- Research article
- Open Access
Exploring the mechanisms of resistance to Teladorsagia circumcincta infection in sheep through transcriptome analysis of abomasal mucosa and abomasal lymph nodes
© The Author(s) 2018
Received: 28 December 2017
Accepted: 28 February 2018
Published: 27 April 2018
The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep.
In Spain, the dairy sheep production of indigenous sheep breeds is based on grazing livestock systems where gastrointestinal nematode (GIN) infections pose a major health problem to adult ewes and cause important economic losses . Ovine resistance to the GIN infection is a highly complex character  and identification of genes influencing increased resistance to GIN infection would be of interest to enhance the efficiency of selection in commercial flocks through the use of molecular information. Hence, several studies have tried to identify QTL influencing indicator traits for GIN infection, such as fecal egg count (FEC), serum levels of IgA and pepsinogen. However, due to the major economic impact of GIN in young animals in countries such as the UK or Australia, most of these QTL studies are focused on lambs [2–5] whereas a limited number of studies have focused on adult sheep [6, 7] or combined data from aged ewes and lambs . In a study carried out with adult sheep, Atlija et al.  suggested that some of the QTL identified that did not show overlapping with previous studies in lambs could be related to specific mechanisms of the immune response that is activated in adult animals. This would support the theory suggested by Stear et al.  that the genetic variation in FEC in lambs is predominantly a consequence of genetic variation in worm length and hence worm fecundity whereas, in contrast, mature sheep may be able to regulate not only fecundity, but also worm number.
As an alternative method to identify genes linked to GIN resistance, studies based on gene expression analysis can identify those genes whose expression may differ between animals and the gene pathways activated depending on the status against infection. In sheep infected by GIN, these kinds of studies based on expression analysis by RT-PCR of specific candidate genes [9–11], the use of microarrays [12, 13] or recently on the RNA-Seq technology [14, 15], have been mainly focused on young animals. For example, for lambs infected with GIN, different studies have shown that the animal status is dependent on the different type of host immune response that is activated against the nematodes in the abomasal (gastric) mucosa and abomasal lymph node [16, 17]. The T. circumcincta infection in lambs has been shown to trigger significant Th2 cytokine changes in mucosa with increased mucosa production of eosinophilia, mastocytosis and neutrophils. Upon GIN infection, the intelectins (ITLN1, ITLN2 and ITLN3) [9, 10] and the interleukin (IL-3, IL-4, IL-5 and IL-13) [18–20] transcripts have been consistently found to be upregulated to induce Th2 response in tissues like abomasal mucosa and lymph nodes of infected lambs whereas this expression was absent in naïve sheep. Irrespective of the breed and infective nematode species, some conserved gene expression responses were identified in relation to early inflammation in resistant lambs and in relation to a chronic inflammatory state in susceptible lambs .
In order to gain knowledge on the different gene expression responses activated in adult sheep between resistant and susceptible animals against T. circumcincta infection, the present study presents a comparative analysis of RNA-Seq datasets obtained from abomasal mucosa and abomasal lymph node obtained from two groups of adult Churra sheep previously classified as resistant and susceptible against this GIN infection. RNA-Seq has been shown as a powerful deep-sequencing technology that can help to elucidate previously inaccessible complexities underlying gene expression responses related to complex quantitative traits such as the resistance to nematode infection . Hence, the study of complete target tissue transcriptomes presented here tries to provide a global picture of the different mechanisms activated as response to infection by GIN in adult sheep.
Materials and methods
Animals and experimental infections
Faecal samples of adult dairy ewes from four Churra dairy sheep flocks reared under semi-intensive management and belonging to the ANCHE breeders’ association (national association of Spanish Churra sheep breeders) were collected to initially assess the GIN infection levels after natural infection. Based on individual FEC, the farm showing the largest range for FEC was selected for additional sampling. In the selected flock, faecal samples were collected from a total of 119 adult ewes 3 months after the last deworming treatment. Based on the individual FEC measures, a total of 18 sheep (age range 6–8 years old) showing the most extreme FEC values were selected for our study. A first experimental infection (EI1) was performed based on a single oral administration of 50 000 T. circumcincta third stage larvae (L3) on these animals and after a treatment with one oral dose of ivermectin (0.2 mg/kg bw, Orame®, Merial, Spain). After this infection, collection of faeces was performed every 2 days, starting from day 14 to day 31 post-infection to calculate the accumulated FEC from each animal. Based on these values, six sheep were classified as susceptible and six sheep as resistant to infection by T. circumcincta. One month after the EI1, all these 12 selected ewes were treated with moxidectin by subcutaneous injection (0.2 mg/kg bw, Cydectin®, Zoetis, Spain) and 3 weeks later were exposed to a second experimental infection (EI2) with a single oral dose of 70 000 T. circumcincta L3. At day 7 after EI2, the animals were sacrificed by an intravenous injection with a lethal dose of 20 mL per sheep of sodium pentobarbital (Dolethal®, Vetoquinol, Spain). At necropsy, abomasal mucosa and lymph node samples for all animals were immediately collected in RNAlater™ Stabilization Reagent (Sigma-Aldrich, St. Louis, MO, USA). These samples were stored overnight at 4 °C and then frozen at −80 °C.
