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
© The Author(s). 2016
Received: 30 November 2015
Accepted: 2 June 2016
Published: 17 June 2016
The 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.
Adult 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.
The 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.
The 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.
KeywordsHaemonchus contortus Helminth β-tubulin Anthelmintic Drug resistance Benzimidazole Albendazole Soft selective sweep Genetic diversity
Anthelmintic resistance in parasitic nematodes is a global threat to sustainable livestock production . However, we still have a limited understanding of how drug selection affects parasite populations and how resistance emerges at the population level. Haemonchus contortus is one of the most economically important helminth parasites of small ruminants worldwide and is an important model for anthelmintic resistance research . This parasite has developed resistance to all the major anthelmintic drug classes and resistance to multiple drug classes occurs, often at high frequency, in many parts of the world [3, 4].
Benzimidazole drugs have been used to control livestock parasites for over 40 years and resistance to this drug class is at an advanced stage in many parts of the world [5, 6]. We have sufficient knowledge of the molecular basis of resistance for this drug class in order to study the emergence and spread of anthelmintic resistance mutations in field populations. Three single nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene have been associated with benzimidazole resistance in H. contortus. The F200Y (TTC to TAC) mutation has been found in every country examined to date and is often present at high frequency [5–9]. We have recently presented genetic evidence that this mutation independently arises multiple times in a geographical region and spreads between sites by migration [5, 10]. The SNPs at codons F167Y (TTC to TAC) and E198A (GAA to GCA) have also been reported in multiple countries but have a more variable occurrence and are generally present at lower frequency than the F200Y (TTC to TAC) mutation [5–7, 11]. We have recently provided genetic evidence to show that E198A (GCA) mutation present in multiple populations in southern India was derived from a single origin .
We are investigating the population genetics of benzimidazole resistance in H. contortus in a variety of different countries to provide insights into its origin and spread [5, 6, 10, 12]. Benzimidazole resistance is at an advanced stage in most countries that we, and others, have examined to date due to the widespread use of this drug class over many years. In places such as the United Kingdom (UK), western Europe, Australia and North America most H. contortus populations have moderate to high levels of benzimidazole resistance [5–7]. In these countries, most parasite populations have at least one of the resistance-associated mutations at high frequency. We have been seeking locations where we can investigate benzimidazole resistance at an earlier stage of its emergence and Pakistan is a potentially interesting country in this respect. Economic constraints mean that most farmers do not use specific anthelmintic treatment regimes, anthelmintics are often diluted before administration and generic drugs of unknown quality are often used. Consequently, we anticipated that benzimidazole resistance might be at a relatively early stage of emergence in animals from small rural farms or owned for subsistence purposes. In contrast, government farms tend to use frequent treatments with benzimidazole drugs providing a potentially interesting contrast in the same geographical region. There are only a few published studies on benzimidazole resistance in Pakistan but there is information to suggest resistance may not yet be widespread [13, 14]. Fecal egg count reduction tests performed on Beetal goats on eighteen farms in the Faisalabad district of the Punjab showed oxfendazole efficacies of between 80–100 % (over 95 % fecal egg count reduction on eleven on the farms) . The sequencing of an isotype-1 β-tubulin gene fragment from 95 adult H.contortus worms collected from abattoirs in the Punjab and North West Frontier provinces revealed just a 7.86 % frequency of the F200Y (TAC) mutation .
In the present paper, we show that the F200Y (TAC) benzimidazole resistance mutation is present at very low frequencies in eighteen H. contortus populations sourced from sheep and goats from rural locations in the Punjab Province of Pakistan. In contrast, this mutation is present at very high frequencies in three government farms that have used frequent and regular anthelmintic treatments for over 30 years. In spite of this difference in drug selection, population genetic analysis, using a panel of microsatellite markers, revealed no significant difference in the overall genetic diversity between the government and pastoral herds. In addition, phylogenetic analysis of isotype-1 β-tubulin haplotypes reveals multiple F200Y(TAC) haplotypes are present in H. contortus on the closed government herds suggesting the presence of soft selective sweeps in the absence of significant contemporary parasite migration.
