tissue type: Serum infection status: Ascaris suum immunization status: No Immunization
Treatment protocol
Experimental pig barrows were obtained from a pig farrowing facility at the Beltsville Agricultural Research Center, Beltsville, MD. Pigs were derived from boars from a four-way crossbred composite BX line (Duroc X maternal Landrace X terminal Landrace X Yorkshire) designed by scientists at the USDA/ARS/US Meat Animal Research Center, Clay Center, NE to be genetically similar to genetics in the commercial swine industry at the time they were born; the genetics of the gilts are predominantly of the BX composite line. Pigs were from a herd screened yearly for porcine reproductive and respiratory syndrome virus (PRRSV), influenza (H1N1 and H3N2), pseudorabies, brucellosis and intestinal worm parasites by the Veterinary Services Group at the Beltsville Agricultural Research Center and have been negative for these infections. They were individually housed in stalls with a non-absorptive concrete floor surface covered with rubber mats with ad libitum access to water and a nutritionally adequate corn/soybean-based diet. All animal experiments and procedures were conducted in accordance with guidelines established and approved by the Beltsville Area Animal Care and Use Committee under protocol 17-019. Pigs in the infection only cohort included 1) three pigs inoculated five times every 10 days with 10,000 T. suis eggs and bled 10 days after the last inoculation, 2) six pigs inoculated eight times every other day with 10,000 A. suum eggs and bled 25 days after the last inoculation, and 3) three pigs inoculated with 10,000 T. suis eggs and bled 53 days later (these pigs were worm free and considered as a resistant phenotype). The second group of six pigs in the vaccination cohort were immunized with 400µg of recombinant protein (80µg each of the five-parasite antigen cocktail in Seppic Montanide ISA 61 VG which was injected subcutaneously). The pigs were immunized a second time four weeks later and again after two weeks later and followed by a challenge infection with either 10,000 infective A. suum or T. suis eggs. The A. suum-infected pigs were bled and euthanized 27 days after inoculation and the T. suis infected pigs 37 after inoculation to collect blood and ileal wash fluid (contents from the ileum were removed and spun at 10,000 rpm and the supernatant fluid decanted and frozen at -80C until used) from the small intestine (3 - immunized/A. suum infected).
Extracted molecule
protein
Extraction protocol
Cloning and expression was performed using standard techniques. All sequences were PCR amplified from cDNA using sequence specific forward and reverse primers that encompassed the mature protein and contained Sac I (forward) and Xho I (reverse) restriction sites for downstream subcloning. Translation stop sites were incorporated into all reverse primers. Amplified sequences were first cloned into pCR2.1-TOPO by TA cloning then transformed into DH5α cells for sequence verification. Validated sequences were restriction enzyme digested and subcloned into the pSUMO bacterial expression vector (Life Sensors) and transformed into BL21 cells for protein production. Expression was performed using overnight cultures to spike 500ml of LB medium containing ampicillin (100 µg/ml) which was induced at OD = 0.7 for 5 hrs. with isopropyl β-D-1-thiogalactopyranoside (0.3 mM final). Pelleted cells were lysed with 1 mg/ml lysozyme, frozen overnight then sonicated. Because all clones formed inclusion bodies during production, the sonicated pellets were first washed 3X with 2% Triton X-100, solubilized in 8M Urea (made fresh) and batch purified by affinity chromatography using 2 ml of Ni-NTA. All mixtures were added to columns, washed with 6M urea (3X), then wash buffer containing 50 mM sodium phosphate, pH 8.0, 300 mM sodium chloride and 20 mM imidazole. Recombinant proteins were eluted with 5 ml of wash buffer containing 500 mM imidazole and 12 mM sodium lauryl sarkosine.
