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Marker genes for activation of the RNA interference (RNAi) pathway in the free-living nematode Caenorhabditis elegans and RNAi development in the ovine nematode Teladorsagia circumcincta

  • T. Tzelos (a1) (a2), J.B. Matthews (a1), B. Whitelaw (a2) and D.P. Knox (a1)

The nematode Teladorsagiacircumcincta is a major cause of parasitic gastroenteritis in sheep in temperate regions. The development of resistance to the major anthelmintic classes used for its control is a threat to small ruminant farming sustainability. Vaccination is a potential alternative control method for this nematode. Gene datasets can be exploited to identify potential vaccine candidates and these validated further by methods such as RNA interference (RNAi) prior to vaccine trials. Previous reports indicate that RNAi in parasitic nematodes is inconsistent and, to date, there are no internal controls that indicate activation of the RNAi pathway in response to double-stranded RNA (dsRNA). The present aims were to determine whether or not the transcription levels of potential marker genes in the RNAi pathway could indicate activation of the pathway in Caenorhabditis elegans and to develop an RNAi platform in T. circumcincta. In C. elegans, transcript levels of three candidate marker genes, Ce-dcr-1 (Dicer), Ce-ego-1 (Enhancer of Glp-One family member) and Ce-rsd-3 (RNAi Spreading Defective), were analysed and results indicated that activation of the pathway had no effect on transcript levels of these genes. In T. circumcincta, two vaccine candidate genes from the Activation-associated Secreted Protein (ASP) family were targets for knockdown. RNAi experiments showed successful silencing of both targets, although inconsistencies in efficacy were observed. After testing a number of parameters that might affect variability, it was found that the length of the storage period of the larvae plays an important role in the consistency of the RNAi results.

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Asojo, O.A., Goud, G., Dhar, K., Loukas, A., Zhan, B., Deumic, V., Liu, S., Borgstahl, G.E.O. & Hotez, P.J. (2005) X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection. Journal of Molecular Biology 346, 801814.
Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363366.
Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 7194.
Britton, C. & Murray, L. (2006) Using Caenorhabditis elegans for functional analysis of genes of parasitic nematodes. International Journal for Parasitology 36, 651659.
Dalzell, J.J., McVeigh, P., Warnock, N.D., Mitreva, M., Bird, D.M., Abad, P., Fleming, C.C., Day, T.A., Mousley, A., Marks, N.J. & Maule, A.G. (2011) RNAi effector diversity in nematodes. PLoS Neglected Tropical Diseases 5, e1176.
Duxbury, M.S. & Whang, E.E. (2004) RNA interference: a practical approach. Journal of Surgical Research 117, 339344.
Elard, L., Comes, A.M. & Humbert, J.F. (1996) Sequences of β-tubulin cDNA from benzimidazole-susceptible and -resistant strains of Teladorsagia circumcincta, a nematode parasite of small ruminants. Molecular and Biochemical Parasitology 79, 249253.
Fire, A., Xu, S.Q., Montgomery, M.K., Kostas, S.A., Driver, S.E. & Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811.
Fischer, S.E.J. (2010) Small RNA-mediated gene silencing pathways in C. elegans. International Journal of Biochemistry & Cell Biology 42, 13061315.
Geldhof, P., Claerebout, E., Knox, D., Vercauteren, I., Looszova, A. & Vercruysse, J. (2002) Vaccination of calves against Ostertagia ostertagi with cysteine proteinase enriched protein fractions. Parasite Immunology 24, 263270.
Geldhof, P., Vercauteren, I., Gevaert, K., Staes, A., Knox, D.P., Vandekerckhove, J., Vercruysse, J. & Claerebout, E. (2003) Activation-associated secreted proteins are the most abundant antigens in a host protective fraction from Ostertagia ostertagi. Molecular and Biochemical Parasitology 128, 111114.
Geldhof, P., Whitton, C., Gregory, W.F., Blaxter, M. & Knox, D.P. (2005) Characterisation of the two most abundant genes in the Haemonchus contortus expressed sequence tag dataset. International Journal for Parasitology 35, 513522.
Geldhof, P., Molloy, C. & Knox, D.P. (2006a) Combinatorial RNAi on intestinal cathepsin B-like proteinases in Caenorhabditis elegans questions the perception of their role in nematode biology. Molecular and Biochemical Parasitology 145, 128132.
Geldhof, P., Murray, L., Couthier, A., Gilleard, J.S., McLauchlan, G., Knox, D.P. & Britton, C. (2006b) Testing the efficacy of RNA interference in Haemonchus contortus. International Journal for Parasitology 36, 801810.
Geldhof, P., Visser, A., Clark, D., Saunders, G., Britton, C., Gilleard, J., Berriman, M. & Knox, D. (2007) RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects. Parasitology 134, 609619.
