Detection of benzimidazole resistance in Haemonchus contortus using RFLP-PCR technique

Area of Science:

• Veterinary Parasitology
• Molecular Diagnostics
• Anthelmintic Resistance

Background:

• Benzimidazole resistance in Haemonchus contortus is a significant veterinary concern.
• This resistance is primarily linked to a specific mutation (Phe to Tyr at codon 200) in the beta-tubulin isotype 1 gene.

Purpose of the Study:

• To develop and validate a novel Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR) technique.
• To detect benzimidazole resistance in Haemonchus contortus by targeting the beta-tubulin isotype 1 gene.

Main Methods:

• A new RFLP-PCR assay using primers AvikaF and AvikaR was designed.
• The technique involves amplifying the target gene region and subsequent restriction digestion.
• Genotyping was performed on 162 adult Haemonchus contortus samples from three different regions.

Main Results:

• The RFLP-PCR successfully differentiated homozygous resistant ('rr'), heterozygous ('rS'), and homozygous susceptible ('SS') individuals based on fragment sizes.
• Genotypic frequencies varied significantly across regions (P<0.001), with Jaipur showing the highest prevalence of resistant 'rr' individuals (98%).
• The overall prevalence of the benzimidazole-resistant allele (r) was 86%, significantly higher than the susceptible allele (S) at 14%.

Conclusions:

• The developed RFLP-PCR is a reliable, reproducible, and cost-effective method for detecting benzimidazole resistance in Haemonchus contortus.
• The technique is suitable for both adult worms and larvae, facilitating broader application.
• This diagnostic tool is valuable for monitoring resistance prevalence and informing anthelmintic drug use in worm control programs.