Briefly, the clone library was created through an in vivo recombination cloning process with PCR-amplified coding sequences from cDNA, and a complementary linearized expressed vector transformed into chemically competent E. coli cells was amplified by PCR and cloned into the pXI vector using a high-throughput PCR recombination cloning method. The cloning methodology is described in detail elsewhere (Davies et al., 2005). All the clones were sequenced (Retrogen, Inc., San Diego, CA), and the results matched the correct target for the selected genes. From each clone, the corresponding protein was expressed using an in vitro transcription and translation (IVTT) system, the E. coli cell-free rapid translation system (RTS) kit (Biotechrabbit, Berlin, Germany), as previously described (Davies et al., 2005). Each expressed protein includes a 5′ polyhistidine epitope tag and a 3′ hemagglutinin (HA) epitope tag. After expressing the proteins according to the manufacturer's instructions, translated proteins were printed onto nitrocellulose-coated glass AVID slides (Grace Bio-Labs, Inc., Bend, OR) using an ArrayJet Marathon Argus robotic microarray non-contact printer (ArrayJet, Roslin, UK). Each slide contained 16 nitrocellulose pads on which the expressed proteins along with controls were printed (this allowed sixteen samples to be probed per slide using sealed chambers that isolate the arrays). Microarray chip printing and protein expression were quality checked by probing random slides with anti-His and anti-HA monoclonal antibodies with fluorescent labeling.
Scan protocol
Probed microarrays (slides) were scanned using a GenePix 4300A high-resolution microarray scanner (Molecular Devices, Sunnyvale, CA), and an image file (.tiff) was saved for each array using GenePix pro 7 software. The signals in the scanned images were quantified using the Mapix software (Innopsys) autogridding feature. For this process, two input files are required: (i) a .gal file that defines the array and subarray layout, and (ii) the .tiff image file for an array. Once the autogridding is complete, the overlays of the mapped array, subarray, and individual spot locations are shown in the graphical user interface (GUI). If the automatic gridding fails to map to the correct positions, the mapping can be manually adjusted using the GUI. The final raw intensity is the foreground intensity minus the local background intensity. The raw signals were automatically extracted and saved as .csv files in data matrix format, with array spots as rows and samples as columns, using R (http://www.R-project.org).
Description
Arrays were incubated overnight at 4°C with agitation, washed three times with Tris-buffered saline (TBS)–0.05% Tween 20, and incubated with Rabbit Anti-Pig IgG (MilliporeSigma, Burlington, MA) diluted 1:500 in blocking buffer at room temperature. Arrays were washed three times with TBS–0.05% Tween 20 and incubated with Cy3 Goat Anti-Rabbit IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:200 in blocking buffer at room temperature, protected from light. Arrays were washed three times with TBS–0.05% Tween 20, three times with TBS, and once with water and then air dried by being centrifuged at 1,000 × g for 4 min and left overnight in a desiccator before scanning. Probed microarrays (slides) were scanned using a GenePix 4300A high-resolution microarray scanner (Molecular Devices, Sunnyvale, CA).
Data processing
Initial processing of the raw array data was performed as previously described (Li et al., 2017). First, raw values were transformed using the base 2 logarithm. Next, the data set was normalized to remove systematic effects by subtracting the median signal intensity of the IVTT control spots for each sample. Since the IVTT control spots carry not only the chip, sample, and batch-level systematic effects, but also antibody background reactivity to the IVTT system, this procedure normalizes the data and provides a relative measure of the specific antibody binding versus the nonspecific antibody binding to the IVTT controls. With the normalized data, a value of 0.0 means that the intensity is no different than that of the IVTT controls, and a value of 1.0 indicates a doubling with respect to IVTT control spots. Log2 relative fluorescence values (relative to the median of the negative control probes) were calculated for each protein and each sample (one sample per array). Principal components analysis (PCA) was performed using the “prcomp” package in R, using only the proteins with relative Log2 fluorescence >0 relative to negative controls. The Protein IDs come from the PRJNA80881 Ascaris suum genome annotation, retrieved from https://parasite.wormbase.org/Ascaris_suum_prjna80881/Info/Index
