A., Evans, G., Mellencamp, M. A., Cia, C., Deeb, N., Wang, L. and Galina-Pantoja, L., Differences in susceptibility to Haemophilus parasuis infection in pigs. Can J Vet Res 2008. 72: 228-235.
Growth protocol
A total of 200 piglets born on a commercial farm in Spain were used for a bacterial challenge experiment. The experimental infection protocol was carried out in 10 separate batches each containing 20 animals over a period of about a year. Each batch of animals contained offspring from 2 different sires; in total 6 sires were used over the 10 batches. The sires (all Landrace-Duroc cross animals) were mated with a Landrace-Large White cross dam line. At birth, piglets were collected from the mother’s birth canal without touching the floor. Their skin was then disinfected using a 2 % iodine solution and they were immediately transferred to a previously cleaned and disinfected barrier isolation unit. For the first 2 days the new-born pigs were fed bovine colostrum ad libitum every 2 hours. Over the next 13 days the piglets were fed a mixture of porridge containing protein and water. The intervals between feeds were increased and the percentage of water in the porridge mix reduced gradually over this period until by day 15 the animals were eating dry food. All piglets were treated with the antibiotic gentamicin for the first 2 days to prevent Escherichia coli (E. coli) infection. Any animals that did succumb to diarrhoea were treated further with gentamicin for 3 days (although treatment was stopped for all animals 8 days prior to challenge), and the animal and surroundings were disinfected to prevent bacteria from spreading. At day 7 nasal swabs were taken from each of the pigs and tested for H. parasuis by PCR. All the pigs were negative for presence of the bacteria at this time. The animals were 21 days old at the start of the challenge experiment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TriReagent (Sigma) according to the manufacturer's instructions. The RNA was then further purified using RNeasy columns (Qiagen) according to the manufacturers instructions. An on column DNase I digest was also performed. RNA quality was determined by chip electrophoresis using a Bioanalyzer in conjunction with the RNA Nano 6000 kit (Agilent). Only samples with a 28:18S rRNA ratio of >1:1 and little visible degradation were taken forward for microarray experiments.
Label
Cy3
Label protocol
Labelled cDNA targets were prepared using a two step process. First, the target population was amplified by RT-PCR using the SMART template switching system (BD Biosciences). Second, the PCR product was labelled with Cy3 or Cy5-dCTP (GE Healthcare) using a Bioprime DNA Labelling System (Invitrogen). A 500 ng amount of total RNA was mixed with 10 pmol of 3′ SMART CDS primer IIA (5′-AAG CAG TGG TAT CAA CGC AGA GTA C-T30VN-3′) and 10 pmol template switching primer [5′-d(AAG CAG TGG TAT CAA CGC AGA GTA CGC)r(GGG)- 3′].Reverse transcription was carried out using Powerscript (BD Biosciences). PCR was performed using the first strand cDNA as template with the following PCR primer (5′ AAG AGT GGT ATC AAC GCA GAG T-3′) and HotStar Taq (Qiagen). The conditions were: 95 ºC for 15 minutes, then 18 cycles of 94 ºC for 30 seconds, 65 ºC for 30 seconds and 68 ºC for 6 minutes. PCR products were then purified using a MinElute PCR purification kit (Qiagen). Purified PCR product in a random primer mix was denatured at 95 ºC for 5 minutes. A low-dCTP mix (5mM dATP, dGTP, and dTTP; 2mM dCTP), Cy3 or Cy5-dCTP (1mM) (GE Healthcare) and 40 units of Klenow polymerase (Invitrogen) were added and the reactions incubated at 37 ºC for 2 hours. Labelled products were purified using Autoseq G-50 columns (GE Healthcare).
