Reliable reference gene selection for quantitative real time PCR in Haemonchus contortus

Lecová, Lenka; Růžičková, Michaela; Laing, Roz; Vogel, Heiko; Szotáková, Barbora; Prchal, Lukáš; Lamka, J.; Vokřál, Ivan; Skálová, Lenka; Matoušková, Petra

doi:10.1016/j.molbiopara.2015.08.001

Cited by 17 publications

(15 citation statements)

References 25 publications

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“…All samples were measured in duplicates with less than 0.5 Cq difference. Relative expression was calculated using ΔΔCt method ( Livak and Schmittgen, 2001 ), using Hc-ama and Hc-gpd as reference genes as recommended by Lecová et al ( Lecova et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

Matoušková

Lecová

Laing

et al. 2018

International Journal for Parasitology: Drugs and Drug Resistan

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UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus.

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Section: Methodsmentioning

confidence: 99%

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

Matoušková

Lecová

Laing

et al. 2018

International Journal for Parasitology: Drugs and Drug Resistan

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“…qRT-PCR was carried out following the Brilliant III Ultra Fast SYBR QPCR Master Mix protocol (Agilent Technologies, UK, 600882) and results analysed using MxPro qPCR Software, Version 4.10. Gene expression was normalised to ama (HCOI01464300) and gpd (HCOI01760600) ( Lecova et al, 2015 ). Primer sequences are listed in Supplementary Table S2 .…”

Section: Methodsmentioning

confidence: 99%

Transcriptomic profiling of nematode parasites surviving vaccine exposure

Sallé

Laing

Cotton

et al. 2018

International Journal for Parasitology

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“…Reference genes superoxide dismutase ( sod ), fatty acid retinol binding protein ( far ) and glyceraldehyde-3P-dehydrogenase ( gpd ) were adopted from Lecova et al (2015) who had shown stable expression of these transcripts in adult H. contortus following varying treatments with albendazole and ivermectin. Primer sequences of both reference and candidate CYP genes can be found in Table S1 along with PCR efficiencies for each primer set.…”

Section: Methodsmentioning

confidence: 99%

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-resistant isolate

Yilmaz

Ramünke

Demeler

et al. 2017

International Journal for Parasitology: Drugs and Drug Resistan

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Benzimidazoles (BZs) remain amongst the most widely used anthelmintic drug classes against gastro-intestinal nematode infections, although their efficacy is increasingly compromised by resistance. The primary underlying mechanisms for BZ resistance are single-nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene causing the substitutions F167Y, E198A or F200Y. However, resistance is believed to be multi-genic and previous studies have shown that isolates carrying 90–100% F200Y can vary considerably in their resistance level in the egg hatch assay (EHA). Cytochrome P450 monooxygenases (CYPs) are associated with drug resistance in mammals and arthropods and have been considered as mediators of anthelmintic resistance. In Caenorhabditis elegans, several members of the CYP34/35 and CYP31 families are BZ and/or xenobiotic inducible and thiabendazole (TBZ) is metabolised by CYP35D1. Here, expression of all 5 CYPs closely related to the C. elegans CYP34/35 and CYP31 families was investigated in fourth-stage larvae of two susceptible and three BZ-resistant Haemonchus contortus isolates following in vitro exposure to TBZ for 3 and 6 h using real-time RT-PCR. The resistance status of all isolates was determined using EHAs and quantification of resistance-associated β-tubulin SNPs using pyrosequencing. While none of the CYPs was TBZ inducible, constitutive expression of CYP34/35 family member HCOI100383400 was significantly 2.4–3.7-fold higher in the multi-drug resistant WR isolate with the strongest BZ resistance phenotype compared to susceptible and intermediate-level BZ-resistant isolates. Although this increase is only moderate, HCOI100383400 might still be involved in high-level BZ resistance by further decreasing susceptibility in isolates already carrying 100% of a β-tubulin SNP causing BZ resistance. Lower transcript levels were observed for all CYPs in the intermediately resistant IRE isolate in comparison to the susceptible HcH isolate, which, except for CYP HCOI01579500, were statistically non-significant. This suggests that none of the investigated CYPs may contribute to protection against TBZ in this particular isolate.

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Reliable reference gene selection for quantitative real time PCR in Haemonchus contortus

Cited by 17 publications

References 25 publications

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

Transcriptomic profiling of nematode parasites surviving vaccine exposure

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-resistant isolate

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