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

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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, Ivermectin, Relative expression, P-glycoprotein, H. contortus, RNA, RT-qPCR

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

Notes:

  1. This protocol assumes a basic knowledge of H. contortus L3 isolation and parasitological techniques. Figure 1 describes the main points considered in the present protocol.

  2. At least two isolates of H. contortus are required to perform a comparison when IVM resistance is suspected with a susceptible isolate as reference.



    Figure 1. The main points in performing the diagnosis of ivermectin (IVM) resistance and expression profiles of P-glycoprotein genes from Haemonchus contortus (L3)


  1. Haemonchus contortus isolates and laboratory conditions

    1. Haemonchus contortus L3 recovery by parasitological techniques

      1. Figure 2 shows the collection of coprological material from donor sheep infected with H. contortus ( Thienpont et al., 2003; Liébano- Hernández et al., 2011). This is confirmed 21 days after infection by a McMaster quantitative technique, and then faecal samples positive to GIN eggs are cultured to recover L3 larvae following this protocol:

        1. The McMaster technique estimates the number of GIN eggs per gram (EPG) from faecal samples by density gradient:

          1. Collect 10 g of faecal sample from infected sheep using a clean plastic bag of 10 cm x 10 cm.

          2. Use a pestle to macerate and homogenize the faeces in the plastic bags.

          3. Weigh 2 g of faeces and place them in a 50-ml plastic beaker or mortar.

          4. Add 28 ml of saturated sodium chloride at 1:24 density and homogenize.

          5. By density, the GIN eggs will be found in the supernatant in few seconds.

          6. Put a piece of gauze (5 x 5 cm) on the surface of the homogenized sample.

          7. Take an aliquot using a pipette or dropper through the gauze.

          8. Use the McMaster chamber to quantify the EPG (Figure 2).

          9. Observe the McMaster chamber using an optical microscope (4x and 10x) to count all nematode eggs between the lines in both compartments.

          10. The result of the count is notified as EPG. Apply the following formula to estimate the EPG:

        2. Faecal culture and sedimentation of infective larvae (Baermann technique) to collect fresh LH. contortus ( Thienpont et al., 2003):

          1. Collect sample faeces (300-500 g) from sheep.

          2. Place the faecal sample in a 250-ml plastic beaker and add tap water to cover.

          3. Homogenize the faecal sample. Add foam rubber and mix it with faecal sample, cover it with aluminium foil and incubate at room temperature (25 °C) for seven days.

          4. After this period prepare 100 g of faecal/foam rubber mixture and place it in a piece of non-sterile gauze (15 x 15 cm). Wrap the sample and tie it up into a ball. Perform the same procedure for remaining faecal samples.

          5. Place each gauze ball into a funnel connected to a 10-ml tube and put it vertically. Add tap water to cover the gauze ball. This is the Baermann parasitology technique.

          6. Wait for 24 h for the larvae to descend to the bottom of the tube.

          7. Recover the tube(s) containing the larvae pellet(s). Store the tube(s) at 4 °C for 2 h to precipitate all larvae. Carefully discard the supernatant using a pipette.

          8. Recover the larvae pellet(s) from tube(s) and mix them in a clean 15-ml plastic tube.

          9. Clean larvae by filtering through an optical lens wipe on the Baermann funnel. Add tap water to cover the wipe and place the H. contortus larvae L3 on the top of the lens wipe.

          10. After 24 h recover H. contortus L3 pellet on the bottom of tube.

          11. Recovering larvae and store at 4 °C.

      2. Cleaning of H. contortus L3 by density gradient:

        1. Prepare a 40% sucrose solution (see Recipes) and add 6 ml to in a 15-ml plastic centrifuge tube.

        2. Centrifuge the recovered L3 larvae at 1,000 x g at room temperature for 3 min. Discard the supernatant.

        3. Place the larvae pellet in the tube containing the sucrose solution. A ring gradient will be formed. Recover the larval ring carefully using a pipette.

        4. Transfer larval ring to a clean 15-ml plastic centrifuge tube. Add distilled water to 15 ml to eliminate sucrose residues.

        5. Centrifuge at 1,000 x g at room temperature for 3 min. Discard supernatant, conserving the larval pellet. Repeat this step twice more.

        6. Re-suspend the clean larval pellet in distilled water. Store at 4 °C.

      3. Removing of H. contortus L3 second moult at 0.187% v/v commercial NaClO:

        1. Prepare 6 ml of 0.187% v/v commercial NaClO solution (see Recipes) in a 15-ml plastic tube.

        2. Centrifuge the clean previously washed larvae. Discard the supernatant. Place the clean larval pellet carefully into the tube containing the NaClO solution.