Pigs were randomly allocated to either a control group or an infection group and kept in separate rooms. The pigs in the infection group were inoculated intra-tracheally with a 3 ml suspension of the pathogenic H. parasuis serovar 5, strain 29755, in phosphate buffered saline (PBS) containing 5 x 10^8 colony forming units (cfu) per ml. Control pigs were inoculated in the same manner with PBS alone. Of the 200 animals that started the experiment, 80.5 % of the animals were still alive at the time of challenge. This meant that 133 animals were inoculated with H. parasuis while 29 animals were designated ‘Uninfected control’ animals. The time-course of the experiment was 72 hours, containing 3 time-points at 24, 48 and 72 hours. At each time-point, 1 control animal and 4-5 challenged animals were euthanized. Pigs were monitored daily for clinical signs of disease and approximately equal numbers of pigs were euthanized at 24, 48, and 72 hours post-infection and scored semi-quantitatively for the presence and severity of lesions associated with Glasser's disease and the presence of H. parasuis by culture and PCR. The four clinical signs measured were: 1) demeanour. 2) dysponea. 3) lameness. 4) tremor. The 9 lesions assessed were: 1) pleuritis. 2) hydrothorax. 3) pericarditis. 4) hydropericardium. 5) pleuritis. 6) ascites. 7) meningitis. 8) arthritis. 9) penumonia. The six sites tested by culture and PCR were: 1) pleura. 2) pericardium. 3) peritoneum. 4) carpal joint. 5) foot joint. 6) meninges. The following tissue samples were also taken for each animal and stored in RNA Later (Ambion): lung, pericardium, carpal joint, foot joint, and meninges. The animals were classified into 4 susceptibility groups based on the extent of disease: 1) Fully Resistant. 2) Less Resistant. 3) Less Susceptible. 4) Fully Susceptible. The aim of the microarray experiment was to compare the extremes of susceptibility at early (24 hours) and late (72 hours) infection time-points. It was decided to use lung tissue to study the infection due to the intra-tracheal nature of the challenge. Consequently, 6 'Fully Resistant' animals were chosen for the experiment, and were matched by sire and time-point to 'Susceptible' animals (4 Fully Susceptible animals and 2 Less Susceptible animals). The Fully Resistant animals were negative for the presence of bacteria by culture and PCR at each of the 6 sites tested, and by RT-PCR in lung. They had clinical signs or lesion scores of no more than 1 in no more than 2 sign/lesion categories or a total clinical sign or lesion score of no more than 3. The Less susceptible animals were positive for H parasuis at 4 or fewer sites tested by culture or PCR and had a total score for clinical signs and lesions of 5 or less. The Fully Susceptible animals were positive for bacteria at 5 or more sites by culture or PCR and had total clinical sign and lesion scores of 3 or more. The scoring system is described in detail in the following paper: Blanco, I., Canals, A., Evans, G., Mellencamp, M. A., Cia, C., Deeb, N., Wang, L. and Galina-Pantoja, L., Differences in susceptibility to Haemophilus parasuis infection in pigs. Can J Vet Res 2008. 72: 228-235.
Growth protocol
A total of 200 piglets born on a commercial farm in Spain were used for a bacterial challenge experiment. The experimental infection protocol was carried out in 10 separate batches each containing 20 animals over a period of about a year. Each batch of animals contained offspring from 2 different sires; in total 6 sires were used over the 10 batches. The sires (all Landrace-Duroc cross animals) were mated with a Landrace-Large White cross dam line. At birth, piglets were collected from the mother’s birth canal without touching the floor. Their skin was then disinfected using a 2 % iodine solution and they were immediately transferred to a previously cleaned and disinfected barrier isolation unit. For the first 2 days the new-born pigs were fed bovine colostrum ad libitum every 2 hours. Over the next 13 days the piglets were fed a mixture of porridge containing protein and water. The intervals between feeds were increased and the percentage of water in the porridge mix reduced gradually over this period until by day 15 the animals were eating dry food. All piglets were treated with the antibiotic gentamicin for the first 2 days to prevent Escherichia coli (E. coli) infection. Any animals that did succumb to diarrhoea were treated further with gentamicin for 3 days (although treatment was stopped for all animals 8 days prior to challenge), and the animal and surroundings were disinfected to prevent bacteria from spreading. At day 7 nasal swabs were taken from each of the pigs and tested for H. parasuis by PCR. All the pigs were negative for presence of the bacteria at this time. The animals were 21 days old at the start of the challenge experiment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TriReagent (Sigma) according to the manufacturer's instructions. The RNA was then further purified using RNeasy columns (Qiagen) according to the manufacturers instructions. An on column DNase I digest was also performed. RNA quality was determined by chip electrophoresis using a Bioanalyzer in conjunction with the RNA Nano 6000 kit (Agilent). Only samples with a 28:18S rRNA ratio of >1:1 and little visible degradation were taken forward for microarray experiments.
Label
Cy5
Label protocol
Labelled cDNA targets were prepared using a two step process. First, the target population was amplified by RT-PCR using the SMART template switching system (BD Biosciences). Second, the PCR product was labelled with Cy3 or Cy5-dCTP (GE Healthcare) using a Bioprime DNA Labelling System (Invitrogen). A 500 ng amount of total RNA was mixed with 10 pmol of 3′ SMART CDS primer IIA (5′-AAG CAG TGG TAT CAA CGC AGA GTA C-T30VN-3′) and 10 pmol template switching primer [5′-d(AAG CAG TGG TAT CAA CGC AGA GTA CGC)r(GGG)- 3′].Reverse transcription was carried out using Powerscript (BD Biosciences). PCR was performed using the first strand cDNA as template with the following PCR primer (5′ AAG AGT GGT ATC AAC GCA GAG T-3′) and HotStar Taq (Qiagen). The conditions were: 95 ºC for 15 minutes, then 18 cycles of 94 ºC for 30 seconds, 65 ºC for 30 seconds and 68 ºC for 6 minutes. PCR products were then purified using a MinElute PCR purification kit (Qiagen). Purified PCR product in a random primer mix was denatured at 95 ºC for 5 minutes. A low-dCTP mix (5mM dATP, dGTP, and dTTP; 2mM dCTP), Cy3 or Cy5-dCTP (1mM) (GE Healthcare) and 40 units of Klenow polymerase (Invitrogen) were added and the reactions incubated at 37 ºC for 2 hours. Labelled products were purified using Autoseq G-50 columns (GE Healthcare).
