Possible mechanisms of host resistance to Haemonchus contortusinfection in sheep breeds native to the Canary Islands

1 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 www.nature.com/scientificreports Possible mechanisms of host resistance to Haemonchus contortus infection in sheep breeds native to the Canary Islands Zhengyu Guo1, Jorge Francisco González2, Julia N. Hernandez2, Tom N. McNeilly3, Yolanda Corripio-Miyar3, David Frew3, Tyler Morrison3, Peng Yu1 & Robert W. Li4 Haemonchus contortus appears to be the most economically important helminth parasite for small ruminant production in many regions of the world. The two sheep breeds native to the Canary Islands display distinctly different resistant phenotypes under both natural and experimental infections. Canaria Hair Breed (CHB) tends to have significantly lower worm burden and delayed and reduced egg production than the susceptible Canaria Sheep (CS). To understand molecular mechanisms underlying host resistance, we compared the abomasal mucosal transcriptome of the two breeds in response to Haemonchus infection using RNAseq technology. The transcript abundance of 711 and 50 genes were significantly impacted by infection in CHB and CS, respectively (false discovery rate <0.05) while 27 of these genes were significantly affected in both breeds. Likewise, 477 and 16 Gene Ontology (GO) terms were significantly enriched in CHB and CS, respectively (P < 1.0 × 10−4). A broad range of mechanisms have evolved in resistant CHB to provide protection against the parasite. Our findings suggest that readily inducible acute inflammatory responses, complement activation, accelerated cell proliferation and subsequent tissue repair, and immunity directed against parasite fecundity all contributed to the development of host resistance to parasitic infection in the resistant breed. The intestinal worm Haemonchus contortus is arguably the most economically important helminth parasite for small ruminant production in many regions of the world. As a voracious blood feeder residing in the mucosal layer of the abomasum, H. contortus causes anaemia and hyper-gastrinaemia and alters abomasal secretion. H. contortus infection results in reduced growth, compromised reproduction, and elevated mortality, due to its ubiquitous distribution and severe pathogenicity. Consequently, H. contortus parasitism represents the primary constraint to profitable production of sheep and goats worldwide. Over the past years, the rapid emergence of drug-resistant H. contortus strains and increasing demands by consumers for inexpensive organic meat and milk products with less drug residues have spurred research on the development of anthelmintic-independent parasite control strategies, such as vaccines1 and novel biologics, nutrient supplements and bioactive compounds, and selective breeding. Among them, selectively breeding sheep and goats with abilities to better resist parasitic infections appears to be a solution to sustainable small ruminant production. Differences in resistance and susceptibility to parasitic infections between sheep breeds have been long documented2. Over the decades, comparative studies have identified at least 19 sheep breeds displaying varying degrees of resistance to parasitic infections3. For example, St. Croix lambs shed significantly fewer eggs and harbor 99% fewer worms in the abomasum than the age-matched Dorset lambs during both natural and experimental infections4. Locally-adapted breeds such as Santa Ines sheep of Brazil have significantly reduced worm 1Department of Electrical and Computer Engineering & TEES-AgriLife Center for Bioinformatics and Genomic Systems Engineering (CBGSE) Texas A&M University, College Station, TX 77843, USA. 2Department of Animal Pathology, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Las Palmas, Spain. 