Bioinformatics investigation of expression changes of abomasal gene transcripts to Heamonchus contortus infection in resistant and sensitive sheep

Document Type : Research Paper


1 Ph.D. Student, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

2 Professor, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

3 Assistant Professor, Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

4 Professor, Department of Animal Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

5 Associate Professor, Department of Medical Parasitology and Mycology, School of Medicine, Guilan University of Medical Science, Rasht, Iran


Introduction: Gastrointestinal nematodes (GIN) represent a major health issue for livestock production systems worldwide. Haemonchus contortus is one of the most pathogenic GIN in small ruminants and causes serious losses to farmers, both in impaired production and in control with anthelmintics. It has long been recognized that differences in host resistance and susceptibility to parasitic infection exist in various sheep breeds and that genetics may play an important role in regulating host resistance, which has spurred efforts to control parasitic infection through selective breeding for naturally resistant sheep. A detailed understanding of the genes and mechanisms involved in expressing a resistant phenotype and the factors that regulate this response would facilitate the identification of candidate genes for selection. Recent advances in high-throughput technology, such as microarrays and RNA sequencing, have made a large number of transcriptome data accessible. As a result, researchers are now able to obtain more reliable results by integrating information from multiple sources. Accordingly, meta-analysis can be used as a useful and powerful tool to identify differential gene expression. It can also find out genes that their products are key molecules in response to the infection and use in animal breeding programs. The purpose of this study was to conduct a systematic review and meta-analysis on data collected from infected sheep with H. contortus and general analysis of changes in abomasal gene transcripts in response to infection using RNA-seq technology and bioinformatics tools.
Materials and methods: In this study, to identify infection-related genes, pathways, and molecular mechanisms underlying host resistance to this parasite, a meta-analysis was performed by combining two different datasets, including 70 samples of sheep H. contortus infection with Rankprod package of R software. After pre-processing, to remove heterogenicity across studies, batch effect correction was performed on gene expression data. The result of the principal component analysis showed that batch correction reduced the batch variation among the datasets. Meta-analysis was carried out and DEGs selected by meta-analysis were further analyzed and characterized. Enrichment analysis, as an efficient method for functional analysis of massive genetic data, was used to determine the biological process, molecular function, and cellular component of DEGs. Moreover, we searched upstream regions of DEGs for over-represented DNA motifs and functional analysis of discovered motifs. To explore the potential interaction network of the DEGs, the protein-protein interaction network among the DEGs was analyzed using the STRING database, which included direct and indirect associations of proteins. After analyzing the result derived from STRING analysis and expression change information for each DEG, the network figure was drawn for the selected DEGs (connected with one or more DEGs) by using the Cytoscape software and hub genes identified with the CytoHubba plugin of Cytoscape.
Results and discussion: Results derived from the meta-analysis showed a total of 1388 differentially expressed genes between resistant and susceptible sheep. Among them, 1137 were significantly upregulated, whereas 251 were downregulated across the datasets. In the identified DEGs, DEGs corresponding to ribosomal protein S3A, lysozyme C-1-like (LOC443320), and heterogeneous nuclear ribonucleoprotein K were the most strongly upregulated ones, while tenascin C and fibromodulin were the most strongly downregulated. Results from enrichment analysis showed these differentially expressed genes (DEGs) were involved in different biological processes such as one-carbon metabolism, translation, cell surface receptor signaling pathway, immune response, metabolic pathways, PPAR signaling pathway, etc. Moreover, searching in upstream regions of DEGs to find DNA motifs, were able to identify eight conserved sequence motifs. The functional analysis of these motifs revealed that they were involved in the positive regulation of gene expression, defense response, positive regulation of immune response, cellular calcium ion homeostasis, etc. Using the protein-protein interaction analysis also identified multiple hub genes such as albumin and CD4 which may show that improved immune response, induced by up-regulation different genes affects the creation of resistance.
Conclusions: The mechanisms of sheep resistance to GIN infections involve complex immune responses. Our results offered overall insight into changes in the transcriptomes of resistant and susceptible sheep and molecular mechanisms of host resistance induced by H. contortus infection. We propose these DEGs as a useful resource of molecular biomarkers and potential candidate genes for breeding programs which can provide a basis for further research on this topic.


