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Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones

  • J. DE GRAEF (a1), J. DEMELER (a2), P. SKUCE (a3), M. MITREVA (a4) (a5), G. VON SAMSON-HIMMELSTJERNA (a2), J. VERCRUYSSE (a1), E. CLAEREBOUT (a1) and P. GELDHOF (a1)...


Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.


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*Corresponding author: Laboratory for Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Tel: +32 (09) 2647517. Fax: +32 (09) 2647496. E-mail:


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Alvinerie, M., Sutra, J. F., Galtier, P., Lifschitz, A., Virkel, G., Sallovitz, J. and Lanusse, C. (1999). Persistence of ivermectin in plasma and faeces following administration of a sustained-release bolus to cattle. Research in Veterinary Science 66, 5761. doi: 10.1053/rvsc.1998.0240.
Ardelli, B. F., Guerriero, S. B. and Prichard, R. K. (2005). Genomic organization and effects of ivermectin selection on Onchocerca volvulus P-glycoprotein. Molecular and Biochemical Parasitology 143, 5866. doi: 10.1016/j.molbiopara.2005.05.006.
Ardelli, B. F., Guerriero, S. B. and Prichard, R. K. (2006). Characterization of a half-size ATP-binding cassette transporter gene which may be a useful marker for ivermectin selection in Onchocerca volvulus. Molecular and Biochemical Parasitology 145, 94100. doi: 10.1016/j.molbiopara.2005.09.011.
Ardelli, B. F. and Prichard, R. (2008). Effects of ivermectin and moxidectin on the transcription of genes coding for multidrug resistance associated proteins and behaviour in Caenorhabditis elegans. The Journal of Nematology 40, 290298.
Ardelli, B. F. and Prichard, R. K. (2007). Reduced genetic variation of an Onchocerca volvulus ABC transporter gene following treatment with ivermectin. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 12231232. doi: 10.1016/j.trstmh.2005.03.019.
Ardelli, B. F., Stitt, L. E. and Tompkins, J. B. (2010). Inventory and analysis of ATP-binding cassette (ABC) systems in Brugia malayi. Parasitology 137, 11951212. doi: 10.1017/S0031182010000120.
Blackhall, W. J., Liu, H. Y., Xu, M., Prichard, R. K. and Beech, R. N. (1998). Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus. Molecular and Biochemical Parasitology 95, 193201.
Blackhall, W. J., Prichard, R. K. and Beech, R. N. (2008). P-glycoprotein selection in strains of Haemonchus contortus resistant to benzimidazoles. Veterinary Parasitology 152, 101107. doi: 10.1016/j.vetpar.2007.12.001.
Bourguinat, C., Ardelli, B. F., Pion, S. D., Kamgno, J., Gardon, J., Duke, B. O., Boussinesq, M. and Prichard, R. K. (2008). P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus. Molecular and Biochemical Parasitology 158, 101111. doi: 10.1016/j.molbiopara.2007.11.017.
Broeks, A., Gerrard, B., Allikmets, R., Dean, M. and Plasterk, R. H. (1996). Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans. The EMBO Journal 15, 61326143.
Broeks, A., Janssen, H. W., Calafat, J. and Plasterk, R. H. (1995). A P-glycoprotein protects Caenorhabditis elegans against natural toxins. The EMBO Journal 14, 18581866.
Coles, G. C., Bauer, C., Borgsteede, F. H., Geerts, S., Klei, T. R., Taylor, M. A. and Waller, P. J. (1992). World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary Parasitology 44, 3544.
Coles, G. C., Watson, C. L. and Anziani, O. S. (2001). Ivermectin-resistant Cooperia in cattle. The Veterinary Record 148, 283284.
De Graef, J., Sarre, C., Mills, B. J., Mahabir, S., Casaert, S., De Wilde, N., Van Weyenberg, M., Geldhof, P., Marchiondo, A., Vercruysse, J., Meeus, P. and Claerebout, E. (2012). Assessing resistance against macrocyclic lactones in gastro-intestinal nematodes in cattle using the faecal egg count reduction test and the controlled efficacy test. Veterinary Parasitology 189, 378382. doi: 10.1016/j.vetpar.2012.04.040.
Demeler, J., Kuttler, U. and von Samson-Himmelstjerna, G. (2010). Adaptation and evaluation of three different in vitro tests for the detection of resistance to anthelmintics in gastro intestinal nematodes of cattle. Veterinary Parasitology 170, 6170. doi: 10.1016/j.vetpar.2010.01.032.