3Moredun Research Institute, Midlothian EH26 0PZ, Scotland, UK. 4United States Department of Agriculture, Agriculture Research Service (USDA-ARS), Animal Genomics and Improvement Laboratory, Beltsville, MD 20705, USA. Correspondence and requests for materials should be addressed to J.F.G. (email: [email protected]) or P.Y. (email: [email protected]) or R.W.L. (email: [email protected]) received: 15 October 2015 Accepted: 28 April 2016 Published: 20 May 2016 OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 burdens and fewer nodular lesions under natural infections than Suffolk and Ile de France lambs on the same pasture5. In Europe, resistance against H. contortus is better developed in Merinoland sheep than in Rhon sheep6. Red Maasai sheep have been shown to be more resistant to Haemonchus infection than the South African Dorper breed during natural exposure to parasites in Kenya7. Moreover, resistance to parasite infection has a significant genetic component. The contribution of the host genome and genetics has been estimated. For example, additive genetic variation accounts for approximately 30% of the overall variation for parasitic infection8. The resistance traits are often polygenic in nature and not influenced by genes with major effects9. Nevertheless, estimates of heritability for parasite indicator traits in small ruminants are phenotype-dependent, ranging from 0.11 to 0.40 for transformed fecal egg counts (EPG) and 0.19 to 0.26 for packed cell volume (PCV) in German Rhon sheep10. In addition, the host age plays a role. A good example is that in Scottish Blackface lambs at the end of the first grazing season, the heritability of adult worm length is very strong at 0.628. While many efforts have been made to identify genetic variants associated with parasite resistance and tolerance in sheep breeds11–13, molecular mechanisms and biological pathways underlying host resistance to parasitic infections in sheep remain largely unknown. Due to unique geographical characteristics of the Canary Islands, indigenous sheep breeds have been exploited by local farmers for centuries. Among them, the Canaria Hair Breed (CHB) and Canaria sheep (CS) are predominately raised for the production of meat and milk, respectively. Previous studies demonstrate that CHB constantly displays better resistance phenotypes to H. contortus infection than CS, including significantly lower levels of fecal egg counts, fewer adult worm counts, lower number of eggs in utero and female worm stunting14. Further studies15 identified significant negative correlations between two effector cells, eosinophils and γ δ /WC1+ T cells, and parasite fecundity in CHB, suggesting that inter-breed difference in regulating immune responses affects Haemonchus infection. In this study, we conducted a RNA-seq based comparative transcriptome analysis in the two indigenous breeds and attempted to understand the molecular basis underlying host resistance. Results Parasitology. The total worms recovered from the infected groups of CHB and CS were 1,109.75 (± 1,547.73, SD) and 3,280.50 (± 2,398.03), respectively. The difference is statistically significant (P < 0.05, Fig.1). Neither Haemonchus worms nor fecal eggs were recovered from the uninfected group of either breed, as expected. EPG values detected from infected CS sheep were 262.50 ± 287.54 (mean ± SD) while no fecal eggs were detectable in the infected group of CHB sheep at 20 days post infection (dpi). No parasite eggs in either group prior to the experimental challenge were observed. Haemonchus infection induced distinctly different transcriptome patterns in the abomasal mucosa of CHB and CS breeds. In this study, approximately 79.91% of raw reads (± 7.08%; SD) were uniquely mapped to the ovine genome. Compared to their respective uninfected controls, the numbers of genes significantly impacted by infection in CHB and CS breeds at a stringent cutoff value (false discovery rate or FDR < 0.05), were 711 and 49, respectively (Fig.2). The abundance of 27 genes was significantly changed by infection in both breeds (Table1). Among them, 25 genes, such as arachidonate 15-lipoxygenase (ALOX15), collagen, type VI, α 5 (COL6A5), and serglycin (SRGN), were significantly upregulated while the expression of