Main Subjects

Abeel T., Saeys Y., Rouze P. and Vande Peer Y. 2008. Pro SOM: Core promoter prediction based on unsupervised clustering of DNA physical profiles. Bioinformatics, 24: 24-31.
Andersson L. 2012. How selective sweeps in domestic animals provide new insight into biological mechanisms. Journal of Internal Medicine, 271(1): 1-14.
Andronicos N. M., Hunt P. and 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. International Journal for Parasitology, 40: 417-429.
Bailey T. L., Boden M., Buske F. A., Frith M., Grant C. E., Clementi L., Ren J., Li W. W. and Nobel W. S. 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research, 37: 202-208.
Bath G. F. 2014. The “BIG FIVE”-A South African perspective on sustainable holistic internal parasite management in sheep and goats. Small Ruminant Research, 118: 48-55.
Benavides M. V., Sonstegard T. S., Kemp S., Mugambi J. M., Gibson J. P., Baker R. L., Hanotte O., Marshall K. and Tassell C. V. 2015. Identification of novel loci associated with gastrointestinal parasite resistance in a Red Maasai x Dorper backcross population. PLoS One, 1: e122797.
Berger S. L. 2002. Histone modifications in transcriptional regulation. Current Opinion in Genetics & Development, 12: 142-148.
Bernard A., Boumsell L. and Hill C. 1984. Joint report of the first international workshop on human leucocyte differentiation antigens by the investigators of the participating laboratories. In: Bernard A., Boumsell L. and Dausset J. (Eds) Leucocyte typing: human leucocyte differentiation antigens detected by monoclonal antibodies: specification, classification, nomenclature. Berlin, Springer. Pp. 45-48.

Bionaz M., Chen S., Khan M. J. and Loor J. J. 2013. Functional role of PPARs in ruminants: potential targets for fine-tuning metabolism during growth and lactation. PPAR Research, 1: e684159.

