Unveiling β-Tubulin Isotype-1 Polymorphisms in Haemonchus contortus from Pakistani Livestock: New Insights into Benzimidazole Resistance and Protein Structure Analysis.

Background: Haemonchus contortus, a gastrointestinal hematophagous parasitic nematode, causes substantial economic losses to the livestock sector across the globe. The widespread use of benzimidazoles in livestock has contributed greatly to the evolution of resistance in H. contortus. To effectively tackle this expanding threat, regular and diligent monitoring is required to identify and manage resistance, ensuring that treatment techniques continue to be effective.

Objective: This study aimed to investigate the genetic and structural variations in β-tubulin isotype-1 of Haemonchus contortus isolated from goats and cattle in Pakistan, with a focus on understanding the mechanisms underlying resistance to benzimidazole.

Methods: A total of 150 goats and 100 cattle from four abattoirs in Rawalpindi were tested for Haemonchus contortus. Faecal samples were cultured for L3 larvae, DNA was isolated, and whole genome sequencing was performed. β-tubulin isotype 1 gene sequence was extracted. Phylogenetic tree with reference genome from GenBank, and protein models were generated via ExPaSy and I-TASSER and validated with SAVESv.6.0.

Results: Six genotypes of Haemonchus contortus isotype1 β-tubulin gene, each with a length of 385 base pairs were identified. The E198A SNP was the most prevalent mutation among the larvae of Haemonchus contortus collected from cattle (62.5%) and goats (50%), followed by the F200Y which was detected in 6 (37.5%) larvae collected from cattle and 9 (37.5%) larvae from goats. SNP F167Y was not identified in any of the samples. The phylogenetic analysis showed that the isolates were closely related to H. contortus from China (KX258930) and H. contortus × H. placei hybrids from Pakistan. Six genotypes of β-tubulin isotype 1 gene were translated into three haplotypes based on protein sequences: wild type/susceptible, E198A-resistant/mutant, and F200Y-resistant/mutant. Structural analysis revealed high model quality and confidence, with the E198A and F200Y resistant/mutants exhibiting different characteristics, with refined quality factors exceeding 91%.

Conclusions: The study demonstrated alterations in tubulin structure and function are crucial for understanding anthelmintic resistance patterns. These findings help for improving therapeutic approaches, comprehending resistance mechanisms, and promoting sustainable parasite control.