transthyretin (TTC) was repressed by infection. Intriguingly, the transcript abundance of cadherin 26 (CDH26) was significantly induced by infection in both breeds (adjusted P value or FDR < 1.63 × 10−10); and is strongly correlated with worm counts only in CS (Fig.3). However, infection had a bidirectional impact on the transcript abundance of a uncharacterized gene containing a unknown microRNA (ENSOARG00000023771), which was significantly upregulated in CHB but downregulated in CS. The genes significantly impacted by infection only in CS included mast cell proteinase-3, γ -glutamyltransferase 5 (GGT5), CD163 as well as those involved in smooth muscle contraction, such as tropomyosin (TPM2), myosin, light chain 9, regulatory (MYL9), and calponin 1, basic, smooth muscle (CNN1). Figure 1. Differences in worm counts between resistant and susceptible sheep breeds under experimental Haemonchus contortus infection. Boxes denote the inter-quartile range between the 1st and 3rd quartiles (25 and 75%, respectively). Black line: mean; Yellow line: median. CHB: Canaria Hair Breed (resistant). CS: Canaria sheep (susceptible). www.nature.com/scientificreports/ 3 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 Among the genes significantly impacted by infection in CHB sheep, several cytokine receptors and chemokines were strongly upregulated. Notable, the transcript of IL17 receptor beta (IL17RB) was 14.4 fold higher in infected animals than uninfected controls in CHB. IL2 receptor beta (IL2B) was also upregulated. Similarly, chemokine CXC ligand 12 (CXCL12) and chemokine (CXC motif) receptor 6 (CXCR6) were upregulated by infection in CHB. Among the well-known Th2 cytokines, the expression of IL6, IL10 and IL13 was upregulated by infection in both breeds. Moreover, while the extent of upregulation of IL6 by infection remained similar in both breeds (~6.8 fold), overexpression of both IL10 and IL13 mRNA molecules was more profound in the resistant breed (CHB) than in CS. On the other hand, the IL5 mRNA was upregulated by infection in CS but barely detectable in CHB at the sequencing depth in this study. The IL4 expression followed the similar trend: it was upregulated approximately 9 fold by infection in CS but was barely detectable in CHB. However, the IL9 mRNA level remained unchanged by infection in both breeds. Several genes involved in arachidonic acids metabolism, including eicosanoids metabolism, were significantly impacted by infection, such as arachidonate 5-lipoxygenase (ALOX5) and its activating protein (ALOX5P), prostaglandin reductase 1 (PTGR1), prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) (PTGS1, COX1), and thromboxane A synthase 1 (TBXAS1), were all strongly upregulated by infection in CHB. In addition, at least 11 genes implicated in complement activation were significantly impacted by infection in CHB, such as complement factor properdin (CFP, 2.8 fold), complement component 7 (C7, 4.2 fold), and complement factor I (CFI, 12.1 fold). Other known genes involved in protective immunity to helminth infection strongly upregulated by infection in CHB included amphiregulin (AREG, 2.2 fold), granzyme genes A and B (GZMA and GZMB, 6.8 and 12.9 fold, respectively). 41 of the 711 genes significantly impacted by infection in CHB are related to extracellular matrix (ECM, Table2). Of them, fibronectin 1 (FN1) was strongly upregulated. At least ten collagen genes were significantly upregulated, such as those from Type I, Type III, Type V, Type VI, and Type XII (Table2). For example, the expression of collagen, type VI, alpha 5 (COL6A5) and collagen, type XII, alpha 1 (COL12A1) was increased 20.8 and 2.4 fold, respectively in CHB, compared to the uninfected controls. Likewise, matrix metallopeptidase 1 (MMP1), MMP2, and MMP14 were significantly up-regulated while the transcript of MMP11 was repressed by infection. Furthermore, several cell adhesion molecules, including integrins, lectins, and cadhesion, were strongly upregulated by infection in CHB, such as conglutinin-like (COLEC8, 451.7 