Black J. C., Van Rechem C. and Whetstine J. R. 2012. Histone lysine methylation dynamics: establishment, regulation, and biological impact. Molecular Cell, 48: 491-507.
Bricarello P. A., Gennari S. M., Oliveira-Sequeira T. C., Vaz C. M., de Goncalves I. G. and Eschevarria F.A. 2004. Worm burden and immunological responses in Corriedale and Crioula Lanada sheep following natural infection with Haemonchus contortus. Small Ruminant Research, 51: 75-83.
Buske F. A., Boden M., Bauer D. C. and Bailey T. L. 2010. Assigning roles to DNA regulatory motifs using comparative genomics. Bioinformatics, 26: 860-866.
Cánovas A., Reverter A., DeAtley K. L., Ashley R. L., Colgrave M. L., Fortes M. R. S., Islas-Trejo A., Lehnert S., Porto-Neto L., Rincon G., Silver G. A., Snelling W. M., Medrano J. F. and Thomas M. G. 2014. Multi-tissue omics analyses reveal molecular regulatory networks for puberty in composite beef cattle. PLoS One, 9: e102551.
Chagas A., Domingues L. F., Gaínza Y. A., Barioni-Junior W., Esteves S. N. and Niciura S. C. 2016. Target selected treatment with levamisole to control the development of AR in a sheep flock. Parasitology Research, 115: 1131-1139.
Chin C., Chen S. H., Wu H. H., Ho C., Ko M. T. and Lin C. 2014. CytoHubba: identifying hub objects and sub-networks from complex interactome. BMC System Biology, 8: e11.
Diez-Tascon C., Keane O. M., Wilson T., Zadissa A., Hyndman D., Baird D. B., Mcewan J. C. and Crawford A. M. 2005. Microarray analysis of selection lines from outbred populations to identify genes involved with nematode parasite resistance in sheep. Physiological Genomics, 21: 59-69.
Dolinská M., Ivanisinova O., Konigova A. and Várady M. 2014. Anthelmintic resistance in sheep gastrointestinal nematodes in Slovakia detected by in-vitro methods. BMC Veterinary Research, 10: e233.
Fu M. and Blackshear P. J. 2017. RNA-binding proteins in immune regulation: A focus on CCCH zinc finger proteins. Nature Reviews Immunology, 17: 130-143.
Gasser R., Schwarz E., Korhonen P. and Young N. 2016. Understanding Haemonchus contortus better through genomics and transcriptomics. Advances in Parasitology, 93: 519-567.
Geurden T., Hoste H., Jacquiet P., Traversa D., Sotiraki S., Regalbono A. F., Tzanidakis N., Kostopoulou D., Gaillac C., Privat S., Giangaspero A., Zanardello C., Noe L., Vanimisetti B. and Bartram D. 2014. Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy. Veterinary Parasitology, 201: 59-66.
Gill H. S. 1994. Cell-mediated immunity in Merino lambs with genetic resistance to Haemonchus contortus. International Journal for Parasitology, 24: 749-756.
Gill H. S., Watson D. L. and Brandon M. R. 1993. Monoclonal antibody to CD4+ T cells abrogates genetic resistance to Haemonchus contortus in sheep. The Journal of Immunology, 78: 43-49.
Gossner A. G., Wilkie H., Joshi A. and Hopkins J. 2013. Exploring the abomasal lymph node transcriptome for genes associated with resistance to the sheep nematode Teladorsagia circumcincta. Journal of Veterinary Parasitology, 44: 1-13.
Gross B., Pawlak M., Lefebvre P. and Staels B. 2017. PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nature Reviews Endocrinology, 13: 36-49.
Hankins J. 2006. The role of albumin in fluid and electrolyte balance. Journal of Infusion Nursing, 29: 260-265.
Hilger D., Masureel M. and Kobilka B. K. 2018. Structure and dynamics of GPCR signaling complexes. Nature Structural & Molecular Biology, 25: 4-12.
Huang D. W., Sherman B. T. and Lempicki R. A. 2009. Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Research, 37: 1-13.
Karanu F. N., McGuire T. C., Davis W. C., Besser T. E. and Jasmer D. P. 1997. CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens. Parasite Immunology, 19: 435-445.
Keane O. M., Dodds K. G., Crawford A. M. and McEwan J. C. 2007. Transcriptional profiling of Ovis aries identifies Ovar-DQA1 allele frequency differences between nematode-resistant and susceptible selection lines. Physiological Genomics, 30: 253-261.
Khan A., Fornes O., Stigliani A., Gheorghe M., Castro-Mondragon J. A., Lee R. V., Bessy A., Cheneby J., Kulkarni S. R., Tan G., Baranasic D., Arenillas D. J., Sandelin A., Vandepoele K., Lenhard B., Ballester B., Wasserman W., Parcy F. and Mathelier A. 2017. JASPAR 2018: update of the open-access database of transcription factor binding profiles and its web framework. Nucleic Acids Research, 46: 260-266.
Knight P. A., Griffith S. E., Pemberton A. D., Pate J. M., Guarneri L., Anderson K., Talbot R. T., Smith S., Waddington D., Fell M., Archibald A. L., Burgess S. T., Smith D. W., Miller H. R. and Morrison I. W. 2011. Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta. Veterinary Research, 42: 78-100.
Koyama K., Tamauchi H. and Ito Y. 1995. The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris. Parasite Immunology, 17: 161-165.
Meyer P., Niedenh I. and Ten Lohuis M. 1994. Evidence for cytosine methylation of non-symmetrical sequences in transgenic petunia hybrida. The Embo Journal, 13: 2084-2088.
Miller J. E., Bahirathan M., Lemarie S., Hembry F. G., Kearney M. T. and Barras S. R. 1998. Epidemiology of gastrointestinal nematode parasitism in Suffolk and Gulf Coast native sheep with special emphasis on relative susceptibility to Haemonchus contortus infection. Veterinary Parasitology, 74: 55-74.
Mpetile Z., Cloete S., Kruger A. and Dzama K. 2015. Environmental and genetic factors affecting faecal worm egg counts in Merinos divergently selected for reproduction. Journal of Animal Science, 45: 510-520.