The Layyah government farm (pop2G) was established in 1980 from angora goats sourced from Turkey which were crossed with locally sourced goats of the “hairy breed”. This herd has been treated with anthelmintics (levamisole HCL and oxfendazole) approximately every 3 months since its establishment and has been closed to animal movement since that time. The Okara (Pop1S) and Jahangirabad (Pop3S) government farms were established using local sheep breeds (lohi and kajli) in 1985 and 1989 respectively. Both these herds have been treated alternately with albendazole and oxfendazole approximately every 3 months since their establishment. There has been some historical movement of sheep between the Okara and Jahangirabad farms but these herds have otherwise been closed to animal movement since they were established.
Genomic DNA isolation and species confirmation
Adult worms were fixed in 70 % ethanol immediately following removal from the host abomasum. The heads of individual worms were dissected and lysed in single 0.2 μl tubes containing 50 μl of proteinase K lysis buffer and stored at -80 °C as previously described [5, 15]. One microliter of 1:5 dilution of neat single worm lysate was used as PCR template and identical dilutions of lysate buffer, made in parallel, were used as negative controls. To prepare pooled lysates of each population, 1 μl aliquots of each individual neat adult worm head lysate were pooled. One microliter of a 1:20 dilution of pooled lysate was used as template for subsequent PCRs.
A combination of gross morphological identification and genotyping of the SNP at position 24 of the ITS-2 rDNA was used to confirm the identity of H. contortus in these populations as previously reported . A minimum of 14 and a maximum of 32 individual adult H. contortus worms were identified per population (560 H. contortus worms in total). Individual H. contortus DNA lysates were pooled for each population for isotype-1 β-tubulin genotyping.
Pyrosequence genotyping to determine the relative frequencies of the isotype-1 β-tubulin F200Y(TAC), E198A(GCA) and F167Y(TAC) benzimidazole resistance-associated SNPs in H. contortus populations
A 328 bp fragment, spanning exons 4 and 5 and intervening intron of the isotype-1 β-tubulin gene was PCR amplified from pooled DNA lysates for each of the 21 H. contortus populations (Additional file 1: Table S1). Final PCR conditions were 1X thermopol reaction buffer, 2 mM MgSO4, 100 μM dNTPs, 0.1 μM forward and reverse primers and 1.25U Taq DNA polymerase (New England Biolabs, Ipswich,USA) and a previously published primer pair was used; forward primer (HcPYRF: 5′-GAC GCA TTC ACT TGG AGG AG-3′) and reverse primer (HcPYRR: 5′-Biotin-CAT AGG TTG GAT TTG TGA GTT-3′) . The thermo-cycling parameters consisted of an initial 95 °C for 5 min followed by 35 cycles of 95 °C for 1 min, 53 °C for 1 min and 72 °C for 1 min with a single final extension cycle of 72 °C for 5 min.
Following PCR amplification, the relative frequency of the F167Y (TAC), E198A (GCA) and F200Y (TAC) isotype-1 β-tubulin SNPs in the amplicons derived from each parasite population were determined by pyrosequence genotyping using the PryoMark ID system using the allele quantification (AQ) mode (Biotage, Uppsala, Sweden). Previously published sequencing primers were used for the F167Y (TAC), E198A (GCA) and F200Y (TAC) mutations; Hcsq167: 5′-ATA GAA TTA TGG CTT CGT-3′, Hcsq198: 5′-ACT GGT AGA GAA CAC CG-3′ and Hcsq200: 5′- TAG AGA ACA CCG ATG AAA CAT-3′, respectively [8, 10, 16]. The base dispensation was set to GATGCTCGT for codon 167, GATGCAGCA for codon 198, and GATGCTGTA for codon 200. Peak heights were measured using the AQ mode of the PSQ 96 single nucleotide position software as previously described .