RNA extraction, sequencing and bioinformatics analysis
mRNA were extracted from the abomasal mucosa and abomasal lymph node samples from the six resistant and six susceptible selected animals, using the Absolutely RNA miRNA Kit from Agilent (La Jolla, CA, USA). RNA integrity (RIN value) was analyzed using the agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Considering both tissue samples the RIN values of the RNA samples ranged between 6.7 and 7.8. The preparation of the libraries and subsequent sequencing was performed with an Illumina HiSeq sequencer 2000, generating stranded paired-end reads of 75 bp with a depth of 30 million reads.
The read quality of each sample was assessed using FastQC V_0.11.5 software . The high quality read samples were later aligned against the ovine reference genome (Oar_v3.1) using the alignment software STAR_v2.5.2b . We used the option –outSAMtype BAM Unsorted to obtain an unsorted bam file instead of the sam file obtained by default with the STAR software. The unsorted aligned reads were indexed and sorted by read names using Samtools_v1.3.1 . The sorted reads were counted for features (genes) in each sample using the HTSeq-count software  with the intersection-strict mode and reverse stranded option and using the information of the reference sheep genome annotation (Oar_v3.1.88.gtf) available at the FTP Download-Ensemble (release 88). This produces a feature list (gene list) with number of raw counts for each sample.
Before performing the differential gene expression analysis, we tried to quantify the abundance of all the annotated genes for each abomasal lymph node and abomasal mucosa sample analyzed. The gene expression levels were normalized by library size and gene length by calculating fragments per kilobase of exon per million fragments mapped (FPKM) with the RSEM software package  using the ensemble genome annotation (Oar_v3.1) as a reference. Initially the reference sheep genome annotation (Oar_v3.1.88.gtf) was preprocessed using the option rsem-prepare-reference (–star). After that, we estimated the gene expression levels for each sample using the rsem-calculate-expression (–star) option of the RSEM program. The genes with at least 0.01 FPKM in each sample were considered as expressed and the mean gene expression across susceptible and resistant samples of each tissue were classified as high (> 500 FPKM), medium (10–500 FPKM) and low (< 10 FPKM) expressed genes based on their FPKM values.
The differential expression analysis was performed using two R based packages, EdgeR  and DESeq , with the raw counts from each sample obtained from HTSeq count. In these two software, the differential analysis of count data was performed in a similar way, but following different approaches to estimate normalization and dispersion. DESeq is, in comparison, less powerful but EdgeR is more sensitive to outliers . Thus, after performing differential expression analysis between the resistant and the susceptible sample groups individually with the two software, only the genes commonly identified as differentially expressed genes (DEGs) by both EdgeR (FDR < 0.05) and DESeq (adjusted P < 0.05) were considered as “GIN-activated” DEGs. This double analysis is expected to reduce the presence of false positive results from our analysis. Based on log fold change (Log2FC) values, the DEGs were further classified as up-regulated in each group. These gene lists were subjected to three types of gene-set enrichment analysis [gene ontology (GO) analysis, KEGG pathway analysis and disease association analysis] using the web-based tool WebGestalt . For these analyses, the human genome was considered as a reference and the parameters considered were the default statistical method Hypergeometric, multiple test adjustment with the BH method (Benjamini–Hochberg FDR), the significant level of adjP < 0.05. In addition, for a term to be considered significantly enriched a minimum of five genes were required for GO and disease association analyses, and a minimum of three genes were required for the KEGG pathway analysis.
Infected sheep status confirmation
Based on the accumulated FEC estimation carried out on the 18 animals subjected to EI1, six animals were classified as “susceptible” (Chu6, Chur9, Chu11, Chu14, Chu17 and Chu19) and six as “resistant” (Chu1, Chu2, Chu7, Chu8, Chu15 and Chu21). Two of the individuals that were classified as “resistant” had zero counts throughout the control phase (Chu7, Chu15). The remaining five animals showed an uncertain profile between the two defined categories and therefore were discarded from further analysis. The mean accumulated FEC in the susceptible group was 5594 ± 2661 eggs per gram (epg) and 308 ± 338 epg in the resistant group.