Hybridization protocol
Aliquots of the purified Cy3 and Cy5-labelled samples to be hybridized to the same array were combined such that the final concentration of each dye in the labelled target in the hybridization buffer would be 0.8 pmol/µl. A 4µl volume of ‘Porcine Hybloc’ (Applied Genetic Laboratories) (1 µg/µl) or 10 µl of Human cot-1 DNA (Invitrogen) (1 µg/µl), 1µl of poly dA (8 µg/µl) and 1 µl of yeast tRNA (4 µg/µl) were then added to block non-specific binding of repetitive sequences in the target to the probe sequences on the microarray during hybridization. Next, the sample volume was adjusted to 100 µl using nuclease-free water, and 10µl of 3 M sodium acetate (pH 4.8) and 250 µl of 100% ethanol were added. The mixture was mixed by inversion and the DNA was precipitated by incubation at -80 ºC for 30 minutes followed by centrifugation at 13,000 x G for 20 minutes at 4 ºC. The supernatant was discarded and 500 µl of 70 % ethanol was added. The pellet was washed by vortex then centrifuged at 13,000 G for 5 minutes. The pellet was air-dried for 5 minutes then re-suspended in 50 µl of ‘OpArray Hyb Buffer’. Re-suspension was aided by heating at 65 ºC for 5 minutes, followed by vortex mixing and brief centrifugation. The ‘target’ DNA was denatured by incubation at 65 ºC for 5 minutes. A 25 x 60 mm ‘Lifterslip’ (Thomas Scientific) was placed over the microarray portion of the slide. The denatured DNA was then applied slowly to the bottom edge of the Lifterslip, from where it was drawn over the array surface by capillary action. The microarray was placed in a Hybridization cassette (Telechem) containing 15 µl of ‘Milli Q’ water in each of its wells. The cassette was sealed and placed on a horizontal surface in a waterbath. The target DNA was hybridized to the microarray at 42 ºC for 15 hours in the dark. After hybridization, each microarray was removed from the hybridization cassette and washed in a slide tray containing Operon Wash Buffer 2 solution at 42 ºC for 10 minutes. They were then transferred to a slide tray containing Operon Wash Buffer 3 and washed for 10 minutes at room temperature. Finally, they were washed twice in Operon Wash Buffer 4 for 5 minutes and dried by centrifugation in at 200xg for 5 minutes. They microarrays were stored in light tight boxes and scanned on the same day.
Scan protocol
The hybridization signal on each microarray was detected by laser induced fluorescence of the Cy3 and Cy5 labelled ‘target’ molecules bound to the individual probe oligonucleotides. The microarrays were scanned using a ‘GenePix 4100A’ scanner (Axon instruments) containing a 532 nm laser and a 635 nm laser, the wavelengths that correspond to the excitation wavelengths of Cy3 and Cy5 respectively. Scan settings were adjusted and scans performed using ‘GenePix Pro version 4.1’ software. First, each microarray was scanned at a resolution of 40 µm in both the Cy3 and Cy5 channels to determine quickly whether the hybridizations were successful and to identify the area corresponding to the central 8 subgrids on the microarray. This area was then scanned in both the Cy3 and Cy5 channels at a resolution of 5 µm. The photo multiplier tube (PMT) gain settings were adjusted during the scan to ensure that: • The complete dynamic range of the scanner was being utilized to capture as broad a range of signal intensities as possible. • The total amount of signal acquired in each channel was approximately the same. The adjusted gain settings for each channel were then used to scan the entire microarray area at a resolution of 5 µm. False colour images for each channel were saved as TIFF files.
Description
n/a
Data processing
Hybridization signal was quantified using ‘Bluefuse’ v3.0 software (BlueGnome). The Cy3 and Cy5 TIFF images for each array, together with a ‘GenePix Array List’ (GAL) file describing the spot layout of the microarray, were loaded into the software. Grid layouts were then placed automatically by the software for each microarray, and checked by eye to ensure they were positioned correctly. The Cy3 and Cy5 signal, and the ratio of expression in the 2 channels, was then quantified. Features with irregularities affecting signal intensity were removed by manual flagging in Bluefuse v3.0. Mean signal intensities for duplicate spots were then calculated, and the data were imported into the ‘R’ based statistical analysis package ‘limma’ (linear models for microarray analysis). The data were normalized using a global Loess algorithm and the data for duplicate spots.
