Microsatellite marker analysis of Haemonchus contortus populations from Pakistan suggests that frequent benzimidazole drug treatment does not result in a reduction of overall genetic diversity

Abstract

BackgroundThe impact of drug selection pressure on the overall genetic diversity of parasitic nematode populations in the field is poorly understood. In this study, we address this issue for the small ruminant parasite Haemonchus contortus in the Punjab, Pakistan. This region provides an opportunity to compare H. contortus populations that have been subjected to a prolonged period of frequent benzimidazole drug treatments on government farms with parasite populations that have been exposed to little or no drug treatment in neighbouring pastoral herds.MethodsAdult H. contortus worms were collected from the abomasa of small ruminants from three government farms frequently using benzimidazole drugs, and closed to animal movement, for over 30 years and also from from eighteen pastoral herds subject to minimal drug selection. The frequency of three known benzimidazole resistance associated mutations was determined in each parasite population. For the seven parasite populations in which resistance mutations were found, the diversity, geographical distribution and phylogenetic relationships of isotype-1 β-tubulin benzimidazole resistance haplotypes were determined. In addition, the genetic diversity of the parasite populations on the three government farms were compared with those from four pastoral herds.ResultsThe F200Y (TAC) resistance mutation was present at a very high frequency in H. contortus populations from government herds, but not from pastoral herds, consistent with their respective drug selection histories. Population genetic analysis, using a panel of microsatellite markers, revealed that there was little genetic differentiation among the parasite populations with no significant difference in the overall genetic diversity between government and pastoral herds. In addition, sequence analysis of the isotype-1 β-tubulin locus revealed multiple F200Y (TAC) haplotypes demonstrating soft selective sweeps even in government herds with little or no contemporary parasite migration.ConclusionsThe results suggest that, although the frequent drug treatment used on government farms has selected for a high frequency of benzimidazole resistance mutations, there has been little or no reduction in the overall genetic diversity of the selected parasite populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1624-0) contains supplementary material, which is available to authorized users.

Benzimidazole resistance survey for Haemonchus , Teladorsagia and Trichostrongylus in three European countries using pyrosequencing including the development of new assays for Trichostrongylus

“…However, codon 198 was not included at that time. A high prevalence of SNPs at codon 200 has been reported from Pakistan (Chaudhry et al., 2016), where no SNPs were obtained in codon F167Y and F167Y & E198A. Similarly, field studies in Canada (Barrere et al., 2013a, Barrere et al., 2013b) revealed no SNPs in codon 198 but in codon 167 some SNPs were found in Haemonchus .…”

“…Data from a recent survey in India and Pakistan confirmed the T A C SNP in codon F200Y as the most prevalent one, but in contrast to the results from previous studies in America and Europe, no mutations were found at codon F167Y (T A C) and only a small number of populations displayed the SNP in codon E198A (G C A) in India (Chaudhry et al., 2015). Microsatellite marker analysis of H. contortus populations from Pakistan suggests that frequent BZ drug treatment does not result in a reduction of overall genetic diversity (Chaudhry et al., 2016). There have been very few molecular genotyping studies performed in cattle parasitic nematodes.…”

Evidence for the F200Y (TAC) mutation conferring benzimidazole resistance in a southern USA cattle population of Haemonchus placei spreading from a single emergence

“…Adult worms were fixed in 80% ethanol immediately following removal from the host abomasum. The heads of individual worms were dissected and lysed in single 0.5ul tube containing 40 μl of proteinase K lysis buffer and stored at −80°C as previously described by Chaudhry et al (2016b). 1 μl of a 1:5 dilution of a neat single worm lysate was used as PCR template and identical dilutions of lysis buffer, made in parallel, were used as negative controls.…”

“…Six previously published microsatellites (Hcms3561, Hcms53265, Hpms43, Hpms52, Hpms53, Hpms102) were selected as a potentially useful markers based on their properties (Chaudhry et al, 2015a; Chaudhry et al, 2016b; Santos et al, 2017). These studies were produce a clear unambiguous genotypes with either a single or double Genescan peak on single worms as anticipated for single markers in both H. placei and H. contortus .…”

The role of drug efflux systems in anthelmintic resistance in parasitic nematodes

“…Although these high-throughput techniques are expensive, researchers are exploiting such techniques for their potential application in exploring the development and spread of AR worldwide (e.g. Chaudhry et al, 2016;Rashwan et al, 2016), and efforts are underway for modifying such techniques to make them cost effective.…”