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

Abstract

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.