Development of PCR-RFLP for the detection of benzimidazole resistance polymorphisms in isotype 1 β-tubulin gene of Trichostrongylus colubriformis

Abstract

Trichostrongylus spp. is a predominant gastrointestinal strongyle in small ruminants and the development of benzimidazole resistance in this species can significantly contribute to the overall resistance status in farms. The objective of the present study was to develop PCR-RFLP for genotyping Trichostrongylus colubriformis larvae for benzimidazole resistance polymorphisms at codons 167, 198 and 200 in the isotype 1 β-tubulin gene. A 246 bp region of the isotype 1 β-tubulin gene containing the three codons was amplified by semi-nested PCR. A modified reverse primer was used in the semi-nested PCR for substituting the third nucleotide downstream of codon 200 (C) with nucleotide T to create a restriction site for identifying the polymorphism at codon 200. Restriction endonuclease mapping of the 246 bp product was done and the restriction enzymes HpyCH4III, HpyCH4V and RsaI were selected for the identification of SNPs at codons 200, 198 and 167, respectively. RFLP with HpyCH4III yielded 147 and 99 bp fragments in the susceptible genotype (SS) and 147, 67 and 32 bp fragments in the resistant genotype (rr) at codon 200. HpyCH4V was expected to cut the 246 bp semi-nested PCR product into 205 and 41 bp fragments in the resistant genotype at codon 198, while the susceptible genotype yielded an uncut 246 bp product. RFLP with RsaI was expected to give two fragments of sizes 199 and 47 bp in the susceptible genotype and three fragments of sizes 199, 25 and 22 bp in the resistant genotype. A total of 150 T. colubriformis larvae were genotyped at the three codons. Benzimidazole resistance-associated SNPs were found at codon 200 while no polymorphism was identified at codons 167 and 198. Twenty eight per cent of the larvae genotyped were found to be homozygous resistant (rr, TAC/TAC), 60.67 per cent homozygous susceptible (SS, TTC/TTC) and 11.33 per cent heterozygous (rS, TTC/TAC) at codon 200 of isotype 1 β-tubulin gene. The overall frequency of the resistant allele (r, TAC) was 0.337 and that of the susceptible allele (S, TTC) was 0.663. The present study forms the first report of PCR-RFLP for genotyping T. colubriformis in goats at the three benzimidazole resistance-associated codons.