Ghosh, K. & Hotez, P.J. (1999) Antibody-dependent reductions in mouse hookworm burden after vaccination with Ancylostoma caninum secreted protein 1. Journal of Infectious Diseases 180, 16741681.
Grishok, A. (2005) RNAi mechanisms in Caenorhabditis elegans. FEBS Letters 579, 59325939.
Halliday, A.M., Lainson, F.A., Yaga, R., Inglis, N.F., Bridgett, S., Nath, M. & Knox, D.P. (2012) Transcriptional changes in Teladorsagia circumcincta upon encountering host tissue of differing immune status. Parasitology 139, 387405.
Hammond, S.M. (2005) Dicing and slicing: The core machinery of the RNA interference pathway. FEBS Letters 579, 58225829.
Hawdon, J.M., Jones, B.F., Hoffman, D.R. & Hotez, P.J. (1996) Cloning and characterization of Ancylostoma-secreted protein - a novel protein associated with the transition to parasitism by infective hookworm larvae. Journal of Biological Chemistry 271, 66726678.
Hawdon, J.M., Narasimhan, S. & Hotez, P.J. (1999) Ancylostoma secreted protein 2: cloning and characterization of a second member of a family of nematode secreted proteins from Ancylostoma caninum. Molecular and Biochemical Parasitology 99, 149165.
Hoogewijs, D., Houthoofd, K., Matthijssens, F., Vandesompele, J. & Vanfleteren, J.R. (2008) Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Molecular Biology 9, .
Hussein, A.S., Kichenin, K. & Selkirk, M.E. (2002) Suppression of secreted acetylcholinesterase expression in Nippostrongylus brasiliensis by RNA interference. Molecular and Biochemical Parasitology 122, 9194.
Issa, Z., Grant, W.N., Stasiuk, S. & Shoemaker, C.B. (2005) Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis. International Journal for Parasitology 35, 935940.
Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D.P., Zipperlen, P. & Ahringer, J. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231237.
Knox, D.P., Geldhof, P., Visser, A. & Britton, C. (2007) RNA interference in parasitic nematodes of animals: a reality check? Trends in Parasitology 23, 105107.
Laing, R., Kikuchi, T., Martinelli, A., Tsai, I., Beech, R., Redman, E., Holroyd, N., Bartley, D., Beasley, H., Britton, C., Curran, D., Devaney, E., Gilabert, A., Hunt, M., Jackson, F., Johnston, S., Kryukov, I., Li, K., Morrison, A., Reid, A., Sargison, N., Saunders, G., Wasmuth, J., Wolstenholme, A., Berriman, M., Gilleard, J. & Cotton, J. (2013) The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery. Genome Biology 14, R88.
Lu, G., Villalba, M., Coscia, M., Hoffman, D. & King, T. (1993) Sequence analysis and antigenic cross-reactivity of a venom allergen, antigen 5, from hornets, wasps, and yellow jackets. Journal of Immunology 150, 28232830.
Maeda, I., Kohara, Y., Yamamoto, M. & Sugimoto, A. (2001) Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current Biology 11, 171176.
Maniar, J.M. & Fire, A.Z. (2011) EGO-1, a C. elegans RdRP, modulates gene expression via production of mRNA-templated short antisense RNAs. Current Biology 21, 449459.
Maule, A.G., McVeigh, P., Dalzell, J.J., Atkinson, L., Mousley, A. & Marks, N.J. (2011) An eye on RNAi in nematode parasites. Trends in Parasitology 27, 505513.
Menon, R., Gasser, R.B., Mitreva, M. & Ranganathan, S. (2012) An analysis of the transcriptome of Teladorsagia circumcincta: its biological and biotechnological implications. Eleventh International Conference on Bioinformatics (InCoB2012): Computational Biology. BMC Genomics 13 (Suppl. 7), S10, doi:10.1186/1471-2164-13-S7-S10
Ministry of Agriculture, Fisheries and Food (1986) Manual of veterinary parasitological laboratory techniques. MAFF Ref. Book 418. London, UK, HMSO.
Nisbet, A.J., Knox, D.P., McNair, C.M., Meikle, L.I., Smith, S.K., Wildblood, L.A. & Matthews, J.B. (2009) Immune recognition of the surface associated antigen, Tc-SAA-1, from infective larvae of Teladorsagia circumcincta. Parasite Immunology 31, 3240.
Nisbet, A.J., Bell, N.E.V., McNeilly, T.N., Knox, D.P., Maizels, R.M., Meikle, L.I., Wildblood, L.A. & Matthews, J.B. (2010a) A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida). Parasite Immunology 32, 503511.