Gene expression level
Results from enrichment analyses performed on the 71 GIN-activated DEGs up-regulated in resistant sheep identified for abomasal lymph node samples
Nb. of genes
KEGG pathway analysis
Glycine, serine and threonine metabolism
ALAS2, PSPH, GLYCTK
C = 32; O = 3; E = 0.03; R = 103.67
rawP = 3.33e−06; adjP = 9.99e−06
PPAR signaling pathway
RXRG, PPARG, AQP7
C = 70; O = 3; E = 0.06; R = 47.39;
rawP = 3.59e−05; a djP = 5.39e−05
CMBL, ALAS2, CKMT1A, PSPH, GLYCTK,
C = 1130; O = 7; E = 1.02; R = 6.85;
rawP = 6.15e−05; adjP = 6.15e−05
Disease association analysis
KRT20, PGC, B4GALNT2, PPARG, LGALS4
C = 354; O = 5; E = 0.32; R = 15.62
rawP = 1.66e−05; adjP = 0.0002
KRT20, PGC, PPARG, SLC22A4, LGALS4
C = 331; O = 5; E = 0.30; R = 16.70
rawP = 1.20e−05; adjP = 0.0002
SFTPD, PPARG, PF4, CLCA1, IL5RA
C = 435; O = 5; E = 0.39; R = 12.71
rawP = 4.43e−05; adjP = 0.0003
KRT20, PGC, PPARG, SLC22A4, LGALS4
C = 413; O = 5; E = 0.37; R = 13.39
rawP = 3.46e−05; adjP = 0.0003
The mechanisms of sheep resistance to GIN infections involve complex immune responses. In relation to the specific infection with T. circumcincta, several studies have previously been carried out in lambs showing that, as a local immune response to T. circumcincta infection, the levels of IgA and eosinophils were increased in the abomasal tissues of infected lambs [31–34]. Based on cDNA microarray-based studies, several up-regulated transcripts were found in abomasal tissues of T. circumcincta infected lambs and confirmed the activation of the Th2-type immune response in mucosa tissue, including eosinophilia and mastocytosis [35–37]. In this study, we tried to decipher the immune mechanisms activated in adult sheep during infection with T. circumcincta through the transcriptome analysis of the two main tissues targeted by the infection, the abomasal mucosa and the abomasal lymph node.
Although our initial purpose was to classify the animals to be studied as resistant and susceptible based on natural infection, the lack of homogeneity regarding the infection level when working with animals in pasture determined the need to perform a first experimental infection (EI1) to ensure similar infection levels among all the animals included in the study.
The results of the differential expression analyses performed in the two tissues studied suggest that at day 7 post-infection there is not a clear differential gene expression response in the abomasal mucosa (due to the lack of DEGs identified by EdgeR). However, a differential response in the abomasal lymph node was clearly observed, based on the identification of 106 genes commonly identified as DEGs by the two software. We acknowledge that the results from the differential expression analyses performed with EdgeR and DESeq may include a similar fraction of false negative, as they both rely on a negative binomial model and use the false discovery rate procedure  to adjust for multiple testing. In this regard, Zhang et al.  suggested that taking the intersection of DEGs from two or more tools is recommended if the number of false positives is a major concern in the study. Following this, we considered for further analyses those genes that were identified as DEGs by the two implemented methods, reducing the initial number of DEGs from 261 and 125, respectively for DESeq and EdgeR, to 106 genes defined as GIN-activated DEGs.
Hence, focusing on the 106 GIN-activated DEGs identified in the complete analysis of the lymph node samples, some of the significant terms identified by the enrichment analyses showed a clear correspondence with immune response mechanisms. In particular, the two GO terms related to cytokines highlighted by the GO analysis of genes upregulated in resistant ewes (cytokine-mediated signaling pathway and cellular response to cytokine stimulus) were related to the same five genes (PALM3, DUOX2, PPARG, PF4, IL5RA) (Table 1). The role of PPARG, DUOX2 and IL5RA genes in relation to the immune response has been previously reported in different nematode infection studies [11, 20, 40]. In our study, the PPARG gene also supported the identification of the PPAR signaling pathway in the KEGG analysis, together with the RXRG and AQP7 genes. The PPARG gene was also linked to many of the significant related terms highlighted by the disease association analysis (gastrointestinal neoplasms, intestinal diseases, inflammation and gastrointestinal diseases, Table 1). The IL5RA gene also supported the identification of the term inflammation as enriched in the disease association analysis (Table 1).