        3. Homogenize larvae with the NaClO solution by pipetting for 10 min.

        4. Observe a 10-μl aliquot using an optical microscope (4x and 10x) in order to confirm the larval unsheathing process. The L3 larvae will release their second moult (sheath).

        5. Add distilled water to the tube containing the larval pellet to 15-ml to eliminate NaClO.

        6. Centrifuge at 1,000 x g at room temperature for 3 min. Discard supernatant, conserving the larval pellet. Repeat this step twice more to wash the pellet.

        7. Suspend the larval pellet in distilled water and count the larvae in 5-10 µl aliquots using a microscope (4x and 10x), and count the larvae in each drop. Calculate number of larvae by volume proportion.



      Figure 2. Schematic representation of H. contortus L3 recovery by parasitological techniques in laboratory


    2. Molecular identification of Haemonchus genus

      1. DNA purification (protocol modified from DNeasy Blood and Tissue® kit, Qiagen):

        Note: The buffers and Proteinase K solution mentioned in this section are included in the kit and are ready for use.

        1. Place H. contortus L3 (washed and unsheathed) in a lysis tube with ceramic or zirconium beads and add 180 μl of buffer ATL.

        2. Disrupt the samples twice using a homogenizer (MagNA Lyser) for 30 s at 6,000 Hz.

        3. Place the lysed larvae in a free-nuclease 1.5-ml tube with 20 μl of proteinase K. Mix thoroughly in a vortex and incubate at 56 °C for 15 min. Homogenize occasionally during incubation.

        4. Add 200 µl of buffer AL to the sample of denatured cellular proteins and then mix thoroughly using a vortex for 30 s.

        5. Add 200 µl of ethanol (96-100%), and mix thoroughly using a vortex.

        6. Pipette the mixture into the DNeasy Mini spin column and place it in the 2-ml tube included in the kit. Centrifuge at 6,000 x g for 3 min. Discard flow-through and collection tube.

        7. Place the DNeasy Mini spin column into a new 2-ml collection tube, add 500 µl buffer AW1, and centrifuge for 3 min at 6,000 x g. Discard flow-through and collection tube.

        8. Place the DNeasy Mini spin column into a new 2-ml collection tube, add 500 µl buffer AW2 and centrifuge for 3 min at 20,000 x g. Discard flow-through and collection tube.

        9. Place the DNeasy Mini spin column into a clean 1.5-ml microcentrifuge tube, and pipette 30 µl buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 3 min at 6,000 x g to elute.

        10. Repeat the previous step once more. Add 30 µl of fresh buffer AE to the DNasy Mini Spin column in order to reach a final elution volume of 60 µL after centrifugation.

        11. Determine concentration and purity of DNA by nanophotometer through optical density ratio at A260/A280 value. A ratio value in the range 1.8-2.0 would indicate acceptable purity.

        12. Load the genomic DNA onto 3% agarose gel (see Recipes) and carry out electrophoresis at 60 V for 30 min in buffer 1x TAE (see Recipes) to confirm the DNA integrity.

        13. Visualize DNA integrity using a transilluminator and an image of a full band at the top of the agarose well gel should be observed. If smearing of the DNA band is seen, the DNA is losing integrity; the level of loss is indicated by the size of the band-smear.

      2. Identification of Haemonchus and other gastrointestinal nematodes from mixture samples.

        1. PCR-tubes of 0.2 ml should be used to mix all reagents (Table 1). Into each tube add the reaction mixture, including the pair of specific nematode primers (Table 2; Zarlenga et al., 2001).


          Table 1. Components for the PCR mix in order to identify genera of nematodes


        2. Table 2. Primer descriptions and nucleotide sequence for genotyping GIN genera

        3. Place PCR tubes in thermocycler (Bio-Rad, C1000 Touch) to perform PCR-end point. The PCR assay conditions are cited in Table 3.


          Table 3. PCR conditions for genotyping of GIN genera

        4. To confirm the PCR fragment size, load 15 µl of the PCR products and 5 µl of DNA ladder onto 3% agarose gel (see Recipes) to confirm the PCR fragment size by electrophoresis at 80 V for 90 min in buffer 1x TAE.

        5. Visualize PCR products in a transilluminator and identify the fragment and correct size based on base-pairs (bp) corresponding to the nematode genus.