Ntuli T. M. 2015. Cell death- autophagy, apoptosis and necrosis (Ed.): Apoptosis and infections. South Africa: IntechOpen. P. 446.

Peña M. T., Miller J. E. and Horohov D. W. 2006. Effect of CD4+ T lymphocyte depletion on resistance of Gulf Coast native lambs to Haemonchus contortus infection. Veterinary Parasitology, 138: 240-246.
Rani T. S., Bhavani S. D. and Bapi R. S. 2007. Analysis of E. coli promoter recognition problem in di-nucleotide feature space. Bioinformatics, 23: 582-588.
Rowe A., Gondro C., Emery D. and Sangster N. 2009. Sequential microarray to identify timing of molecular responses to Haemonchus contortus infection in sheep. Veterinary Parasitology, 161: 76-87.
Saddiqi H. A. 2010. Evaluation of some indegenous breeds of sheep for natural resistance Haemonchus contortus infection. Ph.D. Dissertation, Faisalabad University, Pakistan.
Salehinasab M., Rahimi Mianji Gh., Ebrahimie E. and Ghafouri S. A. 2018. Identification of differentially expressed genes in H5N1 infected chickens using meta-analysis of DNA microarray datasets. Animal Production Research, 7(3): 13-23. (In Persian).
Shannon P., Markiel A., Ozier O., Baliga N. S., Wang J. T., Ramage D., Amin N., Schwikowski B. and Ideker T. 2003. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research, 13: 2498-2504.
Szklarczyk D., Gable A. L., Lyon D. and Junge A. 2018. STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Research, 47: 607-613.

Ulloa A., Zarinan T., Castillo J. A. and Maravillas J. L. 2017. Reference module in neuroscience and biobehavioral psychology. Elsevier SciTech Connect. Pp. 1-10.

Urban J. F., Katona I. M. and Finkelman F. D. 1991. Heligmosomoides polygyrus: CD4+ but not CD8+ T cells regulate the IgE response and protective immunity to mice. Experimental Parasitology, 73: 500-511.
Van Panhuys N., Prout M., Forbes E., Min B., Paul W. E. and Gros G. L. 2011. Basophils are the major producers of IL-4 during primary helminth infection. The Journal of Immunology, 186: 2719-2728.
Vijayasarathi M., Sreekumar C., Venkataramanan R. and Raman M. 2016. Influence of sustained deworming pressure on the AR status in strongyles of sheep under field conditions. Tropical Animal Health and Production, 48: 55-62.
Wang X. and Zhu W. G. 2008. Advances in histone methyltransferases and histone demethylases. Journal of Cancer, 27: 1018-1025.
Zhang Z., Burch P. E., Cooney A. J., Lanz R. B., Pereira F. A., Wu J., Gibbs R. A., Weinstock G. and Wheeler D. A. 2004. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome. Genome Research, 14: 580-590.
Zhao F., Ilbert M., Varadan R., Cremers C. M., Hoyos B., Acin-Perez R., Vinogradov V., Cowburn D., Jakob U. and Hammerling U. 2010. Are zinc-finger domains of protein kinase C dynamic structures that unfold by lipid or redox activation? Antioxidants & Redox Signaling, 14: 757-766.