Gastro-intestinal nematode infection studies that have overlapping genes with our study
Tissue used for study
Yearling Scottish Blackface sheep
Challenged naive sheep
ITLN2, IL4, galectin 14
RT-PCR, Western blot
Immune day 5 vs naïve day 5
cDNA microarray, RT-PCR, QT-PCR
ITLN2, ITLN1, ITLN3 galectin 4, galectin 1
cDNA microarray, RT-PCR, QT-PCR
Naive day 5 vs naive day 0
cDNA microarray, RT-PCR, QT-PCR
6 months old Merino-cross wethers
ITLN2, CLCA1, interleukins
Sequential microarray (across all arrays)
6 months old Merino-cross wethers
Sequential microarray (day 22 vs day 3 biopsies)
Primary challenge vs Tertiary challenge
PPARG, KRT5, SLC31A2, KRT18
micro array data, QT PCR
T. colubriformis, H. contortus
DUOX1, IL2RA, IL10
LYZ, ITLN2, ITLN3, CLCA, KRT10, KRT8, KRT19
SDS-PAGE and Shortgun proteomics
Lambs Scottish Blackface
SLC30A2, galectin 14
1 year old canaria hair breed (CHB) and canaria sheep (CS)
galectin 15, IL5, ALPL, MMP1, MMP11, MMP14, MMP2
1 year old canaria hair breed (CHB) and canaria sheep (CS)
Infected CHB and CS
Lambs Scottish Blackface
Resistant vs Control
IL5RA, IL13, IL13RA2, IL1RL1, IL4, SLC9A4
cDNA microarray, RT-PCR
Lambs (Scottish Blackface x Leicester)
Abomasum, lymph node
ITLN2, ITLN1, ITLN3
Among the list of ten genes commonly identified by our study and other authors to be responsive to GIN infection, PPARG, LYZ, and IL5RA are directly related to inflammatory response. PPARG encodes for the peroxisome proliferator activated receptor gamma, which is a ligand activated transcription factor that regulates adipocyte differentiation and glucose homeostasis, but it has also been recognized as playing a key role in the immune response through its ability to inhibit the expression of inflammatory cytokines and to direct the differentiation of immune cells towards anti-inflammatory phenotypes . In our study the PPARG gene was found to be upregulated in resistant ewes compared with susceptible ewes. A modified expression pattern of the PPARG gene in relation to the infection by T. colubriformis and H. contortus in sheep has already been reported by Andronicos et al.  (Table 2). A study carried out on naïve Perendale lambs suggested this gene plays a role in coordinately regulating genes more highly expressed in the intestine of the susceptible lambs . In mice, high expression of the PPARG gene was found in response to nematode infection and the mice lacking the PPARG gene were unable to mount protective immune response to nematode infection. Hence, PPARG was suggested as a factor that drives type 2 responses in worm infection .
The interleukin gene IL5RA was also a GIN-activated gene up-regulated in resistant sheep. This gene supported the enriched GO terms cytokine-mediated signaling pathway and cellular response to cytokine stimulus and the disease association related term Inflammation. This gene was also found to have an increased expression in resistant Scottish Blackface lambs to T. circumcincta by Gossner et al.  (Table 2). IL5RA is required for the biological activities of IL5 to promote eosinophil-mediated activation and recruitment into tissues in acute inflammatory responses . Increased levels of eosinophils have been classically linked to resistance to Trichostrongyle parasites in sheep. Hence, eosinophilia has been suggested as a marker of resistance to T. circumcincta in Scottish Blackface lambs , whereas a more pronounced eosinophilia has been documented in animals bred for increased resistance to T. colubriformis infection .
The LYZ gene encodes lysozyme, a protein with antibacterial activity. In our study this gene was the most highly up-regulated GIN-activated gene in susceptible sheep. This observation agrees with the work of Knight et al.  who reported, in the abomasal mucosa, down-regulation of members of the gastric lysozyme family (LYZ 1A, 2A, 3A and 4A) in immune versus naïve sheep at days 2 and 5 post-challenge with T. circumcincta (Table 2). Gastric lysozyme genes are highly expressed in the ovine abomasum and are thought to act as a major digestive enzyme of the peptidoglycan cell walls of bacteria entering from the rumen, functioning at low pH. Other studies, however, have found genes of the lysozyme family to be up-regulated in abomasal epithelial extracts from previously infected sheep versus naïve sheep [37, 44] (Table 2). Also the alterations in lysozyme production have been suggested to contribute to the resulting nutritional loss seen in infected animals .
Another GIN-activated gene up-regulated in resistant sheep based on our study, CLCA1, has also been identified as up-regulated in immune lambs in the study reported by Knight et al.  and in the H. contortus challenged yearling lambs analyzed by Rowe et al. . The encoded protein of this gene is thought to act as a multifunctional signaling protein, including an early modulator of immune responses by regulation of cytokines . Proteins of the CLCA family may contribute to parasite expulsion by being responsible for mucus hydration across the gut epithelium and smooth muscle contraction  (Table 2).
Another transcript related to mucous cells, ITLN, was the third most highly up-regulated in resistant sheep in our study. The intelectin 1 and 2 are protein coding genes related to carbohydrate binging. Other studies have already reported an increased expression of this gene in relation to the infection response of lambs to T. circumcincta [9, 37] and H. contortus infection . The early expression of ITLN post-challenge in immune yearling sheep compared with naïve yearling sheep was suggested as a protective role and it may also alter the characteristics of mucus leading to worm entrapment  (Table 2). Up-regulation of ITLN has also been reported in resistant mice in response to Trichuris muris infection . The expression of ITLN1 and ITLN3 was found in lymph node tissues in response to T. circumcincta infection in Scottish Blackface x Leicester lambs but no protein expression was found on immunohistochemistry  (Table 2). In cattle, the expression of ITLN2 was reported in abomasal mucosa tissue of resistant 12 month old Angus cattle . In murine models the intelectin transcript plays a key role in the expression of IL-25, IL23 and has been found to amplify type 2 immune response in asthma and atrophic dermatitis conditions in humans . Overall, our study supports the role of some genes such as ITLN2, CLCA1 in adult sheep GIN resistance. These two genes have been previously reported to be increased in immune lambs and to be up-regulated in resistant adult sheep. Whereas the expression of these genes in lambs was found to be affected by infection in abomasal mucosa, our study did not identify an altered pattern in that tissue but only in the lymph node.