Cloning and sequencing of an isotype-1 β-tubulin fragment encompassing the F200Y (TAC), E198A (GCA) and F167Y (TAC) mutations
The same 328 bp isotype-1 β-tubulin fragment described above was also amplified, cloned and sequenced from the pooled lysates from the seven H. contortus populations in which the F200Y (TAC) was detected (Pop24S, Pop1S, Pop27G, Pop2G, Pop3S, Pop5G and Pop13S). Primers HcPYRF: 5′-GAC GCA TTC ACT TGG AGG AG-3′ and HcPYRR: 5′-CAT AGG TTG GAT TTGTGA GTT-3′ were again used but with the following PCR reaction conditions; 5X Phusion HF reaction buffer, 2 mM MgSO4, 200uM dNTPs, 0.2uM forward and reverse primers and 1U of Phusion high fidelity DNA polymerase (Finenzyme inc., Keilaranta, Finland). The thermo-cycling parameters of isotype-1 β-tubulin consisted of an initial 98 °C for 30 s followed by 35 cycles of 98 °C for 10 s, 59 °C for 30 s and 72 °C for 1 min with a single final extension cycle of 72 °C for 5 min. Amplicons were cloned into PJET 1.2/BLUNT vector (Thermo Scientific, Waltham, USA) and sequenced using previously described standard procedures . Sequences were aligned with the H. contortus isotype-1 β-tubulin sequence (Acc. No. X67489) and edited using Geneious Pro 5.4 software . A previously described approach was used to bioinformatically filter the isotype-1 β-tubulin sequences to remove SNPs occurring only once in the dataset and ensure PCR-induced mutations were not included in the analysis [5, 10].
Phylogenetic network analysis of H. contortus isotype-1 β-tubulin sequences
Networks based on genetic distance were computed using the neighbour-net method employed in SplitsTree4  to produce circular (equal angle) split networks. Median joining networks were generated in Network 4.6.1 software (Fluxus Technology Ltd., Clare, England). A full median network containing all possible shortest trees was generated by setting the epsilon parameter equal to the greatest weighted distance (epsilon = 10). All unnecessary median vectors and links were removed with the MP (Maximum Parsimony) option . A phylogenetic network tree of the isotype-1 β-tubulin haplotypes was reconstructed using maximum likelihood (ML) in MEGA5 . The program jModeltest 12.2.0  was used to select the appropriate model of nucleotide substitutions for ML analysis. According to Bayesian information criterion the best scoring was Hasegawa-Kishino-Yano (HKY + G). The model of substitution was used with parameters estimated from data. Branch supports were obtained by 1,000 bootstraps of the data. The most probable ancestral node was determined by rooting the networks to a closely related outgroup and Haemonchus placei sequence was used to root the H. contortus network.
Microsatellite genotyping and population genetic analysis
Previously described H. contortus microsatellites were screened to produce a panel of eight markers that consistently amplified from the Pakistan populations under study (data not shown) [5, 22, 23]. A summary of primer sequences and allele ranges are given in Additional file 2: Table S2 and the PCR conditions for each marker were as previously described . The forward primer of each microsatellite primer pair was 5′ end labelled with fluorescent dye (IDT, Canada) and the GeneScan ROX 400 internal size standard was used on the ABI Prism 3100 genetic analyser (Applied Biosystems, Carlesbad, California, USA). Individual chromatograms were analysed using Gene Mapper software version 4.0 to accurately size the amplicons and determine genotypes (Applied Biosystems, USA).
