Hostname: page-component-76fb5796d-22dnz Total loading time: 0 Render date: 2024-04-25T17:06:49.046Z Has data issue: false hasContentIssue false
  • English
  • Français

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp

Published online by Cambridge University Press:  12 February 2020

J. A. Scare Open the ORCID record for J. A. Scare [Opens in a new window] ,
P. Dini ,
J. K. Norris ,
A. E. Steuer ,
K. Scoggin ,
H. S. Gravatte ,
D. K. Howe ,
P. Slusarewicz  and
M. K. Nielsen
J. A. Scare*
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
P. Dini
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
J. K. Norris
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
A. E. Steuer
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
K. Scoggin
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
H. S. Gravatte
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
D. K. Howe
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
P. Slusarewicz
Affiliation:
MEP Equine Solutions, 3905 English Oak Circle, Lexington, KY40514, USA
M. K. Nielsen
Affiliation:
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA
*
Author for correspondence: J. A. Scare, E-mail: Jessica.scare@uky.edu
Get access Rights & Permissions [Opens in a new window]

Abstract

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL−1) and IVM at 1.1 μm (1 μg mL−1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Keywords

Anthelminticascaridin vitroParascaristranscriptome
Type
Research Article
Information
Parasitology , Volume 147 , Issue 6 , May 2020 , pp. 659 - 666
Check for updates
Copyright
Copyright © Cambridge University Press 2020