Three genes identified in our study as GIN-activated, and up-regulated in resistant sheep, belong to the galectin family, LOC443162 (Gal-14), LGALS4 (Gal-4) and LOC101102156 (Gal-9). Galectins mediate innate and adaptive immune functions by modulating the activity of complement receptor 3, macrophage and dendrocyte adhesion to lymphocytes . The expression of Gal-14 was found in adult sheep after exposure to an allergen (house dust mites) and may be responsible for eosinophil function and inflammation due to allergy . There was a significant up-regulation of Gal-4 in challenged naïve yearling lambs after infection with T. circumcincta L3 . The expression of Gal-14 was maximum at day 10 post-challenge in yearling Scottish Blackface lambs previously infected with T. circumcincta . In Scottish Blackface lambs, the differential expression of the Gal-14 gene was reported in abomasal lymph node of resistant animals after 14-day post-infection with T. circumcincta  (Table 2). In addition, a role of galectins has been widely reported in reference to different host parasite interactions and these proteins appear to be responsible for adhesion of pathogens to host cells and host adaptive immunity .
Our list of GIN-activated genes also includes some genes belonging to the same gene families reported in other relevant GIN studies. Some of these include genes from the matrix metallopeptidase (MMP) family (MMP28) and solute carrier family genes (SLC22A4 and SLC25A34, SLC38A2). The role of these genes in inflammatory diseases has been previously described. Hence, different matrix metallopeptidase family genes were related to inflammatory response in humans, in particular the MMP28 gene was responsible for altering inflammatory response in mice . Also, the expression of other SLC family members were reported in response to nematode infection in both susceptible and resistant sheep and variants in the SLC22A4 gene have been associated to Inflammatory Bowel disease in humans . Whereas, high expression of SLC30A2 in susceptible sheep in response to T. circumcincta infection has been reported in Scottish Blackface lambs , H. contortus infection shows a significant impact on the expression of the SLC2A3 gene in Canary sheep breeds  (Table 2).
It is noteworthy that there is a list of about 86 genes identified as GIN-activated in our study that have not been reported or that do not belong to gene families considered as responsive to GIN infection in previous studies (Additional file 10). Interestingly, for some of these genes we found connection with the immune response (PGC, SFTPD, TUBA4A, SST, BPIFB1, PF4, B4GALNT2, JCHAIN, AQP7, KLHL25, NEDD4, ANO6). Because most of the previously reported studies considered in our comparative survey (Table 2) are focused on lambs, we think that the genes reported here in Additional file 10 might indicate genes that are specifically activated in adult sheep and not in lambs.
Other genes identified as DEGs by other studies have not been found as GIN-activated genes in the present work. Hence, genes belonging to the chemokine (CCL, CXCL) and the collagen families (COL9A2, COL6A5), or the interferon gamma gene (IFNG), etc. which are reported as DEGs in abomasal lymph node in resistant lambs as a response to T. circumcincta infection [14, 20] were not found as DEGs in our study. This may be due to differential activation of the immune response to T. circumcincta infection in adult sheep compared with young animals although some similar pathways were activated. Also the differences among the different studies regarding the species responsible for gastrointestinal parasite infection, the different experimental approaches (natural vs artificial), the post-infection sample extraction timings, or the environmental conditions (dry vs humid climates) are other major factors that could explain discrepancies related to the specific genes activated during GIN infection.
Also some of these genes may have been identified as DEGs in our study by one of the two DE analyses performed. Some genes such as CENPN, FABP4, HSH2D, KIF2C, KIFC1, MMP1, NDC80, NEK2, PKMYT1, SFN, SPAG5, UBE2C, UHRF1, which have been reported as GIN related by other studies were only identified as DEGs by DESeq but not by EdgeR. Hence, the approach implemented in our paper to avoid false positives, may have determined a restrictive threshold and we may have lost some important genes in our final list of GIN-activated genes.
Our study provides a global picture of the changes that occur in the transcriptome of target tissues, abomasal mucosa and abomasal lymph node, as a response to T. circumcincta infection in resistant and susceptible adult sheep. Whereas at day 7 post-infection, we did not find a differential response between the two compared groups in abomasal mucosa, a total of 106 genes were identified to show a distinct pattern between the two contrasted groups in the lymph node samples. The comparative study of our results with the available literature has shown remarkable coincidences for some of these genes with other gene expression studies related to GIN infection in lambs. Hence, the expression of genes such as ITLN2, CLCA1, galectin 14, etc. appear to be consistently affected by nematode infection, in both lambs and adult sheep. The differential expression of some immune-related genes reported in the present study as a response to T. circumcincta and that are not coincident with previous studies focused on lamb animals could indicate immune mechanisms that are specifically activated in adult animals (e.g. PGC, SFTPD, TUBA4A, SST, BPIFB1, PF4, B4GALNT2, JCHAIN, AQP7, KLHL25, NEDD4, ANO6). The RNA-Seq technology has shown here to be an appropriate platform to investigate the molecular mechanisms underlying the immune response to nematode infection in adult sheep. Future studies will focus on the genetic variability of the GIN-activated genes reported here with the aim of identifying potential candidate mutations that could be directly implemented in selection programs to increase GIN resistance in commercial sheep populations.