Nisbet, A.J., Smith, S.K., Armstrong, S., Meikle, L.I., Wildblood, L.A., Beynon, R.J. & Matthews, J.B. (2010b) Teladorsagia circumcincta: Activation-associated secreted proteins in excretory/secretory products of fourth stage larvae are targets of early IgA responses in infected sheep. Experimental Parasitology 125, 329337.
Nisbet, A.J., Zarlenga, D.S., Knox, D.P., Meikle, L.I., Wildblood, L.A. & Matthews, J.B. (2011) A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses? Parasite Immunology 33, 236243.
Nisbet, A.J., McNeilly, T.N., Wildblood, L.A., Morrison, A.A., Bartley, D.J., Bartley, Y., Longhi, C., McKendrick, I.J., Palarea-Albaladejo, J. & Matthews, J.B. (2013) Successful immunization against a parasitic nematode by vaccination with recombinant proteins. Vaccine 31, 40174023.
Parkinson, J., Mitreva, M., Whitton, C., Thomson, M., Daub, J., Martin, J., Schmid, R., Hall, N., Barrell, B., Waterston, R.H., McCarter, J.P. & Blaxter, M.L. (2004) A transcriptomic analysis of the phylum Nematoda. Nature Genetics 36, 12591267.
Redmond, D.L., Smith, S.K., Halliday, A., Smith, W.D., Jackson, F., Knox, D.P. & Matthews, J.B. (2006) An immunogenic cathepsin F secreted by the parasitic stages of Teladorsagia circumcincta. International Journal for Parasitology 36, 277286.
Samarasinghe, B., Knox, D.P. & Britton, C. (2011) Factors affecting susceptibility to RNA interference in Haemonchus contortus and in vivo silencing of an H11 aminopeptidase gene. International Journal for Parasitology 41, 5159.
Sargison, N.D. (2011) Pharmaceutical control of endoparasitic helminth infections in sheep. Veterinary Clinics of North America. Food Animal Practice 27, 139156.
Sargison, N.D., Jackson, F., Bartley, D.J., Wilson, D.J., Stenhouse, L.J. & Penny, C.D. (2007) Observations on the emergence of multiple anthelmintic resistance in sheep flocks in the south-east of Scotland. Veterinary Parasitology 145, 6576.
Schallig, H.D.F.H. & Van Leeuwen, M.A.W. (1997) Protective immunity to the blood-feeding nematode Haemonchus contortus induced by vaccination with parasite low molecular weight antigens. Parasitology 114, 293299.
Scott, P.R. (2007) Sheep medicine. 1st edn.London, Manson Publishing.
Seaton, D.S., Jackson, F., Smith, W.D. & Angus, K.W. (1989) Development of immunity to incoming radiolabelled larvae in lambs continuously infected with Ostertagia circumcincta. Research in Veterinary Science 46, 241246.
Smith, W.D., Jackson, F., Jackson, E. & Williams, J. (1983) Local immunity and Ostertagia circumcincta: changes in the gastric lymph of immune sheep after a challenge infection. Journal of Comparative Pathology 93, 479488.
Tawe, W., Pearlman, E., Unnasch, T.R. & Lustigman, S. (2000) Angiogenic activity of Onchocerca volvulus recombinant proteins similar to vespid venom antigen 5. Molecular and Biochemical Parasitology 109, 9199.
Taylor, M.A., Coop, R.L. & Wall, R.L. (2007) Veterinary parasitology. 3rd edn.Oxford, Blackwell Publishing.
Tijsterman, M., May, R.C., Simmer, F., Okihara, K.L. & Plasterk, R.H.A. (2004) Genes required for systemic RNA interference in Caenorhabditis elegans. Current Biology 14, 111116.
Vance, V. & Vaucheret, H. (2001) RNA silencing in plants - defense and counterdefense. Science 292, 22772280.
Viney, M.E. & Thompson, F.J. (2008) Two hypotheses to explain why RNA interference does not work in animal parasitic nematodes. International Journal for Parasitology 38, 4347.
Visser, A., Geldhof, P., De Maere, V., Knox, D.P., Vercruysse, J. & Claerebout, E. (2006) Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi. Parasitology 133, 777783.
Wang, J. & Kim, S.K. (2003) Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 16211634.
Wrigley, J., McArthur, M., McKenna, P.B. & Mariadass, B. (2006) Resistance to a triple combination of broad-spectrum anthelmintics in naturally-acquired Ostertagia circumcincta infections in sheep. New Zealand Veterinary Journal 54, 4749.
Zhan, B., Liu, Y., Badamchian, M., Williamson, A., Feng, J., Loukas, A., Hawdon, J.M. & Hotez, P.J. (2003) Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum. International Journal for Parasitology 33, 897907.
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