Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep

Álvarez-Sánchez, M.A.; Pérez-García, J.; Cruz-Rojo, M.A.; Rojo-Vázquez, F.A.

doi:10.1016/j.vetpar.2005.02.004

Cited by 61 publications

(29 citation statements)

References 22 publications

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“…Analysis of isolates characterised with regard to TCBZ susceptibility [3] and field populations will be necessary to establish the importance of the S1144R substitution in the development of the TCBZ-resistant phenotype in locations other than the Netherlands. If changes in this region of Pgp are widely associated with TCBZ resistance it may be possible to develop an allele-specific molecular test analogous to that used for nematodes [17] for potentially TCBZ-resistant populations based upon genomic DNA extracted from fluke eggs. These are a source of F. hepatica genomic DNA readily available for epidemiological surveys.…”

Section: Haplotypementioning

confidence: 99%

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

Wilkinson

Law

Hoey

et al. 2012

Molecular and Biochemical Parasitology

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Control of fasciolosis is threatened by the development of anthelmintic resistance. Enhanced triclabendazole (TCBZ) efflux by ABC transporters such as P-glycoprotein (Pgp) has been implicated in this process. A putative full length cDNA coding for a Pgp expressed in adult Fasciola hepatica has been constructed and used to design a primer set capable of amplifying a region encoding part of the second nucleotide binding domain of Pgp when genomic DNA was used as a template. Application of this primer set to genomic DNA from TCBZ-resistant and -susceptible field populations has shown a significant difference in the alleles present. Analysis of an allele occurring at a three-fold higher frequency in the "resistant" population revealed that it was characterised by a serine to arginine substitution at residue 1144. Homology modelling studies have been used to locate this site in the Pgp structure and hence assess its potential to modify functional activity.

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Section: Haplotypementioning

confidence: 99%

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

Wilkinson

Law

Hoey

et al. 2012

Molecular and Biochemical Parasitology

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“…A similar type of mutation causing resistance at amino acid 167 (Prichard 2001) and another mutation where glutamate is replaced by alanine at amino acid 198 (Ghisi et al 2007) has been identified in the isotype 1 β-tubulin gene. Molecular techniques that are highly sensitive and specific have being explored for diagnosis and genotyping of benzimidazole resistant H. contortus (Elard and Humbert 1999;Silvestre and Humbert 2000;Alvarez-Sanchez et al 2005;Coles et al 2006;Tiwari et al 2006;Tiwari et al 2007;Ghisi et al 2007;Walsh et al 2007). These techniques have several advantages over classical methods.…”

Section: Introductionmentioning

confidence: 99%

Genotyping of benzimidazole susceptible and resistant alleles in different populations of Haemonchus contortus from Himalayan and sub-Himalayan regions of North-West India

Garg

Yadav

2008

Trop Anim Health Prod

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An allele-specific PCR was standardized to diagnose the mutation (Phe to Try) at residue 200 of the isotype 1 beta tubulin gene responsible for benzimidazole resistance in Haemonchus contortus adult and infective larvae. Adult and infective larvae of Haemonchus contortus were collected from sheep under different managemental practices (intensive and extensive) in temperate Himalayan regions (Mukteshwar and Kedarkhatta), sub-himalayan region (Pantnagar) of Uttarakhand state and subtropical region of Uttar Pradesh (Bareilly) in north-west India. Genotyping of adult H. contortus, collected from abomasi of slaughtered sheep reared under extensive management, by AS-PCR revealed that the frequency of resistant (r) alleles was significantly higher (P < 0.001) at Pantnagar (0.57) as compared to Bareilly (0.25) and Mukteshwar (0.08). Also, genotyping of infective larvae of the parasite from intensively managed sheep farms indicated that the frequency of resistant (r) alleles was significantly higher (P < 0.001) at Pantnagar (0.85) as compared to Kedarkattha (0.70) and Bareilly (0.62). The results revealed that managemental practices followed in the areas under study have a direct bearing on the spread of benzimidazole resistant alleles.

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“…The usefulness of this assay was limited in that it was not validated on fecal eggs and was not in a multiplex format, thereby requiring four separate tests for each sample. Additional QPCR assays to differentiate horse strongyles and anthelmintic resistant strains of trichostrongyles have been reported, but these assays were restricted to larval or adult DNA as well (Von Samson-Himmelstjerna et al 2003;Álvarez-Sánchez et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors

Harmon

Williams

Zarlenga³

et al. 2007

Parasitol Res

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The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.

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Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep

Cited by 61 publications

References 22 publications

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

Genotyping of benzimidazole susceptible and resistant alleles in different populations of Haemonchus contortus from Himalayan and sub-Himalayan regions of North-West India

Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors

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