The authors declare that they have no competing interests.
JJA and BGG conceived and designed the study. MMV designed and performed the experimental infections. ASV optimized the bioinformatics workflow and PKC performed the bioinformatics analyses. The manuscript was written by PKC and critically reviewed by JJA, BGG, MMV and ASV. All authors read and approved the final manuscript.
Financial support for this project was received from the LE248U14 project of Junta de Castilla and León Government. P. K. Chitneedi is funded by a predoctoral fellowship from the Junta de Castilla and León Government and the European Social Fund. B Gutiérrez-Gil is funded by the “Ramón y Cajal” Programme (RYC-2012-10230) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). M. Martínez-Valladares is also funded by the “Ramón y Cajal” Programme (RYC-2015-18368) from MINECO.
Ethics approval and consent to participate
The whole experiment was carried out according to the current National Spanish legislation on the protection of animals used in experimentation (Royal Decree 53/2013) and after obtaining the positive report from the subcommittee for experimentation and animal welfare of the University of León (OEBA), and the approval of the competent body of the regional government, Junta de Castilla y León.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Riggio V, Pong-Wong R, Sallé G, Usai MG, Casu S, Moreno CR, Matika O, Bishop SC (2014) A joint analysis to identify loci underlying variation in nematode resistance in three European sheep populations. J Anim Breed Genet 131:426–436View ArticlePubMedPubMed CentralGoogle Scholar
- Silva MVB, Sonstegard TS, Hanotte O, Mugambi JM, Garcia JF, Nagda S, Gibson JP, Iraqi FA, McClintock AE, Kemp SJ, Boettcher PJ, Malek M, Van Tassell CP, Baker RL (2012) Identification of quantitative trait loci affecting resistance to gastrointestinal parasites in a double backcross population of Red Maasai and Dorper sheep. Anim Genet 43:63–71View ArticlePubMedGoogle Scholar
- McRae KM, McEwan JC, Dodds KG, Gemmell NJ (2014) Signatures of selection in sheep bred for resistance or susceptibility to gastrointestinal nematodes. BMC Genomics 15:637View ArticlePubMedPubMed CentralGoogle Scholar
- Riggio V, Matika O, Pong-Wong R, Stear MJ, Bishop SC (2013) Genome-wide association and regional heritability mapping to identify loci underlying variation in nematode resistance and body weight in Scottish Blackface lambs. Heredity 110:420–429View ArticlePubMedPubMed CentralGoogle Scholar
- Sallé G, Jacquiet P, Gruner L, Cortet J, Sauvé C, Prévot F, Grisez C, Bergeaud JP, Schibler L, Tircazes A, François D, Pery C, Bouvier F, Thouly JC, Brunel JC, Legarra A, Elsen JM, Bouix J, Rupp R, Moreno CR (2012) A genome scan for QTL affecting resistance to haemonchus contortus in sheep. J Anim Sci 90:4690–4705View ArticlePubMedGoogle Scholar
- Gutiérrez-Gil B, Pérez J, Álvarez L, Martínez-Valladares M, de la Fuente LF, Bayón Y, Meana A, San Primitivo F, Rojo-Vázquez FA, Arranz JJ (2009) Quantitative trait loci for resistance to trichostrongylid infection in Spanish Churra sheep. Genet Sel Evol 41:46View ArticlePubMedPubMed CentralGoogle Scholar
- Atlija M, Arranz J-J, Martinez-Valladares M, Gutiérrez-Gil B (2016) Detection and replication of QTL underlying resistance to gastrointestinal nematodes in adult sheep using the ovine 50K SNP array. Genet Sel Evol 48:4View ArticlePubMedPubMed CentralGoogle Scholar
- Stear MJ, Strain S, Bishop SC (1999) Mechanisms underlying resistance to nematode infection. Int J Parasitol 29:51–56View ArticlePubMedGoogle Scholar
- French AT, Knight PA, Smith WD, Brown JK, Craig NM, Pate JA, Miller HR, Pemberton AD (2008) Up-regulation of intelectin in sheep after infection with Teladorsagia circumcincta. Int J Parasitol 38:467–475View ArticlePubMedGoogle Scholar
- French AT, Knight PA, David Smith W, Pate JA, Miller HR, Pemberton AD (2009) Expression of three intelectins in sheep and response to a Th2 environment. Vet Res 40:53View ArticlePubMedPubMed CentralGoogle Scholar
- Ingham A, Reverter A, Windon R, Hunt P, Menzies M (2008) Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep. Int J Parasitol 38:431–442View ArticlePubMedGoogle Scholar
- Rowe A, Gondro C, Emery D, Sangster N (2009) Sequential microarray to identify timing of molecular responses to Haemonchus contortus infection in sheep. Vet Parasitol 161:76–87View ArticlePubMedGoogle Scholar
