All animal care handling techniques and procedures as well as the procedures for experimental infection, tissue sampling and slaughtering were approved by the French Ethic Committee n°069 (Comité d’Ethique en Matière d’Expérimentation Animale des Antilles et de la Guyane, CEMEAAG) authorized by the French Ministry of Higher Education, Research and Innovation. The experiment was performed at the INRA Experimental Facilities PTEA (Plateforme Tropicale d’Expérimentation sur l’Animal) according to the certificate number A 971–18–02 of authorization to experiment on living animals issued by the French Ministry of Agriculture.
Animals and experimental design
The breeding value (BV) of each goat of the experimental of flock of PTEA was estimated since 1995 by using FEC at 11 months of age (when available) following natural mixed infection on pasture and taking into account the FEC of its ascendants and pedigree. The Eight 9-month old Creole kids were chosen on the basis of their extreme estimated BV in their cohort. Before the experiment the kids were reared at pasture with a limited level of GIN contamination (FEC < 500). The FEC of the 8 kids (n = 4 resistant and n = 4 susceptible), were not statistically different. The averages BV of the 2 groups of animals were distant by 1.04 genetic standard deviation. The animals were drenched with moxidectine (Cydectine®, Fort Dodge Veterinaria S. A., Tours, France, 300 µg/kg) and housed indoors under worm-free conditions in a single pen, one month before the start of the experiment. Kids were orally infected with a single dose of 10,000 H. contortus third-stage larvae (L3) in two consecutive challenges. Each challenge lasted for 5 weeks with 8 weeks interval between the end of challenge 1 and the start of challenge 2. Four weeks before the second experimental infection a fistula was surgically implanted in the abomasum of each animal to allow abomasal mucosa sampling at 0, 8, 15 and 35 days post infection (dpi).
The custom designed abomasal cannula consisted of a flexible plastic tube with a length of 7 cm and a diameter of 2 cm with a rounded base of 4 cm in diameter. This flexible plastic was chosen to limit the possibility of mechanical abrasion of the mucosal surface of the abomasum. The animals were fasted 16 h before cannula insertion surgery. The animals were premedicated with ketamine (2mg/kg IV, Le Vet Pharma, Wilgenweg, Neitherlands), xylazine (0.2mg mg/kg IM, Le Vet Pharma, Wilgenweg, Neitherlands) and oxytetracycline (20 mg/kg IM, Eurovet Animal Health, Handelsweg, Neitherlands). The animals were positioned in left lateral recumbency. Skin over the surgical site was shaved and prepped with povidone iodine (Vétédine, Laboratoire Vetoquinol S. A., Lure, France). A ventral midline incision was made to locate and exteriorize the abomasum. A 3 cm purse-string suture (Silk 2–0) was placed midway between the lesser and greater curvature and a stab incision was made in the center to insert the cannula. Then, the purse-string suture was tightened and tied off. To maintain the abomasum in an anatomically correct position, another stab incision was made in the abdominal wall at 10cm from the laparotomy incision on the right paramedian area to enable the cannula to be passed freely through. An external flange was placed over the external part of cannula and fixed with adhesive fabric plaster strip. A sterile compress was inserted into the cannula as stopper. After the surgical procedure, all the animals were housed individually with free access to fresh water and hay.
Biopsy sampling procedure
Biopsy specimens were taken from the abomasal mucosa using a flexible endoscope (FG–24V, Pentax, France). The biopsies samples of 2×2×2mm taken with the endoscopic forceps with window (model KW1815S) were quickly snap frozen into liquid nitrogen and stored at –80°C until RNA extraction. The animals were restrained in a harness made with a surgical drape allowing animal legs to protrude and which exposed the cannula. No sedation was used since no signs of discomfort or pain were observed during or after the procedure. The sterile compress inserted into the cannula was removed and the abomasal contents collected. The endoscope was introduced into the abomasal lumen and 3 biopsies per animal and per time points were taken from the abomasal folds of the fundic mucosa. At each time point the whole fundic mucosa was observed and no sign of mucosal injury due to the previous sampling was observed.
RNA extraction and sequencing
Total RNA was extracted using the NucleoSpin® RNA isolation kit (Macherey-Nagel, Hoerdt, France) following the manufacturer’s instructions, except that DNase digestion was performed with twice the indicated amount of enzyme. The total RNA concentration was measured with NanoDrop 2000 (ThermoScientific TM, France). The RNA integrity was verified using an Agilent Bioanalyzer 2100 (Agilent Technologies, France) with a RNA Integrity Number of > 7.5. The extracted total RNA was stored at –80°C until sequencing.
High-quality RNA from all samples was processed for the preparation of cDNA libraries using an Illumina TruSeq RNA sample prep kit for mRNA analysis following the Illumina’s protocols. After quality control and quantification, cDNA libraries were pooled in groups of 6 and sequenced on 5 lanes on the HiSeqTM 2000 (Illumina® NEB, USA) to obtain approximatively 30 million reads (100 bp paired-end) for each sample with insert sizes ranging from 200 to 400 base pairs.
The quality control check on raw reads in FASTQ format was processed using FASTQC. The remaining reads were aligned to the Capra hircus genome (assembly ARS1 from NCBI) using the Burrows-Wheeler Aligner (BWA). Genomic variants including SNPs as well as small insertions and deletions (indels) were detected using mpileup in SAMtools. Variant filtering criterion: A detected variant was kept only if met four criteria: the read depth was more than 10, the quality score was over 20, the minor allele frequency was over 0.05 and the variants present at least in 50% of the group individuals and replicates.
Variant statistics and functional annotation
SNPs, insertions and deletions were compared between samples from the resistant and susceptible groups. Common variants were excluded and only different variants between the two groups were kept for the subsequent analysis. Variant information for distribution among genes was calculated within the R program after merging variants with the respective annotated genes. The effect of the variants (SNPs, insertions and deletions) on genes were determined using variant effect predictor (VEP) web interface tool provided by Ensemble online tools (https://www.ensembl.org/info/docs/tools/vep/index.html) within the goat genome reference (Assembly: ARS1) and results were extracted as.txt file for graphical interface using the R program. Genes containing variants were annotated with Gene Ontology (GO) terms under the categories of biological processes, molecular functions, and cellular components using clusterProfiler R package . The Bonferroni-corrected P-value ≤ 0.05 was used as the threshold. Additionally enriched KEGG pathways for genes containing variants were identified using the same package. The Pathview package was used for visualization . The R version 3.5.1 was used.