Fixation index (pairwise FST) values were calculated from the multi-locus microsatellite genotype data, by random permutation in Arlequin 3.11 . Exact test was used to statistically evaluate deviations from Hardy-Weinberg equilibrium for all populations . Significance levels were adjusted using the sequential method of Bonferroni for multiple comparisons in the same dataset . There was a significant departure from Hardy-Weinberg equilibrium, even after Bonferroni correction, for 10 out of the 56 loci combinations for H. contortus (Additional file 3: Table S3). The presence of null alleles for microsatellite loci has been previously reported and is the likely reason for these departures from Hardy-Weinberg equilibrium [22, 27–29]. Analysis of molecular variance (AMOVA) was estimated through partition of microsatellite diversity between and within populations . Log-likelihood ratio test statistic (G-test), used to estimate the linkage disequilibrium analysis, was carried out by Arlequin 3.11 . Expected and observed heterozygosity (He and Ho), allele richness and estimation of inbreeding coefficient (FIS) for each locus were calculated using Arlequin 3.1 (Additional file 3: Table S3) . As the number of alleles and expected heterozygosity are dependent on sample size, in order to compare populations, expected heterozygosity (Hexpc) and allele richness (Ac) were also calculated per locus for each population with correction for unequal sample size using POPTREE2  and MSA 4.05 , respectively. Differences in these measures of genetic diversity between populations were tested for statistical significance using a Wilcoxon rank sum test .
The F200Y (TAC) isotype-1 β-tubulin mutation was found at high frequency in H. contortus populations on government farms but only rarely from animals from pastoral locations
A 328 bp fragment of the isotype-1 β-tubulin gene was PCR amplified from 21 H. contortus populations (3 populations from government farms and 18 populations from pastoral locations) and the relative frequencies of the three currently known benzimidazole resistance-associated SNPs F167Y (TAC), E198A (GCA) and F200Y (TAC) was determined by pyrosequence genotyping (using pools of 14 to 32 worms per population; see Additional file 1: Table S1). The benzimidazole resistance-associated F200Y (TAC) SNP was found at high frequency (81–100 %) in all three parasite populations from government farms. In contrast, it was not detected in 14 out of the 18 pastoral populations and only detected at low frequency (2–6 %) in the remaining four (Fig. 1 and Additional file 1: Table S1). The F167Y (TAC) and E198A (GCA) SNPs were not detected in any of the populations.
Genetic diversity and population structure of H. contortus populations
Pairwise FST values based on genotyping 18 individual worms from each of seven H. contortus populations with eight microsatellite markers
Sequence diversity and phylogenetic relationships of H. contortus isotype-1 β-tubulin haplotypes from government farms and pastoral locations
The 328 bp isotype-1 β-tubulin fragment was PCR amplified, cloned and sequenced from pooled DNA from each of the seven parasite populations in which the F200Y (TAC) resistance mutation was detected; three populations from government farms (Pop1S, Pop2G and Pop3S) and four populations from rural locations (Pop24S, Pop27G, Pop5G and Pop13S). Following bioinformatic filtering (see Methods), a total of 21 different isotype-1 β-tubulin haplotypes (GenBank accession numbers KR269920–KR269940) were identified in the 89 sequences generated from the seven populations. Six out of the twenty one haplotypes encoded the F200Y(TAC) resistance polymorphism. Summary statistics of the isotype-1 β-tubulin fragment diversity are shown in Additional file 4: Table S4.
Geographical distribution of F200Y (TAC) isotype-1 β-tubulin benzimidazole resistance haplotypes
The three parasite populations from government farms (Pop1S, Pop3S and Pop2G) and one of the parasite populations from a pastoral location (Pop27G) each contained multiple resistance haplotypes. The three remaining parasite populations from rural locations (Pop24S, Pop5G, and Pop13S) contained just a single resistance haplotype (Additional file 6: Figure S1). All four parasite populations from rural locations (Pop24S, Pop27S, Pop5S and Pop13S) showed a high degree of susceptible haplotype diversity whereas the three parasite populations from government farms (Pop1S, Pop3S and Pop2G) did not (Additional file 6: Figure S1).