  2. In vitro evaluation of lethal effect of IVM

    1. Preparation of IVM stock solution (Reyes- Guerrero et al., 2020):

      1. Dissolve 20 mg of IVM in 1 ml of 2 % Triton X-100-ethanol solution (final concentration 22.84 mM).

      2. Homogenize the IVM until completely dissolved.

      3. Evaporate the ethanol at 78.8 °C for 30-60 min, leaving the IVM and Triton X-100 as a clear solution.

      4. Add distilled water (approx. 960 µl) to adjust the final volume to 20 mg/ml final concentration.

      5. Prepare a 2% Triton X-100 solution as in vitro assay control.

    2. In vitro assays:

      Notes:

      1. Perform the in vitro assays with unsheathed larvae previously cited.

      2. Evaluation and standardization of in vitro assays are cited by Reyes-Guerrero et al. (2020).

      3. The experimental design with the concentrations and controls are given in Table 4.


        Table 4. Experimental design of in vitro evaluation of lethal effect of ivermectin

      1. Use 96-well microtitre plates and perform serial dilutions of IVM for each H. contortus isolate to evaluate as shown in Figure 3.

        1. Leave empty wells A, B and C of column 1 (wells 1A, B and C).

        2. Add 50 µl of distilled water to wells A, B, and C of columns 2-5.

        3. Add 50 µl of 2% Triton X-100 solution (control) into wells 6A, B and C.

        4. Place 100 µl of IVM stock solution (22.84 mM) into wells 1A, B and C.

        5. Pipette 50 µl from wells A, B and C of column 1 containing IVM stock solution to column 2A, B and C wells. Homogenize by pipetting.

        6. Make serial dilutions with same volume (50 µl) from wells 2A, B and C into wells 3A, B and C and 4A, B and C and homogenize by pipetting.

        7. Discard 50 µl from the last wells (4A, B and C).

        8. Add 50 µl of distilled water containing 100 L3 larvae from wells 1ABC to wells 6ABC. Use IVM concentrations of 11.42 mM (1ABC), 5.71 mM (2ABC), 2.85 mM (3ABC) and 1.43 mM (4ABC) as serial dilutions. The two columns 5ABC and 6ABC contain distilled water and Triton X-100 controls, respectively.

        9. Homogenize each well carefully by pipetting.



        Figure 3. Schematic representation of the process to perform in vitro assays


      2. After IVM dilution incubate the 96-well microtitre plate at room temperature for 72 h.

      3. Count and record the total numbers of dead and active larvae in each well, placing 10 µl aliquots on a microscope slide and visualizing samples by optical microscope at 4x or 10x magnification.

      4. Perform at least three repetitions per replicate (n = 3) of each dilution and control assays.


  3. Relative gene expression

    1. RNA purification (modified from Trizol Reagent protocol):

      1. Place H. contortus L3 into lysis tube containing ceramic beads and 500 μl of TRIzolTM.

      2. Disrupt the sample using a homogenizer (MagNA Lyser) with two sets of 30 s at 6,000 Hz, and then incubate sample for 5 min at room temperature.

      3. Treat the lysed sample with 500 μl of TRIzolTM in a 1.5-ml microcentrifuge tube.

      4. Add 0.2 ml of chloroform per 1 ml of TRIzolTM and incubate for 3 min at room temperature.

      5. Centrifuge the sample for 15 min at 12,000 x g at 4 °C.

      6. Transfer the aqueous phase containing the RNA to a new tube.

      7. Add 0.5 ml of isopropanol to the aqueous phase and incubate for 10 min at room temperature.

      8. Centrifuge for 10 min at 12,000 x g at 4 °C. Total RNA precipitate forms a white gel-like pellet at the bottom of the tube. Carefully discard the supernatant with a micropipette.

      9. Suspend the pellet in 1 ml 75% ethanol stock solution.

      10. Homogenize the sample for a short period using a vortex, then centrifuge for 5 min at 7,500 x g at 4 °C. Discard the supernatant with a micropipette.

      11. Leave the RNA pellet to air-dry for 20 min at room temperature.

      12. Re-suspend the pellet in 30 µl of RNase-free water by pipetting up and down.

      13. Incubate in a heat block set at 57.5 °C for 15 min.

      14. Estimate RNA concentration and purity using a nanophotometer by optical density ratio at A260/A280 value. A ratio value in the range of 1.8-2.0 would indicate acceptable purity.

      15. Confirm the RNA integrity by electrophoresis in 3% agarose gel (see Recipes) at 60 V for 30 min in buffer 1x TAE.

      16. Visualize the integrity of the RNA using a transilluminator. Two separate clear bands of 28S and 18S rRNA should be observed. Intense and well-defined bands indicate the RNA integrity.