fold), integrin, α 11 (ITGA11, 3.4 fold), and lectin, galactoside-binding, soluble, 15 (LGALS15, 340.1 fold). Of note, approximately 15% of the genes significantly impacted by infection are cell-cycle related. The expression of these cell cycle related genes was predominantly enhanced by Haemonchus infection in CHB. As Table3 shows, at least 92 genes were significantly upregulated by infection, such as cyclin A2 (CCNA2), cyclin B3 (CCNB3), various centromere proteins (CENPL, CENPN, CENPT, and CENPW) and kinesin family (KIF) members, and at least 5 minichromosome maintenance complex (MCM) components (MCM3, MCM4, MCM5, Figure 2. (A) Venn Diagram showing the number of genes with significant differences in transcript abundance induced by infection in two sheep breeds compared to their respective uninfected controls at a false discovery rate (FDR) cutoff < 0.05. CHB: Canaria Hair Breed (resistant). CS: Canaria Sheep (susceptible). (B,C) Scatter plot of log2 ratio (fold change) vs mean. The red color indicates genes detected as differentially expressed between the infected group and uninfected controls at a false discovery rate (FDR) < 0.05 in resistant Canaria Hair Breed (B) and susceptible Canaria Sheep (C). www.nature.com/scientificreports/ 4 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 MCM6, and MCM10). Nevertheless, the infection was also able to repress cell cycle related genes, such as cyclin G1 (CCNG1), regulator of cell cycle (RGCC), and synaptonemal complex protein 3 (SYCP3). Moreover, at least five transcription factors, such as the oncogene MYB, SMAD family members 6 and 9 (SMAD6 and SMAD9), and histone decetylase 5 (HDAC5), were significantly affected by infection in CHB. Intriguingly, four genes known to regulate abomasal acid secretion and gastric function16 were downregulated by Haemonchus infection in CHB, including ATPase, H+ /K+ exchanging, alpha polypeptide (ATP4A), progastricsin (pepsinogen C, PGC), appetite-regulating hormone precursor (GHRL), and forkhead box A2 (FOXA1). However, the transcript abundance of these four genes remained unchanged by infection in CS. Gene_ID Symbol Fold change FDR CHB CS CHS CS ENSOARG00000000338 ABCA2 2.86 2.40 2.28% 1.29% ENSOARG00000008480 ALOX15 10.02 11.45 2.06% 0.00% ENSOARG00000015249 CDH26 150.19 54.19 0.00% 0.00% ENSOARG00000018133 CFTR 4.33 2.38 1.21% 3.67% ENSOARG00000014842 COL6A5 20.79 6.28 1.67% 0.04% ENSOARG00000007787 FCER1A 20.69 18.23 0.00% 0.00% ENSOARG00000019163 HBBB 11.47 43.75 1.63% 0.00% ENSOARG00000008994 IGHE 22.73 35.02 0.00% 0.00% ENSOARG00000013111 IL1RL1 9.73 5.26 0.00% 0.00% ENSOARG00000016842 MCTP1 3.89 2.95 1.26% 1.03% ENSOARG00000002234 SLC2A3 4.39 3.06 0.00% 0.03% ENSOARG00000005322 SRGN 4.25 3.60 0.45% 0.00% ENSOARG00000012855 ST3GAL4 2.44 3.69 0.84% 0.94% ENSOARG00000009990 SYNM 2.16 6.18 2.99% 0.09% ENSOARG00000005941 TNC 3.00 4.01 2.80% 3.19% ENSOARG00000014689 TPSAB1 6.96 9.43 0.00% 0.00% ENSOARG00000006342 TTR 0.28 0.37 0.00% 3.17% ENSOARG00000000857 8.41 7.84 0.00% 1.29% ENSOARG00000002036 38.84 44.81 0.00% 0.00% ENSOARG00000002629 13.34 23.51 0.00% 0.00% ENSOARG00000002942 7.16 7.83 0.00% 0.00% ENSOARG00000002964 12.61 29.65 0.01% 0.00% ENSOARG00000006087 13.49 26.69 0.00% 0.00% ENSOARG00000013005 3.00 3.53 4.13% 0.94% ENSOARG00000013263 71.33 91.67 0.00% 0.01% ENSOARG00000017398 5.73 7.34 2.15% 0.65% ENSOARG00000023771 0.46 3.00 0.19% 1.55% Table 1. Genes significantly impacted by Haemonchus contortus infection in both CHB and CS breeds. Figure 3. Nonlinear regression between worm counts and normalized transcript abundance per million mapped reads of the gene cadherin 26 (CDH26) in susceptible Canaria Sheep (CS). Dotted lines: 95% confidence interval. www.nature.com/scientificreports/ 5 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 The RNAseq results of selected genes were validated by real-time RT-PCR (Fig.4). For example, the expression of CFI, CXCR6, LGALS15, and MMP1 was significantly upregulated while TFF2 mRNA level was significantly repressed by infection only in the resistant breed (CHB), in a