Genotyping of benzimidazole resistant and susceptible isolates of Haemonchus contortus from sheep by allele specific PCR

Abstract

Extensive and indiscriminate use of the benzimidazole class of drugs has led to the onset of anthelmintic resistance. In tropical countries like India, Haemonchus contortus is the most pathogenic parasite infecting sheep and goats. The widespread presence of resistant helminths (especially H. contortus) threatens the livestock farming. The use of various drugs has led to single nucleotide polymorphism that causes specific amino acid substitutions in b-tubulin protein of H. contortus to confer resistance. This emphasizes the need for a survey on the present status of resistance in India. In this study, allele specific PCR was employed to screen the presence of a SNP, a thymine-toadenine transversion which leads to substitution of amino acid in codon 200 of b-tubulin gene that is correlated specifically with BZ resistance. Third stage larvae (L3) from pooled faecal cultures of four organized sheep farms served as a source of genomic DNA for identification of H. contortus and further genotype analysis. A total of 1000 larvae was screened, out of which 673 larvae were identified as H. contortus. Among 673 H. contortus larvae, 539 larvae (80 %) were genotyped as homozygous resistant (rr) and remaining 134 (20 %) were heterozygous susceptible (Sr) by allele specific PCR. The concluded resistance status reasons out the failure of anthelmintic drug in treating ruminants. Immediate steps are needed to avoid further aggravation of the problem. Target selective treatment by reviewing the resistance status of individual drugs, appropriate use of anthelmintic drugs and other control strategies will provide a pragmatic option for delaying the further spread of anthelmintic resistance.

Structural Diversity in Beta-Tubulin Isotype 1 Protein of Benzimidazole Resistant and Susceptible Haemonchus Contortus

“…The results show that SNP at 200 is more prevalent than the other two SNPs in India. The result of the present study is in accordance with the previous reports of Mohanraj et al (2017) and other researchers, who reported mutation at codon 200 of β-tubulin by allele-specific PCR in H. contortus of Tamil Nadu and Uttar Pradesh [9,10,30]. H. contortus, Bunostomum trigonocephalum, T. circumcincta, and Trichostrongylus colubriformis β-tubulin isotype 1 of small ruminants of Uttarakhand showed a prevalence of SNP at position 200 of β-tubulin isotype 1 by allele-specific PCR [31,32,33].…”

First identification of the benzimidazole resistance-associated F200Y SNP in the beta-tubulin gene in Ascaris lumbricoides

“…These hypotheses are consistent with the results obtained previously by our group when we evaluated codons 167 and 198 of some of these same samples [11]. If these populations had been subjected to frequent treatments, probably SNPs linked to benzimidazole resistance would be more frequent, as described for other parasite populations [29,30].…”

“…The prevalence observed in our results is lower than the prevalence reported for veterinary parasites, as in H . contortus [29,30]. However, in some studies that analyzed these SNPs in ascarids of veterinary importance (i.e P .…”

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

“…Molecular diagnostic tests have been demonstrated for multiple benzimidazole (BZ) resistance SNPs in parasitic nematodes using various methodologies, including allele specific (AS)-PCR, restriction fragment length polymorphism (RFLP), and pyrosequencing ( Winterrowd et al, 2003 ; Tiwari et al, 2006 ; von Samson-Himmelstjerna et al, 2009 ; Mohanraj et al, 2017 ). A recent development in large scale surveillance is the ‘Nemabiome’ approach, which makes use of deep amplicon sequencing of barcoded PCR products ( Avramenko et al, 2015 ).…”