Field evidence of multiple anthelmintic resistance in gastrointestinal nematodes of small ruminants in Punjab, India, with comparative evaluation of molecular diagnostic tools for benzimidazole resistance detection in Haemonchus contortus.

Anthelmintic resistance in gastrointestinal nematodes (GINs) of small ruminants is an escalating global threat to livestock health and productivity, one that demands urgent, evidence-based diagnostic strategies. Benzimidazole (BZ) resistance, typically the first to emerge, is now alarmingly prevalent in Haemonchus contortus populations worldwide, including India, where resistant alleles have been documented across a significant proportion of treated flocks. This study evaluated resistance against three major anthelmintic classes i.e. benzimidazoles, macrocyclic lactones, and salicylanilides using multiple Faecal Egg Count Reduction Test (FECRT) formulas, alongside three molecular diagnostic approaches for detecting BZ resistance via β-tubulin isotype 1 genotyping in adult H. contortus worms from sheep and goats. Allele-specific PCR (AS-PCR), restriction fragment length polymorphism PCR (RFLP-PCR), and direct sequencing were compared for sensitivity and practical utility in identifying the F200Y resistance mutation. FECRT results revealed widespread multiple anthelmintic resistance, with egg count reductions falling below efficacy thresholds for albendazole, ivermectin, and closantel. Post-treatment larval cultures confirmed H. contortus as the dominant surviving species across all treatments. Molecularly, BZ-resistant alleles were detected in 47% of samples by AS-PCR and 48% by RFLP-PCR. Critically, RFLP-PCR and direct sequencing both identified heterozygous resistant genotypes that AS-PCR missed, demonstrating superior sensitivity for detecting mixed-resistance populations. These findings make a compelling case for integrating RFLP-PCR into routine resistance surveillance, offering a reliable, cost-effective, and technically accessible complement to FECRT, particularly in resource-limited settings where sequencing infrastructure remains out of reach.