- Andronicos N, Hunt P, Windon R (2010) Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus. Int J Parasitol 40:417–429View ArticlePubMedGoogle Scholar
- McRae KM, Good B, Hanrahan JP, McCabe MS, Cormican P, Sweeney T, O’Connell MJ, Keane OM (2016) Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance. Vet Parasitol 224:96–108View ArticlePubMedGoogle Scholar
- Guo Z, González JF, Hernandez JN, McNeilly TN, Corripio-Miyar Y, Frew D, Morrison T, Yu P, Li RW (2016) Possible mechanisms of host resistance to Haemonchus contortus infection in sheep breeds native to the Canary Islands. Sci Rep 6:26200View ArticlePubMedPubMed CentralGoogle Scholar
- Muñoz-Guzmán MA, Cuenca-Verde C, Valdivia-Anda G, Cuéllar-Ordaz JA, Alba-Hurtado F (2012) Differential immune response between fundic and pyloric abomasal regions upon experimental ovine infection with Haemonchus contortus. Vet Parasitol 185:175–180View ArticlePubMedGoogle Scholar
- Terefe G, Lacroux C, Andréoletti O, Grisez C, Prevot F, Bergeaud JP, Penicaud J, Rouillon V, Gruner L, Brunel JC, François D, Bouix J, Dorchies P, Jacquiet P (2007) Immune response to Haemonchus contortus infection in susceptible (INRA 401) and resistant (Barbados Black Belly) breeds of lambs. Parasite Immunol 29:415–424View ArticlePubMedGoogle Scholar
- Lacroux C, Nguyen THC, Andreoletti O, Prevot F, Grisez C, Bergeaud JP, Gruner L, Brunel JC, Francois D, Dorchies P, Jacquiet P (2006) Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response. Vet Res 37:607–622View ArticlePubMedGoogle Scholar
- Pernthaner A, Cole SA, Morrison L, Hein WR (2005) Increased expression of interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha genes in intestinal lymph cells of sheep selected for enhanced resistance to nematodes during infection with Trichostrongylus colubriformis. Infect Immun 73:2175–2183View ArticlePubMedPubMed CentralGoogle Scholar
- Gossner A, Wilkie H, Joshi A, Hopkins J (2013) Exploring the abomasal lymph node transcriptome for genes associated with resistance to the sheep nematode Teladorsagia circumcincta. Vet Res 44:68View ArticlePubMedPubMed CentralGoogle Scholar
- Costa V, Aprile M, Esposito R, Ciccodicola A (2013) RNA-Seq and human complex diseases: recent accomplishments and future perspectives. Eur J Hum Genet 21:134–142View ArticlePubMedGoogle Scholar
- Bioinformatics B (2011) FastQC A quality control tool for high throughput sequence data. UK Babraham Inst, CambridgeGoogle Scholar
- Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21View ArticlePubMedGoogle Scholar
- Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25:2078–2079View ArticlePubMedPubMed CentralGoogle Scholar
- Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169View ArticlePubMedGoogle Scholar
- Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform 12:323View ArticleGoogle Scholar
- Robinson MD, McCarthy DJ, Smyth GK (2009) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140View ArticlePubMedPubMed CentralGoogle Scholar
- Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106View ArticlePubMedPubMed CentralGoogle Scholar
- Anders S, McCarthy DJ, Chen Y, Okoniewski M, Smyth GK, Huber W, Robinson MD (2013) Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat Protoc 8:1765–1786View ArticlePubMedGoogle Scholar
- Wang J, Duncan D, Shi Z, Zhang B (2013) WEB-based gene set analysis toolkit (WebGestalt): update 2013. Nucleic Acids Res 41:W77–W83View ArticlePubMedPubMed CentralGoogle Scholar
- Martínez-Valladares M, Vara-Del Río MP, Cruz-Rojo MA, Rojo-Vázquez FA (2005) Genetic resistance to Teladorsagia circumcincta: IgA and parameters at slaughter in Churra sheep. Parasite Immunol 27:213–218View ArticlePubMedGoogle Scholar
- Strain SA, Bishop SC, Henderson NG, Kerr A, McKellar QA, Mitchell S, Stear MJ (2002) The genetic control of IgA activity against Teladorsagia circumcincta and its association with parasite resistance in naturally infected sheep. Parasitology 124:545–552View ArticlePubMedGoogle Scholar
- Balic A, Bowles VM, Liu YS, Meeusen ENT (2003) Local Immune responses in sensitized sheep following challenge infection with Teladorsagia circumcincta. Parasite Immunol 25:375–381View ArticlePubMedGoogle Scholar