Demeler, J., Van Zeveren, A. M., Kleinschmidt, N., Vercruysse, J., Hoglund, J., Koopmann, R., Cabaret, J., Claerebout, E., Areskog, M. and von Samson-Himmelstjerna, G. (2009). Monitoring the efficacy of ivermectin and albendazole against gastro intestinal nematodes of cattle in Northern Europe. Veterinary Parasitology 160, 109115. doi: 10.1016/j.vetpar.2008.10.030.
Dicker, A. J., Nath, M., Yaga, R., Nisbet, A. J., Lainson, F. A., Gilleard, J. S. and Skuce, P. J. (2011 a). Teladorsagia circumcincta: the transcriptomic response of a multi-drug-resistant isolate to ivermectin exposure in vitro. Experimental Parasitology 127, 351356. doi: 10.1016/j.exppara.2010.08.019.
Dicker, A. J., Nisbet, A. J. and Skuce, P. J. (2011 b). Gene expression changes in a P-glycoprotein (Tci-pgp-9) putatively associated with ivermectin resistance in Teladorsagia circumcincta. International Journal for Parasitology 41, 935942. doi: 10.1016/j.ijpara.2011.03.015.
Edmonds, M. D., Johnson, E. G. and Edmonds, J. D. (2010). Anthelmintic resistance of Ostertagia ostertagi and Cooperia oncophora to macrocyclic lactones in cattle from the western United States. Veterinary Parasitology 170, 224229. doi: 10.1016/j.vetpar.2010.02.036.
El-Abdellati, A., Charlier, J., Geldhof, P., Levecke, B., Demeler, J., von Samson-Himmelstjerna, G., Claerebout, E. and Vercruysse, J. (2010 a). The use of a simplified faecal egg count reduction test for assessing anthelmintic efficacy on Belgian and German cattle farms. Veterinary Parasitology 169, 352357. doi: 10.1016/j.vetpar.2010.01.015.
El-Abdellati, A., De Graef, J., Van Zeveren, A., Donnan, A., Skuce, P., Walsh, A., Wolstenholme, A., Tait, A., Vercruysse, J., Claerebout, E. and Geldhof, P. (2011). Altered avr-14B gene transcription patterns in ivermectin-resistant isolates of the cattle parasites, Cooperia oncophora and Ostertagia ostertagi. International Journal for Parasitology 41, 951957. doi: 10.1016/j.ijpara.2011.04.003.
El-Abdellati, A., Geldhof, P., Claerebout, E., Vercruysse, J. and Charlier, J. (2010 b). Monitoring macrocyclic lactone resistance in Cooperia oncophora on a Belgian cattle farm during four consecutive years. Veterinary Parasitology 171, 167171. doi: 10.1016/j.vetpar.2010.03.003.
Elsworth, B., Wasmuth, J. and Blaxter, M. (2011). NEMBASE4: the nematode transcriptome resource. International Journal for Parasitology 41, 881894. doi: 10.1016/j.ijpara.2011.03.009.
Huang, Y. J. and Prichard, R. K. (1999). Identification and stage-specific expression of two putative P-glycoprotein coding genes in Onchocerca volvulus. Molecular and Biochemical Parasitology 102, 273281.
James, C. E. and Davey, M. W. (2009). Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans. International Journal for Parasitology 39, 213220. doi: 10.1016/j.ijpara.2008.06.009.
Kaplan, R. M. (2004). Drug resistance in nematodes of veterinary importance: a status report. Trends in Parasitology 20, 477481. doi: 10.1016/
Lespine, A., Alvinerie, M., Vercruysse, J., Prichard, R. K. and Geldhof, P. (2008). ABC transporter modulation: a strategy to enhance the activity of macrocyclic lactone anthelmintics. Trends in Parasitology 24, 293298. doi: 10.1016/
Lespine, A., Ménez, C., Bourguinat, C. and Prichard, R. (2012). P-glycoproteins and other multidrug resistance transporters in the pharmacology of anthelmintics: Prospects for reversing transport-dependent anthelmintic resistance. International Journal for Parasitology: Drugs and Drug Resistance 2, 5875.
Lifschitz, A., Virkel, G., Imperiale, F., Sutra, J. F., Galtier, P., Lanusse, C. and Alvinerie, M. (1999). Moxidectin in cattle: correlation between plasma and target tissues disposition. Journal of Veterinary Pharmacology and Therapeutics 22, 266273.
Lincke, C. R., Broeks, A., The, I., Plasterk, R. H. and Borst, P. (1993). The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells. The EMBO Journal 12, 16151620.