good agreement with the RNAseq analysis. A strong correlation in log2 transformed fold values between the two platforms, qPCR and RNAseq, was evident (a correlation coefficient R = 0.946; Fig.5). Gene Ontology (GO) implicated in host resistance. Among 477 and 16 GO terms significantly enriched in CHB and CS at a P value cutoff 1.0 × 10−4, respectively, five were significantly enriched in both breeds (Table4). Select GO terms that may be implicated in the development of host resistance to Haemonchus infection are listed in Table5. Several GO related to complement activation (both classical and alternative pathways) and its regulation were significantly enriched only in CHB. Numerous cell cycle related GO were significantly enriched as well (Fig.6). GO related to secretory granule and gastric acid secretion were also enriched, suggesting that the ability to regulate secretory and gastric function of the host may be involved in the development of host resistance. Furthermore, the regulation of inflammation at the site of infection (mucosa), including arachidonic acid GeneID Symbol Fold change P value FDR ENSOARG00000013782 ALB 0.20 0.0000 0.10% ENSOARG00000008507 ALPL 10.59 0.0000 0.00% ENSOARG00000005139 APLP1 0.54 0.0001 0.49% ENSOARG00000018738 BMP2 1.87 0.0004 1.11% ENSOARG00000012877 CFP 2.78 0.0000 0.19% ENSOARG00000004871 COL1A1 2.18 0.0005 1.24% ENSOARG00000001508 COL1A2 1.87 0.0029 4.42% ENSOARG00000016476 COL3A1 2.09 0.0000 0.21% ENSOARG00000002129 COL5A1 2.39 0.0002 0.55% ENSOARG00000016440 COL5A2 1.93 0.0001 0.28% ENSOARG00000012810 COL6A1 3.72 0.0000 0.00% ENSOARG00000012880 COL6A2 3.25 0.0000 0.17% ENSOARG00000019080 COL6A3 2.65 0.0003 0.83% ENSOARG00000014842 COL6A5 20.79 0.0008 1.67% ENSOARG00000006410 COL12A1 2.37 0.0005 1.26% ENSOARG00000009670 CPXM2 2.10 0.0007 1.63% ENSOARG00000017328 F3 3.03 0.0000 0.06% ENSOARG00000019404 FBLN1 1.53 0.0022 3.54% ENSOARG00000017189 FBN2 0.41 0.0000 0.01% ENSOARG00000016733 FGA 0.38 0.0009 1.93% ENSOARG00000019329 FN1 4.77 0.0000 0.07% ENSOARG00000018483 ITGA11 3.37 0.0015 2.75% ENSOARG00000016642 ITGB7 1.93 0.0032 4.70% ENSOARG00000010344 LTBP1 2.34 0.0000 0.16% ENSOARG00000005315 MMP1 11.03 0.0001 0.47% ENSOARG00000013161 MMP11 0.41 0.0000 0.21% ENSOARG00000019414 MMP14 1.85 0.0001 0.25% ENSOARG00000018035 MMP2 1.99 0.0000 0.12% ENSOARG00000008537 NAV2 0.57 0.0022 3.52% ENSOARG00000010519 OLFML2B 2.26 0.0004 1.03% ENSOARG00000006153 PDGFA 0.51 0.0009 1.85% ENSOARG00000005685 PLOD2 3.06 0.0011 2.15% ENSOARG00000010041 POSTN 6.73 0.0000 0.00% ENSOARG00000005275 PXDN 1.94 0.0010 1.98% ENSOARG00000004813 SDC2 1.95 0.0006 1.39% ENSOARG00000005209 SDC4 0.57 0.0009 1.93% ENSOARG00000006391 SERPINB5 3.99 0.0000 0.00% ENSOARG00000020413 SERPINE2 1.78 0.0003 0.80% ENSOARG00000015081 TGFBI 3.12 0.0000 0.13% ENSOARG00000005941 TNC 3.00 0.0016 2.80% ENSOARG00000008334 VEGFA 0.44 0.0000 0.05% Table 2. 41 extracellular matrix (ECM) related genes significantly affected by Haemonchus contortus infection in the abomasal mucosa of the Canaria Hair Breed sheep (CHB). www.nature.com/scientificreports/ 6 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 Gene ID Locus (chr:start:end) Gene symbol Fold (I/C) P value FDR ENSOARG00000004361 4:60803768:60856028 ANLN 5.01 5.05E-05 0.0022 ENSOARG00000019052 11:27395740:27400878 AURKB 2.94 7.80E-05 0.0031 ENSOARG00000012875 18:20942195:21007732 BLM 2.46 8.65E-04 0.0185 ENSOARG00000018738 13:48462231:48472599 BMP2 1.87 4.29E-04 0.0111 ENSOARG00000004835 11:42540868:42607539 BRCA1 3.09 6.51E-04 0.0152 ENSOARG00000020126 7:32810618:32861855 BUB1B 3.38 9.57E-05 0.0037 ENSOARG00000020247 7:33203355:33258031 CASC5 3.52 2.94E-03 0.0442 ENSOARG00000014176 6:3657736:3663110 CCNA2 3.95 2.00E-07 0.0000 ENSOARG00000009908 X:51894389:51938870 CCNB3 4.54 1.46E-03 0.0269 ENSOARG00000010352 3:209738381:209761224 CCND2 2.44 1.88E-04 0.0060 ENSOARG00000004318 25:16420008:16437119 CDC2 3.96 3.23E-04 0.0089 ENSOARG00000020542 1:17815061:17818632 CDC20 3.07 5.86E-04 0.0140 ENSOARG00000001274 13:50726485:50734786 CDC25B 1.82 3.24E-03 0.0476 