In most regions of the world examined to date, benzimidazole resistance is at an advanced stage in H. contortus with at least one of the known resistance mutations being present at a high frequency in most populations [5–7]. It is difficult to disentangle the population genetics of resistance and draw conclusions about its origins and spread in such regions. In part, this is because of the difficulty of finding natural field populations that have been subject to minimal drug selection. The Punjab region of Pakistan provides an interesting study region in this respect. Small ruminants in pastoral herds in the rural regions of Punjab, Pakistan are rarely treated with effective anthelmintic drugs due to economic and logistical constraints. In contrast, small ruminants on government farms are treated very frequently. This provides the interesting situation in which parasite populations that have been subject to intense drug selection pressure over many years are in close geographic proximity to parasite populations that have been subject to little or no drug selection pressure. We identified three government herds which were founded from locally derived breeding stock over 30 years ago and have been subsequently closed to animal movement. In the case of the Okara (Pop1S) and Jahangirabad (Pop3S) herds, they were derived exclusively from local breeding stock and, although there has been some limited movement of stock between these two farms, they have been otherwise closed to animal movement since their foundation. Further, the sheep on these farms have been treated with benzimidazole drugs approximately every three months over the last 30 years with no history of any other drug class being used. The third government farm in Layyah (Pop2G) was originally established from locally derived goats crossed with angora goats imported from Turkey and has also been closed to animal movement since its foundation. We have compared these government farms with those the surrounding pastoral herds in order to investigate the effects of benzimidazole selection on the genetic diversity of H. contortus populations.
As expected from the known patterns of drug use, we found that the F200Y (TAC) resistance mutation was present at high frequencies on all three government farms but was absent, or present a very low frequency, in all the pastoral parasite populations sampled. This study thus provides further clear evidence associating the F200Y (TAC) mutation with benzimidazole resistance in H. contortus; the only three farms out of the 21 examined which had a history of intensive benzimidazole use are also the only ones with a high frequency of this mutation. It is noteworthy that the F167Y (TAC) and E198A (GCA) mutations were not detected in any of the H. contortus populations, including the three government farms subject to intense benzimidazole selection pressure over many years. This is consistent with previous studies from different regions the world that have shown the F200Y (TAC) mutation to be essentially ubiquitous in drug selected populations whereas the other two mutations occur more sporadically. For example, in our recent work in southern India the F167Y (TAC) mutation was apparently absent and the E198A (GCA) mutation, although present at multiple locations, appears to have been derived from a single origin in the region . In the study reported here, the F200Y (TAC) mutation was present on multiple haplotypes on each of the government farms; Hr12/Hr22/Hr23 for Layyah (Pop2G), Hr22/Hr31/Hr23 for Jahangirabad (Pop3S) and HR22/Hr31 for Okara (Pop1S). These resistance haplotypes are phylogenetically distant to each other in the haplotype network, being more closely related to one or more susceptible haplotypes than to any of the other resistant haplotypes. This suggests that they were derived from independent origins with the possible exception of haplotypes Hr22 and Hr31 since these are more closely related (Fig. 4). Overall, these new data from Pakistan provide further support for the hypothesis that F200Y (TAC) mutations occur commonly and repeatedly whereas the F167Y (TAC) and E198A (GCA) mutations are much rarer, possibly due to differences in fitness costs between the different mutations.
The occurrence of the F200Y (TAC) mutations on multiple divergent haplotypes on each government farm suggests that a soft selective sweep has occurred at the isotype-1 β-tubulin locus in each population (Fig. 4 and Additional file 6: Figure S1). Previous work has demonstrated that soft selective sweeps can occur at the isotype-1 β-tubulin locus in both H. contortus and Teladorsagia circumcincta [5, 10]. However, these previous studies in the UK and India, were in situations where animal movement was high. Consequently, in those cases the resistance haplotype diversity found at each location could have been due, at least in part, to the migration of resistance haplotypes between farms [5, 10]. However, the government farms in this study have been closed to animal movement for over 30 years and so the soft selective sweeps must have occurred in the absence of significant contemporary migration of resistance alleles between farms.