Lincke, C. R., The, I., van Groenigen, M. and Borst, P. (1992). The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences. Journal of Molecular Biology 228, 701711.
Lindblom, T. H. and Dodd, A. K. (2006). Xenobiotic detoxification in the nematode Caenorhabditis elegans. Journal of Experimental Zoology Part A: Comparative Experimental Biology 305, 720730. doi: 10.1002/jez.a.324.
Maglich, J. M., Parks, D. J., Moore, L. B., Collins, J. L., Goodwin, B., Billin, A. N., Stoltz, C. A., Kliewer, S. A., Lambert, M. H., Willson, T. M. and Moore, J. T. (2003). Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. The Journal of Biological Chemistry 278, 1727717283. doi: 10.1074/jbc.M300138200.
Martin, J., Abubucker, S., Heizer, E., Taylor, C. M. and Mitreva, M. (2012). update 2011: addition of data sets and tools featuring next-generation sequencing data. Nucleic Acids Research 40(Database issue), D720728. doi: 10.1093/nar/gkr1194.
Ménez, C., Mselli-Lakhal, L., Foucaud-Vignault, M., Balaguer, P., Alvinerie, M. and Lespine, A. (2012). Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line. Biochemical Pharmacology 83, 269278.
Nunes, F., Wolf, M., Hartmann, J. and Paul, R. J. (2005). The ABC transporter PGP-2 from Caenorhabditis elegans is expressed in the sensory neuron pair AWA and contributes to lysosome formation and lipid storage within the intestine. Biochemical and Biophysical Research Communications 338, 862871. doi: 10.1016/j.bbrc.2005.10.023.
Prichard, R. K. and Roulet, A. (2007). ABC transporters and beta-tubulin in macrocyclic lactone resistance: prospects for marker development. Parasitology 134, 11231132. doi: 10.1017/S0031182007000091.
Schroeder, L. K., Kremer, S., Kramer, M. J., Currie, E., Kwan, E., Watts, J. L., Lawrenson, A. L. and Hermann, G. J. (2007). Function of the Caenorhabditis elegans ABC transporter PGP-2 in the biogenesis of a lysosome-related fat storage organelle. Molecular Biology of the Cell 18, 9951008. doi: 10.1091/mbc.E06-08-0685.
Synold, T. W., Dussault, I. and Forman, B. M. (2001). The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Naure Medicine 7, 584590. doi: 10.1038/87912.
Van Zeveren, A. M., Visser, A., Hoorens, P. R., Vercruysse, J., Claerebout, E. and Geldhof, P. (2007). Evaluation of reference genes for quantitative real-time PCR in Ostertagia ostertagi by the coefficient of variation and geNorm approach. Molecular and Biochemical Parasitology 153, 224227. doi: 10.1016/j.molbiopara.2007.03.005.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, RESEARCH0034.
Wei, P., Zhang, J., Egan-Hafley, M., Liang, S. and Moore, D. D. (2000). The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature, London 407, 920923. doi: 10.1038/35038112.
Williamson, S. M., Storey, B., Howell, S., Harper, K. M., Kaplan, R. M. and Wolstenholme, A. J. (2011). Candidate anthelmintic resistance-associated gene expression and sequence polymorphisms in a triple-resistant field isolate of Haemonchus contortus. Molecular and Biochemical Parasitology 180, 99105. doi: 10.1016/j.molbiopara.2011.09.003.
Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R. and Prichard, R. (1998). Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog. Molecular and Biochemical Parasitology 91, 327335.
Yan, R., Urdaneta-Marquez, L., Keller, K., James, C. E., Davey, M. W. and Prichard, R. (2012). The role of several ABC transporter genes in ivermectin resistance in Caenorhabditis elegans. Veterinary Parasitology 190, 519529. doi: 10.1016/j.vetpar.2012.06.038.
Zhao, Z., Thomas, J. H., Chen, N., Sheps, J. A. and Baillie, D. L. (2007). Comparative genomics and adaptive selection of the ATP-binding-cassette gene family in caenorhabditis species. Genetics 175, 14071418. doi: 10.1534/genetics.106.066720.


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Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones

  • J. DE GRAEF (a1), J. DEMELER (a2), P. SKUCE (a3), M. MITREVA (a4) (a5), G. VON SAMSON-HIMMELSTJERNA (a2), J. VERCRUYSSE (a1), E. CLAEREBOUT (a1) and P. GELDHOF (a1)...


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