Phenotypic and genotypic characterisation of benzimidazole susceptible and resistant isolates of Haemonchus contortus

Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Kosice, Slovak Republic.
Veterinary Parasitology (Impact Factor: 2.46). 08/2010; 172(1-2):155-9. DOI: 10.1016/j.vetpar.2010.04.022
Source: PubMed


The study was designed to compare the in vitro egg hatch test (EHT) and molecular allele-specific polymerase chain reaction PCR (AS-PCR) methods as tools for detection of benzimidazole resistance in Haemonchus contortus, a nematode parasite of small ruminants. Comparisons were made during the course of an experimental infection and changes in EHT and AS-PCR were monitored to measure the correlation between in vitro and molecular tests. Both methods were carried out according to World Association for the Advancement of Veterinary Parasitology (WAAVP) recommendations. The molecular test was used to discriminate TAC/TTC polymorphism in the beta-tubulin 200 codon of eight (four resistant and four susceptible) isolates of H. contortus. Using DNA from 100 third-stage larvae, the polymerase chain reaction (PCR)-based analysis revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals in all resistant isolates. Both methods showed comparable and reliable results with regard to detection of benzimidazole resistance. The molecular test has an advantage over the EHT because of its higher sensitivity. On the other hand, EHT is less time-consuming, allows reliable detection of <10% resistance allele frequency and is fairly reliable for the detection of benzimidazole resistance under field conditions.

Download full-text


Available from: Marián Várady, Jan 10, 2014
  • Source
    • "Effective monitoring of resistance is vital for maintaining a high efficacy of the currently available anthelmintics and for preventing further selection for resistance, especially in areas where AR is present in only a small proportion of the worm population. The use of a discrimination/delineating dose (DD) in the EHT provides a good estimate of genotypic resistance [14]. The egg hatch discrimination dose test (EHDDT) is less time-consuming, allows the reliable detection of a frequency of resistance alleles below 10% and is fairly reliable for the detection of BZ resistance under field conditions [14]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background The intensive use of anthelmintics for the control of helminthic infections has resulted in the development of anthelmintic resistance, which has become a major practical problem in many countries. A variety of tests are available to monitor anthelmintic resistance but most of them are expensive, laborious and time consuming and therefore unpractical for large field surveys. The main aim of this survey was thus to detect the occurrence of benzimidazole (BZ) and macrocyclic lactone resistance on sheep farms in Slovakia by using novel and modified in vitro methods that are inexpensive, easy to use and quick and therefore practical for large surveys.ResultsBZ-resistant gastrointestinal nematodes were found on all 27 farms. Two farms (7.4%) had high levels of resistance (>40% of hatching), and 22 farms had low levels (<20% of hatching) of resistant nematodes. IVM-resistant populations were found on 14 of 49 sheep farms. The prevalence of BZ and IVM resistance has slightly increased on Slovak sheep farms during the last two decades.Conclusions Both the BZ and IVM surveys indicated that resistance against anthelmintics was present on Slovak sheep farms. Resistance against the BZ class of anthelmintics had been stable for two decades, but a slight increase on IVM resistance was confirmed. Farmers must thus observe the preventive measures to avoid a faster onset of IVM resistance, otherwise the presence of resistant parasites and ineffective treatment may harm the economy of their farms.
    Full-text · Article · Oct 2014 · BMC Veterinary Research
  • Source
    • "Other relevant clade V nematodes include Oesophagostomum spp. of cattle and pigs (Gerwert et al., 2002; Condi et al., 2009) and cyathostomins of horses (Kaplan, 2002). For many years, studies of anthelmintic resistance mechanisms have focused on a candidate gene approach targeting key proteins presumed to be involved in anthelmintic action; more than 30 studies have explored these targets to assess genetic differences between phenotypically susceptible and resistant parasites (Beech et al., 1994; Elard et al., 1996; Pape et al., 2003; Drogemuller et al., 2004; Cudekova et al., 2010; Martínez-Valladares et al., 2012a). The MLs ivermectin (IVM) and moxidectin (MOX) are the anthelmintics of choice for the control of the majority of parasites, resulting in extensive study of potential targets, in particular the glutamate gated chloride channels (GluCl, Wolstenholme, 2011, 2012), the ABC transporters (James and Davey, 2009) and b-tubulin molecules (de Lourdes Mottier and Prichard, 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generated, which were assembled into 26,910 contigs. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, 27% of the transcriptome was annotated. Further in-depth analysis was carried out by comparing the C. goldi dataset with the next generation transcriptomes and genomes of other clade V nematodes, with the Oesophagostomum dentatum transcriptome and the Haemonchus contortus genome showing the highest levels of sequence identity with the cyathostomin dataset (45%). The C. goldi transcriptome was mined for genes associated with anthelmintic mode of action and/or resistance. Sequences encoding proteins previously associated with the three major anthelmintic classes used in horses were identified, with the exception of the P-glycoprotein group. Targeted re-sequencing of the glutamate gated chloride channel α4 subunit (glc-3), one of the primary targets of the macrocyclic lactone anthelmintics, was performed for several cyathostomin species. We believe this study reports the first transcriptome dataset for an equine helminth parasite, providing the opportunity for in-depth analysis of these important parasites at the molecular level. Sequences encoding enzymes involved in key processes and genes associated with levamisole/pyrantel and macrocyclic lactone resistance, in particular the glutamate gated chloride channels, were identified. This novel data will inform cyathostomin biology and anthelmintic resistance studies in future.
    Full-text · Article · Aug 2013 · International journal for parasitology
  • Source
    • "The frequency of the resistance genotype (RR) ranged from 0 to 66.7% and that of the resistance allele (R) varied from 9 to 74% in flocks with H. contortus phenotypically resistant by FECRT to albendazole, ivermectin and/or moxidectin (Table 1). The frequency of F200Y polymorphism in parasite populations varies according to the resistance status to benzimidazole: populations susceptible to benzimidazole present low resistance genotype frequencies, while the frequencies are highly variable, as reported here, in resistant populations (Elard et al., 1999; Álvarez- Sánchez et al., 2005; ˇ Cudeková et al., 2010). A similar pattern of F200Y polymorphism frequency was observed in populations according to resistance status to macrocyclic lactones (Eng et al., 2006; Mottier and Prichard, 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Haemonchus contortus is the most prevalent and pathogenic nematode of sheep in tropical areas. The objectives of this study were to assess the frequency of the F200Y polymorphism on the β-tubulin gene in third-stage larvae of H. contortus from 33 sheep flocks in São Paulo state, Brazil, and to associate this frequency to risk factors based on farm management practices. The resistance allele frequency varied from 9 to 74%, and the resistance genotype frequency varied from 0 to 66.7%. Resistance genotype frequencies higher than 40% were associated with multiple risk factors - new sheep farming enterprises, the absence of farm records, the use of Dorper and Suffolk breeds, rotational grazing, the lack of wetlands on farms, pasture sharing with cattle or horses, frequent incorporation of animals into the flock, semi-intensive farming systems, whole-flock treatment, failure to use the FAMACHA method, lack of the dose-and-move practice, anthelmintic rotation after each application, visual estimation of animal weight for treatment, and lack of drug combination use. It can be concluded that genotyping the F200Y polymorphism can be used to monitor the resistance in sheep flocks and the knowledge of management strategies at the farm level is important to identify drug resistance related factors.
    Full-text · Article · Jul 2012 · Veterinary Parasitology
Show more