ENSOARG00000014063 11:40114667:40125179 CDC6 4.24 2.32E-08 0.0000 ENSOARG00000009851 2:39869389:39909559 CDCA2 2.74 1.11E-03 0.0218 ENSOARG00000005652 3:207550908:207552937 CDCA3 2.65 2.88E-05 0.0015 ENSOARG00000019830 1:12275289:12289262 CDCA8 3.70 7.07E-05 0.0029 ENSOARG00000011059 6:21798087:21861426 CENPE 3.68 1.30E-05 0.0008 ENSOARG00000012529 12:53214185:53224480 CENPL 2.72 6.91E-04 0.0158 ENSOARG00000008206 14:7100459:7127206 CENPN 3.59 6.95E-05 0.0029 ENSOARG00000003186 14:34675201:34680944 CENPT 2.71 1.54E-03 0.0276 ENSOARG00000007744 8:12047574:12055213 CENPW 3.21 1.43E-04 0.0049 ENSOARG00000003158 22:14531509:14550251 CEP55 4.11 2.07E-06 0.0002 ENSOARG00000009704 5:17254276:17278480 CHAF1A 2.25 1.82E-04 0.0059 ENSOARG00000009289 10:21841538:21861932 CKAP2 3.27 2.54E-03 0.0403 ENSOARG00000007721 2:23987476:23992086 CKS2 3.46 1.08E-06 0.0001 ENSOARG00000019542 1:10450581:10480905 CLSPN 2.00 1.47E-03 0.0269 ENSOARG00000021089 7:64546752:64587073 DLGAP5 3.93 6.21E-04 0.0147 ENSOARG00000017620 1:186409:193575 DTYMK 1.92 2.03E-03 0.0339 ENSOARG00000007334 2:242210461:242229702 E2F2 3.47 4.09E-05 0.0019 ENSOARG00000008807 21:25034831:25052009 E2F8 4.39 3.03E-08 0.0000 ENSOARG00000020622 3:199025742:199151626 EPS8 2.36 6.02E-05 0.0025 ENSOARG00000005908 X:61406551:61410223 ERCC6L 4.54 7.42E-07 0.0001 ENSOARG00000014845 2:100985329:101014711 ESCO2 3.40 5.21E-05 0.0022 ENSOARG00000000644 13:67321805:67342479 FAM83D 3.04 9.58E-05 0.0037 ENSOARG00000005211 19:16642527:16690458 FANCD2 2.84 2.96E-05 0.0015 ENSOARG00000003968 8:76466657:76471151 FBXO5 3.65 7.37E-05 0.0030 ENSOARG00000015633 21:39647404:39648523 FEN1 2.10 9.02E-04 0.0191 ENSOARG00000011054 3:210883196:210893395 FOXM1 3.42 6.81E-04 0.0158 ENSOARG00000007717 13:52921512:52934511 GINS1 3.70 8.22E-06 0.0006 ENSOARG00000004495 22:15498816:15537211 HELLS 3.82 1.95E-09 0.0000 ENSOARG00000019189 1:6959524:6970726 HJURP 3.11 1.52E-04 0.0051 ENSOARG00000020743 7:42727941:42735567 KIAA0101 4.06 1.23E-06 0.0001 ENSOARG00000004780 19:16405186:16463300 KIF15 3.29 2.62E-03 0.0413 ENSOARG00000015211 15:56874125:56943420 KIF18A 2.92 1.68E-03 0.0295 ENSOARG00000015873 5:46994289:47001867 KIF20A 3.56 6.85E-05 0.0028 ENSOARG00000005591 24:26450684:26467845 KIF22 2.77 7.56E-05 0.0030 ENSOARG00000018647 7:16078138:16122235 KIF23 2.92 1.63E-03 0.0288 ENSOARG00000001102 1:19196065:19216520 KIF2C 3.45 2.73E-04 0.0079 ENSOARG00000009637 20:7678567:7686289 KIFC1 2.48 5.81E-04 0.0139 ENSOARG00000020216 7:33016321:33025738 KNSTRN 2.15 1.66E-03 0.0292 ENSOARG00000009349 17:52444861:52510658 KNTC1 2.66 1.04E-03 0.0208 ENSOARG00000015347 11:48337064:48345443 KPNA2 2.30 1.21E-04 0.0044 ENSOARG00000015665 6:5610960:5622911 MAD2L1 2.76 1.99E-04 0.0062 ENSOARG00000005416 13:26912745:26937031 MCM10 2.02 3.35E-03 0.0487 ENSOARG00000014143 20:24477536:24494074 MCM3 2.06 3.46E-05 0.0017 Continued www.nature.com/scientificreports/ 7 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 metabolism, cyclooxygenase pathway, and positive regulation of MAPK cascade, as well as leukocyte migration were also implicated in host resistance. On the other hand, four of the 11 GO unique to CS were related to muscle contraction. Discussion Parasite resistance refers to the ability of the host to avert infection, resulting in reduced worm burden17. Numerous factors affect this trait. Among them, host genetics play a predominant role in controlling the development of resistance while host sex, age, and prior exposure are also important18. Differences in parasite resistance and susceptibility existing in various sheep breeds have been long recognized3. Moreover, interand intra-host variations in resistance are evident in certain sheep populations18. Identifying genetics components controlling inter-, and intra-breed differences in parasite resistance has both pragmatic and theoretical implications. Towards Gene ID Locus (chr:start:end) Gene symbol Fold (I/C) P value FDR ENSOARG00000012797 9:32400919:32411149 MCM4 2.57 1.32E-06 0.0001 ENSOARG00000018527 3:178690575:178707822 MCM5 2.32 1.72E-05 0.0010 