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Armstrong, SK, Woodgate, RG, Gough, S, Heller, J, Sangster, NC and Hughes, KJ (2014) The efficacy of ivermectin, pyrantel and fenbendazole against Parascaris equorum infection in foals on farms in Australia. Veterinary Parasitology 205, 575–580.CrossRefGoogle ScholarPubMed
Beech, RN, Skuce, P, Bartley, DJ, Martin, RJ, Prichard, RK and Gilleard, JS (2011) Anthelmintic resistance: markers for resistance, or susceptibility? Parasitology 138, 160–174.CrossRefGoogle ScholarPubMed
Chelladurai, JJ and Brewer, MT (2019) Detection and quantification of Parascaris P-glycoprotein drug transporter expression with a novel mRNA hybridization technique. Veterinary Parasitology 267, 75–83.CrossRefGoogle Scholar
Cvilink, V, Lamka, J and Skálová, L (2009) Xenobiotic metabolizing enzymes and metabolism of anthelmintics in helminths. Drug Metabolism Reviews 41, 8–26.CrossRefGoogle ScholarPubMed
Feyereisen, R (1999) Insect P450 enzymes. Annual Review of Entomology 44, 507–533.CrossRefGoogle ScholarPubMed
Hanser, Elena, Mehlhorn, Heinz, Hoeben, Dagmar and Vlaminck, Kathleen (2003) In vitro studies on the effects of flubendazole against Toxocara canis and Ascaris suum. Parasitology Research 89(1), 63–74. doi: http://dx.doi.org/10.1007/s00436-002-0668-6CrossRefGoogle ScholarPubMed
Hu, Y, Ellis, BL, Yiu, YY, Miller, MM, Urban, JF, Shi, LZ and Aroian, RV (2013) An extensive comparison of the effect of anthelmintic classes on diverse nematodes. PLoS ONE 8, 1–12.Google ScholarPubMed
Ikenouchi, J and Umeda, M (2010) FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex. Proceedings of the National Academy of Sciences 107, 2–7.CrossRefGoogle ScholarPubMed
James, CE, Hudson, AL and Davey, MW (2009) Drug resistance mechanisms in helminths: is it survival of the fittest? Trends in Parasitology 25, 328–335.10.1016/j.pt.2009.04.004CrossRefGoogle ScholarPubMed
Janssen, IJI, Krücken, J, Demeler, J, Basiaga, M, Kornaś, S and von Samson-Himmelstjerna, G (2013) Genetic variants and increased expression of Parascaris equorum P-glycoprotein-11 in populations with decreased ivermectin susceptibility. PLoS ONE 8, 1–10. doi: 10.1371/journal.pone.0061635CrossRefGoogle ScholarPubMed
Janssen, IJI, Krücken, J, Demeler, J and von Samson-Himmelstjerna, G (2015) Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans. International Journal for Parasitology: Drugs and Drug Resistance 5, 44–47.Google ScholarPubMed
Jourdan, PM, Lamberton, PHL, Fenwick, A and Addiss, DG (2018) Soil-transmitted helminth infections. Lancet 391, 252–265.10.1016/S0140-6736(17)31930-XCrossRefGoogle ScholarPubMed
Kaplan, RM (2004) Drug resistance in nematodes of veterinary importance: a status report. Trends in Parasitology 20, 477–481.10.1016/j.pt.2004.08.001CrossRefGoogle ScholarPubMed
Kilpinen, O, Roepstorff, A, Permit, A, Norgaard-Nielsen, G, Lawson, LG and Simonsen, HB (2005) Influence of Dermanyssus gallinae and Ascaridia Galli infections on behavior and health of laying hens (Gallus Gallus domesticus). British Poultry Science 46, 26–34.10.1080/00071660400023839CrossRefGoogle Scholar
Kotze, AC, Hunt, PW, Skuce, P, von Samson-Himmelstjerna, G, Martin, RJ, Sager, H, Krücken, J, Hodgkinson, J, Lespine, A, Jex, AR, Gilleard, JS, Beech, RN, Wolstenholme, AJ, Demeler, J, Robertson, AP, Charvet, CL, Neveu, C, Kaminsky, R, Rufener, L, Alberich, M, Menez, C and Prichard, RK (2014) Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions. International Journal for Parasitology: Drugs and Drug Resistance 4, 164–184.Google ScholarPubMed
Krücken, Jürgen, Fraundorfer, Kira, Mugisha, Jean Claude, Ramünke, Sabrina, Sifft, Kevin C., Geus, Dominik, Habarugira, Felix, Ndoli, Jules, Sendegeya, Augustin, Mukampunga, Caritas, Bayingana, Claude, Aebischer, Toni, Demeler, Janina, Gahutu, Jean Bosco, Mockenhaupt, Frank P and von Samson-Himmelstjerna, Georg (2017) Reduced efficacy of albendazole against Ascaris lumbricoides in Rwandan schoolchildren. International Journal for Parasitology: Drugs and Drug Resistance 7(3), 262–271. doi: http://dx.doi.org/10.1016/j.ijpddr.2017.06.001Google ScholarPubMed
Lacey, E (1988) The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. International Journal for Parasitology 18, 885–936.10.1016/0020-7519(88)90175-0CrossRefGoogle ScholarPubMed
Laurent, GS, Shtokalo, D, Tackett, MR, Yang, Z, Vyatkin, Y, Milos, PM, Seilheimer, B, McCaffrey, TA and Kapranov, P (2013) On the importance of small changes in RNA expression. Methods 63, 18–24.10.1016/j.ymeth.2013.03.027CrossRefGoogle Scholar
Lecová, L, Růžičková, M, Laing, R, Vogel, H, Szotáková, B, Prchal, L, Lamka, J, Vokřál, I, Skálová, L and Matoušková, P (2015) Reliable reference gene selection for quantitative real time PCR in Haemonchus contortus. Molecular and Biochemical Parasitology 201, 123–127.10.1016/j.molbiopara.2015.08.001CrossRefGoogle ScholarPubMed
Lesage, F and Lazdunski, M (2000) Molecular and functional properties of two-pore-domain potassium channels. American Journal of Physiology-Renal Physiology 279, F793–F801.10.1152/ajprenal.2000.279.5.F793CrossRefGoogle ScholarPubMed