    2. RNA decontamination (following manufacturer’s instructions of RQ RNase-Free DNase Kit®)

      1. In PCR tubes (0.2 ml) perform the DNase digestion reaction as follows (Table 5):


        Table 5. Components for RNA decontamination

      2. Incubate at 37 °C for 30 min.

      3. Add 1 µl of RQ1 DNase Stop Solution to stop the reaction.

      4. Incubate at 65 °C for 10 min to inactivate the DNase.

    3. Reverse transcription reaction (modified from ImProm-II Reverse Transcription System Kit®):

      1. Working on ice, add 1 µl of solution from the tube flagged as “random primers” to decontaminate previously obtained RNA. The random primers are used to prime messenger RNA (mRNA) with or without poli(A) tail in order to synthesize complementary DNA (cDNA).

      2. Place PCR-tubes into a pre-heated 70 °C block for 5 min, and then immediately chill in ice. Spin each tube for 10 s.

      3. Mix the following components (Table 6) in the same PCR-tube on ice.


        Table 6. Components for performing the reverse transcription

      4. Place the tube, containing 26 µl final volume, in a heated block at 25 °C, incubate for 5 min, and repeat the incubation twice, at 42 °C for 60 min and at 70 °C for 15 min.

      5. Determine the concentration and purity of cDNA by nanophotometer. An A260/A280 ratio value in the range of 1.8-2.0 would indicate acceptable purity.

    4. qPCR assays for P-gp gene amplification

      1. In PCR tubes (0.2 ml) mix the reagents indicated in Table 7. In each tube, perform the reaction with a pair of P-gp primers: the sequences are shown in Table 8 (Williamson and Wolstenholme, 2012; Issouf et al., 2014; Reyes- Guerrero et al., 2020).


        Table 7. Components for the qPCR mix for transcription level assays

        Table 8. Primer descriptions and nucleotide sequence for qPCR assays

      2. Place the tubes with the final mix in the Rotor-Gene 6000 and perform the PCR with the following conditions shown in Table 9 below:


        Table 9. Real-time PCR conditions for transcription level assays

      3. Optionally, perform an electrophoresis of the qPCR products on 3% agarose gel (see recipes) at 80 V for 60 min in buffer TAE 1x. However, it is important considered that the PCR product contains fluorophores. 

      4. Visualize the PCR products using a transilluminator. The sizes of the fragments of each P-gp gene are shown in Figure 4.



    Figure 4. Electrophoresis simulation developed with SnapGene Software of PCR products of H. contortus P-gp genes (1, 2, 3, 4, 9, 10, 11, 12, 14 and 16).

            The size of each gene fragment is indicated.

Data analysis

  1. Statistical analysis of mortality percent of H. contortus isolates in vitro assays (Reyes- Guerrero et al., 2020 ):

    1. Bi-factorial analysis of variance (multilevel factorial design) is used to analyze the in vitro data. An example is given below using MiniTab software (version 17).

    2. Calculate percentage larval mortality from each IVM treatment and control.

    3. Record all data in a spreadsheet to organize the information from H. contortus isolates (susceptible and resistant), treatments (IVM dilutions and controls) and mortality percentage, following the example in Figure 5.



      Figure 5. Example of data obtained from in vitro assays


    4. As an example, using the Minitab software (version 17) to perform the bi-factorial analysis, the results are shown in Figure 6 (citied below).



      Figure 6. Example of bi-factorial analysis showing results performed in Minitab software


    5. Interpretation of suspicious of IVM resistance is based on the comparison between negative controls and IVM data: if both in vitro results show similar active motility and percentage with significance level of 95% (P ≤ 0.05), this is an indication that IVM is losing toxicity.

    6. The recommendation is to perform PCR to confirm IVM resistance problems.


  2. Gene expression analysis of P-gp of H. contortus (Reyes- Guerrero et al., 2020)

    1. From each isolate under study, the Threshold Cycle (Ct) value is obtained as raw data to analyze the relative expression from the Rotor-Gene Q-Pure detection software (version 1.7). A threshold value of 0.05 is used to eliminate any noise from fluorophores. Figure 7 shows systematically how to obtain the Ct values for P-gp genes.



      Figure 7. Schematic representation of the process to obtain the Ct values


    2. Data are recorded in an Excel spreadsheet and uploaded to the Qiagen® GeneGlobe Data Analysis Center web platform to perform the comparison using the ΔΔCt (double delta Ct) as follow:

      1. Enter https://geneglobe.qiagen.com/analyze/ into web browser and select “PCR” analysis tool option (Figure 8). A registered user account is necessary for access.