ENSOARG00000010614 2:173834090:173868287 MCM6 2.25 3.91E-05 0.0019 ENSOARG00000011541 2:51686232:51755300 MELK 3.87 1.90E-06 0.0002 ENSOARG00000009575 18:53870504:53915925 MIS18BP1 3.74 3.46E-03 0.0499 ENSOARG00000014562 22:46439178:46468099 MKI67 3.15 6.36E-04 0.0149 ENSOARG00000014901 8:60237288:60271515 MYB 3.06 2.71E-05 0.0014 ENSOARG00000003547 13:71800636:71832436 MYBL2 3.10 1.78E-07 0.0000 ENSOARG00000004016 6:37256547:37333851 NCAPG 3.36 5.28E-06 0.0004 ENSOARG00000007995 4:118730937:118799615 NCAPG2 2.64 6.03E-05 0.0025 ENSOARG00000009604 23:37208584:37244969 NDC80 3.71 4.15E-06 0.0003 ENSOARG00000011466 12:69915815:69927483 NEK2 3.45 4.25E-05 0.0019 ENSOARG00000011189 1:113098396:113135220 NUF2 2.47 9.75E-04 0.0199 ENSOARG00000005282 1:26531082:26561083 ORC1 2.82 4.59E-05 0.0021 ENSOARG00000014858 2:101015056:101043656 PBK 4.32 1.36E-05 0.0008 ENSOARG00000017133 13:46615170:46619285 PCNA 2.05 1.17E-04 0.0043 ENSOARG00000010890 12:52045755:52079590 PDPN 2.08 1.43E-03 0.0264 ENSOARG00000015691 17:29540624:29557094 PLK4 1.97 2.02E-03 0.0338 ENSOARG00000020607 7:39763421:39793166 POLE2 2.83 3.61E-04 0.0096 ENSOARG00000012267 18:20787183:20802103 PRC1 3.88 9.65E-04 0.0198 ENSOARG00000020254 7:33275919:33306843 RAD51 3.12 7.61E-05 0.0031 ENSOARG00000010707 24:15349788:15350439 RAN 1.79 1.72E-03 0.0300 ENSOARG00000017221 13:65583237:65642844 RBL1 2.12 2.02E-03 0.0338 ENSOARG00000017883 13:58047850:58061508 RBM38 2.35 3.13E-03 0.0464 ENSOARG00000015333 3:19185137:19191043 RRM2 5.12 5.48E-09 0.0000 ENSOARG00000017493 6:91484955:91532036 SEPT11 2.18 2.04E-04 0.0063 ENSOARG00000018302 2:239211366:239212113 SFN 2.06 2.68E-03 0.0420 ENSOARG00000007399 2:18870299:18918858 SMC2 2.60 1.89E-03 0.0324 ENSOARG00000001001 11:19659072:19677569 SPAG5 2.45 1.79E-04 0.0058 ENSOARG00000017725 5:13248115:13254108 SPC24 2.98 3.22E-04 0.0089 ENSOARG00000002888 16:1518544:1544794 SPDL1 1.95 2.92E-03 0.0442 ENSOARG00000011578 18:20124947:20166662 TICRR 2.83 1.61E-03 0.0286 ENSOARG00000001419 13:60597535:60651659 TPX2 2.52 6.32E-04 0.0149 ENSOARG00000016302 7:3740229:3789354 TRIM36 10.24 1.19E-14 0.0000 ENSOARG00000007151 8:6885831:6925302 TTK 3.59 9.22E-06 0.0006 ENSOARG00000018884 3:136887716:136929972 TUBA4A 2.02 1.14E-04 0.0042 ENSOARG00000006520 13:74125916:74129104 UBE2C 3.13 1.25E-05 0.0008 ENSOARG00000008530 5:16805815:16842579 UHRF1 3.99 2.60E-07 0.0000 ENSOARG00000004351 8:81449297:81450503 1.84 8.66E-04 0.0185 ENSOARG00000006991 19:59822300:59844027 2.08 2.93E-04 0.0084 ENSOARG00000005764 2:240064407:240068660 2.37 2.89E-05 0.0015 ENSOARG00000006571 21:17310180:17310609 2.66 3.78E-06 0.0003 ENSOARG00000005759 16:10486354:10496192 3.81 1.78E-04 0.0058 ENSOARG00000000647 22:13731293:13771253 3.92 4.05E-04 0.0106 Table 3. 100 cell-cycle related genes significantly affected by Haemonchus contortus infection in the resistant breed (CHB). Fold is expressed as infected/uninfected controls. FDR: false discovery rate. www.nature.com/scientificreports/ 8 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 Figure 4. Real-Time RT-PCR analysis (qPCR) of selected genes. Relative expression levels calculated from standard curves were normalized to the endogenous control gene RPL19. Numbers represent mean values plus standard error. Cont.: uninfected controls; Inf.: 20 days post infection by Haemonchus contortus. CS: Canaria Sheep; CHB: Canaria Hair Breed. CFI: Complement factor I; CXCR6: Chemokine (C-X-C motif) receptor 6; GHRL: Ghrelin/obestatin prepropeptide; LGALS15: Galectin 15; IL5: Interleukin 5. MMP1: Matrix metallopeptidase 1; TFF2: Trefoil factor 2. **P < 0.001; ***P < 0.0001. www.nature.com/scientificreports/ 9 Scientific RepoRts | 6:26200 | DOI: 10.1038/srep26200 this end, numerous efforts have been made over the decades to unravel genes and/or genetic variants responsible for resistance, partially driven by strong desires to breed farm animals with strong resistance traits. Traditional QTL analysis and Genome-wide Association Studies (GWAS) have led to reports of dozens of QTL or markers on almost every ovine