Lloberas, M, Alvarez, L, Entrocasso, C, Ballent, M, Virkel, G, Luque, S, Lanusse, C and Lifschitz, A (2015) Comparative pharmacokinetic and pharmacodynamic response of single and double intraruminal doses of ivermectin and moxidectin in nematode-infected lambs. New Zealand Veterinary Journal 63, 227–234.10.1080/00480169.2015.1015645CrossRefGoogle ScholarPubMed
Lyons, ET, Drudge, JH and Tolliver, SC (1990) Prevalence of some internal parasites found (1971–1989) in horses born on a farm in Central Kentucky. Journal of Equine Veterinary Science 10, 99–107.10.1016/S0737-0806(06)80114-0CrossRefGoogle Scholar
Lyons, ET, Tolliver, SC, Ionita, M and Collins, SS (2008) Evaluation of parasiticidal activity of fenbendazole, ivermectin, oxibendazole, and pyrantel pamoate in horse foals with emphasis on ascarids (Parascaris equorum) in field studies on five farms in Central Kentucky in 2007. Parasitology Research 103, 287–291.10.1007/s00436-008-0966-8CrossRefGoogle ScholarPubMed
Lyons, ET, Tolliver, SC, Kuzmina, TA and Collins, SS (2011) Further evaluation in field tests of the activity of three anthelmintics (fenbendazole, oxibendazole, and pyrantel pamoate) against the ascarid Parascaris equorum in horse foals on eight farms in Central Kentucky (2009–2010). Parasitology Research 109, 1193–1197.CrossRefGoogle Scholar
Martin, RJ (1997) Modes of action of anthelmintic drugs. Veterinary Journal 154, 11–34.10.1016/S1090-0233(05)80005-XCrossRefGoogle ScholarPubMed
Martin, F, Höglund, J, Bergström, TF, Karlsson Lindsjö, O and Tydén, E (2018) Resistance to pyrantel embonate and efficacy of fenbendazole in Parascaris univalens on Swedish stud farms. Veterinary Parasitology 264, 69–73.CrossRefGoogle ScholarPubMed
Meng, W and Takeichi, M (2009) Adherens junction: molecular architecture and regulation. Cold Spring Harbor Perspectives in Biology 1, a002899.CrossRefGoogle ScholarPubMed
Mi, H, Muruganujan, A, Casagrande, JT and Thomas, PD (2013) Large-scale gene function analysis with the PANTHER-classification system. Nature Protocols 8, 1551–1566.CrossRefGoogle ScholarPubMed
Mi, H, Huang, X, Muruganujan, A, Tang, H, Mills, C, Kang, D and Thomas, PD (2017) PANTHER Version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Research 45, D183–D189.10.1093/nar/gkw1138CrossRefGoogle ScholarPubMed
Nielsen, MK (2016) Evidence-based considerations for control of Parascaris Spp. infections in horses. Equine Veterinary Education 28, 224–231.10.1111/eve.12536CrossRefGoogle Scholar
Overgaauw, PAM (1997) Aspects of Toxocara epidemiology: toxocarosis in dogs and cats. Critical Reviews in Microbiology 23, 233–251.CrossRefGoogle ScholarPubMed
Peregrine, AS, Molento, MB, Kaplan, RM and Nielsen, MK (2014) Anthelmintic resistance in important parasites of horses: does it really matter? Veterinary Parasitology 201, 1–8.10.1016/j.vetpar.2014.01.004CrossRefGoogle ScholarPubMed
Perez de la Cruz, I, Levin, JZ, Cummins, C, Anderson, P and and Horvitz, HR (2003) sup-9, sup-10, and unc-93 may encode components of a two-pore K+ channel that coordinates muscle contraction in Caenorhabditis elegans. Journal of Neuroscience 23, 9133–9145.CrossRefGoogle Scholar
Scare, JA, Steuer, AE, Shaffer, CL, Slusarewicz, P, Mousley, A and Nielsen, MK (2018) Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris Spp. in vitro. Parasitology 18, 1–9.Google Scholar
Stehbens, SJ, Paterson, AD, Crampton, MS, Shewan, AM, Ferguson, C, Akhmanova, A, Parton, RG and Yap, AS (2006) Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts. Journal of Cell Science 119, 1801–1811.CrossRefGoogle ScholarPubMed
Thamsborg, SM, Nejsum, P and Mejer, H (2013) Impact of Ascaris suum in livestock. In Holland, Celia (ed.), Ascaris the Neglected Parasite. Waltham, MA: Elsevier, pp. 363–381. doi: 10.1016/B978-0-12-396978-1.00014-8.CrossRefGoogle Scholar
Wang, J, Gao, S, Mostovoy, Y, Kang, Y, Zagoskin, M, Sun, Y, Zhang, B, White, LK, Easton, A, Nutman, TB, Kwok, PY, Hu, S, Nielsen, MK and Davis, RE (2017) Comparative genome analysis of programmed DNA elimination in nematodes. Genome Research 27, 2001–2014.10.1101/gr.225730.117CrossRefGoogle ScholarPubMed
Wolstenholme, AJ (2012) Glutamate-gated chloride channels. The Journal of Biological Chemistry 287, 40232–40238.CrossRefGoogle ScholarPubMed
Yazwinski, T.A., Tucker, C.A., Wray, E., Jones, L. and Clark, F.D. (2013) Observations of benzimidazole efficacies against Ascaridia dissimilis, Ascaridia galli, and Heterakis gallinarum in naturally infected poultry. Journal of Applied Poultry Research 22(1), 75–79. doi: http://dx.doi.org/10.3382/japr.2012-00606CrossRefGoogle Scholar
Yilmaz, E, Ramünke, S, Demeler, J and Krücken, J (2017) Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of Haemonchus contortus isolates with different drug susceptibility identifies one gene with high constitutive expression in a multi-resistance. International Journal for Parasitology: Drugs and Drug Resistance 7, 362–369.Google Scholar
Supplementary material: File

Scare et al. supplementary material

Scare et al. supplementary material 1

Download Scare et al. supplementary material(File)
File 631.1 KB
Supplementary material: File

Scare et al. supplementary material

Scare et al. supplementary material 2

Download Scare et al. supplementary material(File)
File 18.1 KB