      2. Upload the data recorded in a data-sheet (Figure 8).



        Figure 8. Homepage of “PCR” analysis tool in the Qiagen® GeneGlobe Data Analysis Center web platform


      3. Select groups under study, including controls and housekeeping genes (Figure 9).



        Figure 9. Example of the selection of groups, controls and housekeeping genes


      4. Carry out the expression normalization (Figure 10), using at least two housekeeping genes of H. contortus: these could be GAPDH and β-Tubulin. Table 8 shows oligonucleotide sequences of the housekeeping genes used.



        Figure 10. Example of data normalization


      5. The platform (Qiagen® GeneGlobe Data Analysis Center) displays fold-change values where:

        1. Fold-change values of > 1 indicate a positive or an up-regulation. The fold-regulation that is equal to the fold-change appears in the Qiagen GeneGlobe menu screen.

        2. Fold-change values of < 1 indicate a negative or down-regulation: the fold-regulation is the negative inverse of the fold-change.

        3. Fold-change values of > 2 are indicated in red; fold-change values of < 0.5 in blue and fold-change values of 0.5-2 in black.

        4. The p values are calculated based on a Student’s t-test of the replicate 2^(-ΔCt) values for each gene in the control group and treatment groups. p-values of < 0.05 are indicated in red and values of > 0.05 in black.

    Figure 11 shows an example of relative expression calculated by the ΔΔCt method on the Qiagen platform system.




    Figure 11. Example of relative expression and interpretation using the Qiagen platform system

Recipes

  1. Buffer 50x TAE

    242 g Tris base (FW = 121.14)

    57.1 ml glacial acetic acid

    100 ml 0.5 M EDTA (pH 8.0)

    Adjust the final volume to 1 liter with deionized water.

  2. 2% Triton X-100 solution

    0.2 ml Triton X-100

    0.98 ml Ethanol

  3. 3% agarose gel

    1.06 g agarose

    45 ml Buffer 1x TAE

  4. 40% sucrose solution

    20 g sucrose

    50 ml distilled water

  5. 0.187% NaClO solution

    0.183 ml CLORALEX

    5.813 ml distilled water

Acknowledgments

This protocol was derived from an original research paper (Reyes- Guerrero et al., 2020, https://doi.org/10.1016/j.molbiopara.2020.111281), which received financial support from the Mexican grants institutions CONACYT-SEP and CENID-SAI, INIFAP under grant numbers 287598 and 20454534898, respectively.

Competing interests

The authors declare no conflict of interest.

Ethics

The criteria for care and handling of experimental animals were set forth in the Official Mexican Standard NOM-062-ZOO-1999.

References

  1. Liébano-Hernández, E., López-Arellano, M.E. and Mendoza-De-Gives G.P. and Aguilar- Marcelino, L. (2011). Manual de diagnóstico para la identificación de larvas de nematodos gastrointestinales de rumiantes. Publicación Especial Vol. 2. México, INIFAP, 1-44.
  2. Reyes-Guerrero, D. E., Cedillo-Borda, M., Alonso-Morales, R. A., Alonso-Diaz, M. A., Olmedo-Juarez, A., P., Mendoza-de-Gives and Lopez-Arellano, M. E. (2020). Comparative study of transcription profiles of the P-glycoprotein transporters of two Haemonchus contortus isolates: Susceptible and resistant to ivermectin. Mol Biochem Parasitol 238: 111281.
  3. Whittaker, J. H., Carlson, S. A., Jones, D. E. and Brewer, M. T. (2017). Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance. J Vet Pharmacol Ther 40(2): 105-115.
  4. Williamson, S. M. and Wolstenholme, A. J. (2012). P-glycoproteins of Haemonchus contortus: development of real-time PCR assays for gene expression studies. J Helminthol 86(2): 202-208.
  5. Thienpont, D., Rochette, F. and Vanpararijs, O.F.J. (2003). Diagnosing Helminthiasis by Coprological Examination. Third edition, Janssen Animal Health, Beerse, Belgium.
  6. Zarlenga, D. S., Barry Chute, M., Gasbarre, L. C. and Boyd, P. C. (2001). A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle. Vet Parasitol 97(3): 199-209.
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How to cite: Cedillo-Borda, M., López-Arellano, M. E. and Reyes-Guerrero, D. E. (2020). In vitro assessment of ivermectin resistance and gene expression profiles of P-glycoprotein genes from Haemonchus contortus (L3). Bio-101: e3851. DOI: 10.21769/BioProtoc.3851.
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