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In vitro assessment of ivermectin resistance and gene expression profiles of P-glycoprotein genes from Haemonchus contortus (L3)

Marilyn Cedillo-Borda Marilyn Cedillo-Borda
ML Ma. Eugenia López-Arellano
David E. Reyes-Guerrero David E. Reyes-Guerrero

Published: Dec 20, 2020 DOI: 10.21769/BioProtoc.3851 Views: 1698

Reviewed by: Matthias RieckherMichael Enos

Original research article

The authors used this protocol in:

http://www.ncbi.nlm.nih.gov/pubmed/32434064

May 2020

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Abstract

The aim of the present protocol is to improve the methodology to identify resistance to ivermectin (IVM), one of the main anthelmintic drugs against parasitic nematodes of ruminants, using the blood-feeding nematode Haemonchus contortus as biological model. The infective larvae (L3) of H. contortus are frequently selected to perform in vitro and molecular assays to analyze problems with drug resistance. This protocol describes the procedures to conserve the integrity and quality of H. contortus larvae in different experimental assays. Two nematode isolates, resistant and susceptible to IVM, are compared to estimate the lethal effect using IVM at 1.43, 2.85, 5.71 and 11.42 mM dilutions in the non-ionic detergent Triton X-100 to conserve the activity of control larvae during the experimental period (from 24 to 72 h). In the last decades, the importance of the use of IVM and other anthelmintic drugs diagnoses requires the confirmation of susceptible isolates to evaluate the best strategies of control. More sensitive methods of diagnosis are provided by PCR techniques and other molecular tools, such as sequencing. In this protocol, the analysis of relative expression, using P-glycoprotein, one of the main genes involved in problems with IVM resistance in nematodes, is evaluated using 10 main P-glycoprotein genes (1, 2, 3, 4, 9, 10, 11, 12, 14 and 16) identified in the resistance of H. contortus to IVM in comparison with a nematode isolate susceptible to IVM, using reverse transcription and real-time PCR (RT-qPCR).

Keywords: In vitro search Ivermectin search Relative expression search P-glycoprotein search H. contortus search RNA search RT-qPCR search

Background

Due to problems in the detection of anthelmintic resistance in parasitic nematodes, it is necessary to have a strict procedure and more accurate methodologies to perform specific anthelmintic resistance diagnoses to support the strategies of control in order to preserve the high level of anthelmintic commercial drug toxicity used in the livestock industry. One of the resistance problems is the efflux of xenobiotic molecules, such as ivermectin (IVM), out of the nematode cells by the transmembrane transporter P-glycoproteins (P-gps). IVM is an anthelmintic drug with wide-ranging activity against ecto- and endoparasites in the livestock industry (Whittaker et al., 2017; Reyes-Guerrero et al., 2020). The present protocol describes the methodology to identify IVM resistance in the laboratory in the blood-feeding nematode Haemonchus contortus, both resistant and susceptible to IVM, a biological model that represents other gastrointestinal nematodes (GINs) with IVM resistance problems. The first study is conducted to determine the lethal effect of IVM on H. contortus infective larvae (L3) in order to improve the standardization of the in vitro assay conditions, using a specific IVM diluent to preserve the motility of control larvae and the toxicity of IVM during the experimental assays. The second study is to establish the relative expression analysis of 10 P-gp genes of H. contortus through RT-qPCR (Reyes-Guerrero et al., 2020). The H. contortus nematode obtained from the donor sheep is used as biological material to perform parasitological techniques to achieve better nucleic acid purification. Unsheathed H. contortus L3 are used with the understanding that other protocols work with L3 after the second moult.