A variety of studies suggest that laboratory strains of H. contortus generally retain high levels of genetic diversity, even if subject to drug selection . However, there is little information on the impact of anthelmintic drug selection on the overall genetic diversity of parasite populations in the field . A comparison of parasite populations on closed government farms in the Punjab, Pakistan, which have a history of frequent benzimidazole drug treatment, with those from neighbouring pastoral herds, that have been subject to little or no drug treatment, provides a valuable opportunity to explore this question. We used a panel of eight previously characterised microsatellite markers to compare the genetic diversity of H. contortus populations from three government farms with those from four pastoral herds. There was a high level of allelic polymorphism for all of the microsatellite markers in all seven populations consistent with the high level of genetic diversity expected for this parasite [5, 29, 35]. The parasite populations on the three government farms had a similarly high level of genetic diversity as those from the pastoral herds (Fig. 2). Indeed, there was no statistically significant difference in the overall level of genetic diversity, as measured by microsatellite marker polymorphism, between any of the populations. These results suggest that frequent benzimidazole use over the last 30 years on the three government farms has not resulted in an overall reduction in genetic diversity of the parasite populations despite the loss of virtually all susceptible isotype-1 β- tubulin haplotypes on these three farms. There was also very low population sub-structuring among the seven parasite populations with the pairwise microsatellite Fst values being non-significant between the government farms (Table 1 and Additional file 3: Table S3). This suggests there has been little population bottlenecking or genetic drift, even for those parasite populations under intensive drug selection and closed to contemporary gene flow. This is likely due to the very large effective population sizes of this parasite species maintaining high genetic diversity . One important implication of these results is that genetic changes associated with drug selection, such as loss of polymorphism and linkage disequilibrium, are likely to be largely confined to the regions of the genome surrounding the loci under selection. Although further work, using larger numbers of genome-wide markers, is needed to confirm this hypothesis, it bodes well for the use of genome-wide scans to identify genetic loci underlying anthelmintic drug resistance for this and related parasites.
The F200Y(TAC) benzimidazole resistance mutation was present at very high frequencies in H. contortus populations from three government farms, but not from eighteen pastoral herds, in the Punjab region of Pakistan, consistent with known differences in drug treatment history. Multiple F200Y(TAC) haplotypes were present in H. contortus populations from each of the “closed” government herds suggesting soft selective sweeps have occurred even in the absence of significant contemporary parasite migration. The selection for a high frequency of benzimidazole resistance alleles on government farms did not lead to a loss in overall genetic diversity, based on microsatellite marker genotyping. This observation has a number of important implications. Firstly, the retention of high genetic diversity in drug resistance parasite populations suggests they are likely to retain adaptive capacity to other selective pressures including treatment with other drug classes. Secondly, genetic changes associated with drug selection, such as loss of polymorphism and linkage disequilibrium, may be largely confined to the regions of the genome surrounding the loci under selection.
We are grateful to the Vice Chancellor (Prof. Dr. Talat Naseer Pasha) of the University of Veterinary and Animal Science Lahore Pakistan for his support in supplying parasite material.
We would like to thank Natural Sciences and Engineering Research Council of Canada (NSERC) (Grant number RGPIN/371529-2209), NSERC-CREATE Host Pathogen Interactions (HPI) graduate training program at the University of Calgary as well as the BBSRC for funding support (Grant number BB/E018505/1).
Availability of data and materials
All sequence data are publicly available: GenBank accession numbers KR269920–KR269940.
Dr JG directed the project, coordinated and managed the study and co-wrote the manuscript. Dr UNC performed the laboratory work, analysed the data and co-wrote the manuscript. Dr ER assisted with the microsatellite genotyping and the data analysis. Field samples (adult worms) were collected from the abomasum of ruminants from different abattoirs by Dr KA, Dr SA, Dr MIR and Dr MZS (University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan). All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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