- Henderson NG, Stear MJ (2006) Eosinophil and IgA responses in sheep infected with Teladorsagia circumcincta. Vet Immunol Immunopathol 112:62–66View ArticlePubMedGoogle Scholar
- Keane O, Zadissa A, Wilson T, Hyndman DL, Greer GJ, Baird DB, McCulloch AF, Crawford AM, McEwan JC (2006) Gene expression profiling of naive sheep genetically resistant and susceptible to gastrointestinal nematodes. BMC Genomics 7:42View ArticlePubMedPubMed CentralGoogle Scholar
- Craig NM, Miller HRP, Smith WD, Knight PA (2007) Cytokine expression in naïve and previously infected lambs after challenge with Teladorsagia circumcincta. Vet Immunol Immunopathol 120:47–54View ArticlePubMedGoogle Scholar
- Knight PA, Griffith SE, Pemberton AD, Pate JM, Guarneri L, Anderson K, Talbot RT, Smith S, Waddington D, Fell M, Archibald AL, Burgess STG, Smith DW, Miller HRP, Morrison IW (2011) Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta. Vet Res 49:78View ArticleGoogle Scholar
- Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B 57:289–300Google Scholar
- Zhang ZH, Jhaveri DJ, Marshall VM, Bauer DC, Edson J, Narayanan RK, Robinson GJ, Lundberg AE, Bartlett PF, Wray NR, Zhao QY (2014) A comparative study of techniques for differential expression analysis on RNA-seq data. PLoS One 9:e103207View ArticlePubMedPubMed CentralGoogle Scholar
- Chen T, Tibbitt CA, Feng X, Stark JM, Rohrbeck L, Rausch L, Sedimbi SK, Karlsson MC, Lambrecht BN, Hedestam GB, Hendriks RW, Chambers BJ, Nylén S, Coquet JM (2017) PPAR-γ promotes type 2 immune responses in allergy and nematode infection. Sci Immunol 2:eaal5196View ArticlePubMedGoogle Scholar
- Martin H (2010) Role of PPAR-gamma in inflammation. Prospects for therapeutic intervention by food components. Mutat Res 690:57–63View ArticlePubMedGoogle Scholar
- Pease JE, Williams TJ (2001) Eotaxin and asthma. Curr Opin Pharmacol 1:248–253View ArticlePubMedGoogle Scholar
- Woolaston RR, Manueli P, Eady SJ, Barger IA, Le Jambre LF, Banks DJ, Windon RG (1996) The value of circulating eosinophil count as a selection criterion for resistance of sheep to trichostrongyle parasites. Int J Parasitol 26:123–126View ArticlePubMedGoogle Scholar
- Athanasiadou S, Pemberton A, Jackson F, Inglis N, Miller HR, Thévenod F, Mackellar A, Huntley JF (2008) Proteomic approach to identify candidate effector molecules during the in vitro immune exclusion of infective Teladorsagia circumcincta in the abomasum of sheep. Vet Res 39:58View ArticlePubMedGoogle Scholar
- Erickson NA, Mundhenk L, Giovannini S, Glauben R, Heimesaat MM, Gruber AD (2016) Role of goblet cell protein CLCA1 in murine DSS colitis. J Inflamm 13:5View ArticleGoogle Scholar
- Datta R, DeSchoolmeester ML, Hedeler C, Paton NW, Brass AM, Else KJ (2005) Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite. Infect Immun 73:4025–4033View ArticlePubMedPubMed CentralGoogle Scholar
- Li RW, Rinaldi M, Capuco AV (2011) Characterization of the abomasal transcriptome for mechanisms of resistance to gastrointestinal nematodes in cattle. Vet Res 42:114View ArticlePubMedPubMed CentralGoogle Scholar
- Yi L, Cheng D, Zhang K, Huo X, Mo Y, Shi H, Di H, Zou Y, Zhang H, Zhao J, Xu Y, Erle DJ, Zhen G (2017) Intelectin contributes to allergen-induced IL-25, IL-33, and TSLP expression and type 2 response in asthma and atopic dermatitis. Mucosal Immunol 10:1491–1503View ArticlePubMedPubMed CentralGoogle Scholar
- Vasta GR, Quesenberry M, Ahmed H, O’Leary N (1999) C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev Comp Immunol 23:401–420View ArticlePubMedGoogle Scholar
- Dunphy JL, Barcham GJ, Bischof RJ, Young AR, Nash A, Meeusen EN (2002) Isolation and characterization of a novel eosinophil-specific galectin released into the lungs in response to allergen challenge. J Biol Chem 277:14916–14924View ArticlePubMedGoogle Scholar
- Vasta GR (2009) Roles of galectins in infection. Nat Rev Microbiol 7:424–438View ArticlePubMedPubMed CentralGoogle Scholar
- Manicone AM, McGuire JK (2008) Matrix metalloproteinases as modulators of inflammation. Semin Cell Dev Biol 19:34–41View ArticlePubMedGoogle Scholar
- McCann MJ, Johnston S, Reilly K, Men X, Burgess EJ, Perry NB, Roy NC (2014) The effect of turmeric (Curcuma longa) extract on the functionality of the solute carrier protein 22 A4 (SLC22A4) and interleukin-10 (IL-10) variants associated with inflammatory bowel disease. Nutrients 6:4178–4190View ArticlePubMedPubMed CentralGoogle Scholar