Materials and Reagents

  1. Optical Lens Wipes (Carolina Biol. Supply Co., catalog number: 634000 )

  2. 96 well-microplate, flat-bottom, clear and sterile (Greiner Bio-One, catalog number: 655161 )

  3. Polypropylene beakers of 50, 250 and 500 ml plastic (MediLab, catalog numbers: 12002/02a , 12002/04a and 12002/05a )

  4. Policemen stirring rods, PP, Ø – 6 mm x h – 245 mm (MediLab, catalog number: 12110/01 )

  5. Pasteur plastic pipette of 1 ml (MediLab, catalog number: 121227/01 )

  6. Polypropylene funnel Ø150 mm (Corning, catalog number: 6120P-150 )

  7. Plastic hose assembly (Fisher Scientific, catalog number: 02-594-1F )

  8. Teasing needle straight of 150 mm x 1.2 mm with handle strong plastic (Lab Connections, catalog number: B235-1-012 )

  9. Gauze

  10. 15-ml plastic centrifuge tube (Corning, catalog number: 430052 )

  11. MagNa Lyser Green Beads tubes filled (Roche, catalog number: 0 3358941001 ) or BeadBug prefilled tubes (Sigma, catalog number: Z763780-50EA )

  12. DNeasy Blood and Tissue Kit® (Qiagen, catalog number: 69504 )

  13. Sodium chloride (commercial food grade)

  14. Sucrose (Sigma-Aldrich, catalog number: MFCD00006626 )

  15. NaClO (Sodium hypochlorite) (Cloralex-Grupo AlEn-México)

  16. Ethidium bromide 10 mg/ml (Bio-Rad Labs, catalog number: 1610433 )

  17. GoTaq® Green Master Mix 2x (Promega, catalog number: M7123 )

  18. GeneRuler Low Range DNA Ladder, ready-to-use (Thermo Scientific, catalog number: SM1193 )

  19. Analytical grade ivermectin (Sigma-Aldrich, catalog number: 18898-1G )

  20. Triton X-100 (Sigma-Aldrich, catalog number: MFCD00128254 )

  21. Ethanol (Hycel, catalog number: 1822-500 )

  22. TRIzol® Reagent (Thermo Fisher Scientific, catalog number: 15596026 )

  23. Chloroform (J.T. Baker, catalog number: 9180 )

  24. Isopropyl alcohol (Sigma-Aldrich, catalog number: W292907 )

  25. RQ RNase-Free DNase Kit® (Promega, catalog number: M6101 )

  26. ImProm-II Reverse Transcription System Kit® (Promega, catalog number: A3800 )

  27. GoTaq® qPCR Master Mix 2x (Promega, catalog number: A6001 )

  28. Nuclease-free water (Promega, catalog number: P1193 )

  29. Agarose (Bio-Rad, catalog number: 1613100 )

  30. Buffer 50x TAE (see Recipes)

  31. 2% Triton X-100 solution (see Recipes)

  32. 3% agarose gel (see Recipes)

  33. 40% sucrose solution (see Recipes)

  34. 0.187% NaClO solution (see Recipes)

Equipment

  1. Centrifuge (Thermo Fisher Scientific, model: Sorvall ST 8R, catalog number: 75007204 )

  2. Microscopy (Zeizz, Primo Star Hal/Led Full-Köhler ERc5s, catalog number: 415500-0057-000 )

  3. PCR Workstation (UVP, HEPA/UV3, catalog number: 95-0438-01 115V )

  4. Nanophotometer (Implen, NP80 )

  5. Nucleic acid Electrophoresis chamber (Any brand/model)

  6. PowerPAc (Bio-Rad, 300, catalog number: 165-5050 )

  7. Imagine System (UVP, EC3, catalog number: 81-020901 )

  8. Thermal cycler to perform conventional PCR (Bio-Rad, C1000 Touch, catalog number: 10000068706 )

  9. Digital Dry Bath (Labnet Int., Inc., AccuBlockTM, catalog number: D1100)

  10. MagNA Lyser Homogenizer (Roche, catalog number: 03 358 968 001) or BeadBug Microtube Homogenizer (Benchmark Scientific, catalog number: D1030)

  11. Mixer (Benchmark Scientific, VortexTM, catalog number: BV101-P)

  12. Thermal cycler 6000 to perform real time (quantitative) PCR (Qiagen, Corbett Rotor-Gene 6000)

  13. Micropipettes (different volumes, any brand/model)

Software

  1. Statistical software SAS (Version 8.0, USA) or other (i.e., Minitab 17)

  2. Rotor-Gene Q – Pure detection Software (version 1.7)

  3. GeneGlobe Data Analysis Center of Qiagen® (https://geneglobe.qiagen.com/analyze/)

Procedure

Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > in vivo model > Protozoan

Molecular Biology > RNA > qRT-PCR

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