chromosome that are associated with various resistance phenotypes, such as fecal egg counts, packed cell volume, and parasite-specific antibody titers9,11,13. Nevertheless, the development of parasite resistance relies upon the precise control of expression of the host genome. Understanding these regulatory elements will be crucial towards unraveling their functional relevance. As a result, while much progress has been made to identify genes associated with nematode resistance in sheep during the past few years19,20, an in-depth comparison and characterization of transcriptome responses of various breeds and populations, especially those local indigenous breeds harboring varying degrees of parasite resistance and susceptibility, is urgently needed. The two indigenous breeds of sheep native to the Canary Islands, CHB and CS, display unique and distinct differences in parasite resistance and susceptibility. When co-grazing together on the same pasture under natural infections, differences in fecal trichostrongylid egg counts between CHB and CS are consistently observed14. Under experimental infections with H. contortus, CHB has a significantly lower, by approximately 50%, worm burden than CS, a undeniable trait of parasite resistance14,15, which is confirmed in this study. Moreover, worms recovered in CHB tend to have significantly shorter body length than those in CS. A significantly lower EPG value is consistently observed in the feces of CHB sheep than those of CS animals during experimental infection. For example, at 27dpi, the mean EPG in CS is 5 fold higher than in CHB14. CHB sheep not only shed significantly fewer parasite eggs but also tend to have a delayed egg production, indicating an anti-fecundity effect of the immune response in this breed. The results from this study show that at 20 dpi, no parasite eggs were recovered in the feces of infected CHB animals while EPG in the feces of infected CS sheep reached 262.50 (± 287.54, SD). This observation is in agreement with the previous findings14. Haemonchus contortus infection generally elicits a potent Th2 immune response in small ruminants. A strong upregulation of several well-known Th2 cytokines by infection in CHB were observed in this study. Previous studies in the Canary Island breeds suggest that divergence in immune response mechanisms exist between CHB and CS. Among various immune cells, abomasal eosinophil numbers are 2 fold higher in CHB than in CS, suggesting that CHB sheep may have developed abilities for enhanced recruitment of eosinophils to the site of infection (abomasal mucosa). Furthermore, CHB sheep have evolved mechanisms attacking the adult stage of the Haemonchus parasite, especially its reproduction, as evidenced by the fact that fecundity is negatively correlated with eosinophils and γ δ T cells in the abomasal mucosa15. However, the precise molecular mechanisms of the parasite resistance in CHB breed remain largely unclear. In this study, we identified a total of 477 and 16 Gene Ontology (GO) terms that are significantly enriched in the transcriptome of resistant and susceptible sheep breeds in responses to Haemonchus infection, respectively. Figure 5. Linear regression analysis of fold changes calculated from qPCR and RNAseq analysis. Blue dots represent log2 transformed fold change values of a single gene in an infected sample obtained from qPCR (X-axis) and RNAseq analysis (Y-axis). Dashed lines: 99% Confidence Interval. R: correlation coefficient. GO_ID Description Z Score P value Resistant Susceptible Resistant Susceptible GO:0002250 adaptive immune response 4.65 5.09 4.59E-05 7.37E-04 GO:0046456 eicosanoid biosynthetic process 6.92 6.84 3.81E-07 6.59E-04 GO:0006691 leukotriene metabolic process 7.22 9.67 9.37E-07 9.52E-05 GO:1901570 fatty acid derivative biosynthetic process 6.92 6.84 3.81E-07 6.59E-04 GO:0006636 unsaturated fatty acid biosynthetic process 7.02 6.43 2.12E-07 9.16E-04 Table 4. Gene Ontology (GO) biological processes (BP) significantly enriched in both resistant (CHB) and susceptible (CS) breeds.