Molecular analysis of selection for benzimidazole resistance in the sheep parasite Haemonchus contortus (original) (raw)
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Veterinary Parasitology, 2003
Three anthelmintic classes with distinct mechanisms of action are commercially available. Selection of nematode populations resistant to all these drugs has occurred, particularly in trichostrongyloid parasites of sheep. Anthelmintic resistance in cattle parasites has only recently been recognized and appears to be less pronounced, even though very similar species infect both hosts. To understand the bases for differences in the rate of resistance development in sheep versus cattle parasites, it is important to first demonstrate that the same kinds of resistance alleles exist in both. The benzimidazoles (BZ), which have been used for more than 40 years, were chosen as an example. BZ-sensitive (BZ S ) and BZ-resistant (BZ R ) nematodes that parasitize sheep have been distinguished at the molecular level by a single nucleotide change in the codon for amino acid 200 of a -tubulin gene, a switch from TT C (phenylalanine) to TĀ C (tyrosine). PCR primers were designed to completely conserved regions of trichostrongyloid -tubulin genes and were used to amplify DNA fragments from Haemonchus contortus (cDNA from a BZ S and a BZ R library) as positive controls. The technique was then extended to the cattle parasites, Cooperia oncophora and Ostertagia ostertagi (from genomic DNA). Sequence analysis proved the presence of amplified BZ S alleles in all three species and BZ R alleles in the BZ R population of H. contortus. Based on these data, nested PCR primers using the diagnostic T or Ā as the most 3 nucleotide were designed for each species. Conditions for selective PCR were determined. To demonstrate feasibility, genomic DNA was recovered from individual H. contortus L 3 larvae from both BZ S and BZ R populations. Genomic DNA was also isolated from >70 individual adult male C. oncophora collected from a cattle farm in New Zealand with reported BZ resistance. Allele-specific PCR discriminated among heterozygotes and homozygotes in both species. This method could find utility in studying the molecular epidemiology of BZ resistance in cattle parasites and for defining the variables that limit the development and spread of anthelmintic resistance in this host.
Small Ruminant Research, 2017
Haemonchus contortus is the most prevalent parasitic nematode in tropical areas, and anthelmintic resistance is a global problem. Our objective was to characterize benzimidazole (BZ) resistance in gastrointestinal nematode populations in Ceará State, Brazil, using the egg hatch test (EHT) and in H. contortus populations using quantitative real-time polymerase chain reaction (qPCR). Twenty locations were surveyed, and fecal samples were collected from a minimum of 40 animals from each farm and pooled. Five thousand L3 from each farm were used to infect single animals at Embrapa (Brazilian Agricultural Research Company) to provide a source of eggs for both phenotypical and molecular tests. The mean EHT was 2.46 g/ml (±0.58 g/ml), and BZ resistance was detected at all surveyed locations. The mean resistant allelic frequencies at positions F200Y and F167Y were 34.16% (±12.13%) and 58.31% (±18.89%), respectively. The resistant allelic frequencies at F167Y were higher than those at F200Y in most studied locations. We also investigated the possibility that specific BZ utilization may influence resistant allelic frequencies. We selected three nematode populations based on the resistant SNP prevalence at F200Y and F167Y as follows: higher frequency at SNP F200Y, higher frequency at SNP F167Y and similar frequencies at both positions. Anthelmintic treatments included two BZs (oxfendazole and albendazole) and ivermectin. Three animals per population per treatment were infected with 5000 L3, and nematode eggs were collected for molecular test before and after anthelmintic treatments. The results showed preferential selection of SNP F167Y in response to oxfendazole, an increase in resistant SNP frequencies in general in response to albendazole and little change in relation to pre-treatment situations in response to ivermectin. Our results confirm that BZ resistance is common. The resistant allele at SNP F167Y in H. contortus prevails in Ceará State, and we provide evidence that this result may be due to the utilization of oxfendazole in recent years.
Benzimidazole is a synthetic anthelmintic against which nematode resistance especially in Haemonchus contortus, is emerging at a alarming speed. The mechanism of benzimidazole resistance appears to involve mutations in the gene encoding β-tubulin isotype 1 (β-tubulin-1). The present study was carried out to find out the variation existing in β-tubulin-1 which is directly involved with drug binding capacity involving microtubules polymerization. DNA of adult nematode H. contortus was extracted, amplified and sequenced. Out of 50 worms investigated, 37 showed benzimidazole susceptible gene while 13 were resistant indicating single nucleotide mutation at amino acid 200 TTC/TAC. In addition, 12 worms showed several regions of consistent difference indicating single nucleotide polymorphism (SNPs) at various positions in coding region. It has been concluded that resistant alleles conferring anthelmintic resistance is prevalent in the local population of H. contortus of northeast Punjab, Pakistan.
Background: Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer), to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubu-lin gene of Haemonchus contortus. Methods: There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleotide polymorphism (SNP) at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer), in which the nucleotide T at the position 443 was substituted through a nucleotide A creating a restriction site for restriction endonuc-lease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp) was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achieving a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respectively. Results: All worms had two alleles encoding for phenylalanine (BZ ss homozygote) for both codons. Conclusion: Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.
Molecular and Biochemical Parasitology, 2009
Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drugresistant parasites that carry point mutations in -tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the -tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate 198 to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine 198 encoding alleles in the -tubulin isotype 1 gene pool of H. contortus in the field.
Journal of Veterinary parasitology, 2020
A study was undertaken to determine the polymorphism at codon 200 of β-tubulin isotype 1 gene associated with benzimidazole (BZ) resistance, by PCR-RFLP (Polymerase chain reaction linked restriction fragment length polymorphism) in Haemonchus contortus from sheep across eight districts of Andhra Pradesh. RFLP using Taa I revealed three distinct patterns named as ‘rr’ (homozygous resistant), ‘rS’ (heterozygous susceptible) and ‘SS’ (homozygous susceptible). The overall genotype frequencies for SS, rS and rr were 0.26, 0.44 and 0.30, respectively. The allelic frequencies of ‘r’ and ‘S’ were 0.52 and 0.48, respectively. The prevalence of benzimidazole resistance allele (r) was significantly (P<0.001) higher than susceptible allele in the study area indicating inconsistency with Hardy-Weinberg equilibrium (HWE). The expected heterozygosity (He) and observed heterozygosity (Ho) estimates were 0.50 and 0.44, respectively. The effective population size (Ne), polymorphic information content (PIC) and fixation index (FIS) were 1.99, 0.37 and 0.13, respectively. The analysis revealed significant heterozygote deficiency. The study revealed that BZ resistance could be a future threat in the area as high frequency of heterozygous worms occur in different districts of Andhra Pradesh.
Molecular and Biochemical Parasitology, 2007
Human hookworms (Ancylostoma duodenale, Necator americanus) are a major cause of malnutrition and anemia, particularly in children, and high worm burdens can lead to stunted growth and mental retardation. Mass drug administration (MDA) with benzimidazole (BZ) anthelmintics has the potential to greatly reduce morbidity and infection prevalence. However, such treatment strategies may apply significant selection pressure on resistance alleles. In several Strongylid parasites of livestock, resistance to BZ drugs is associated with single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codons 167 and 200. As an initial investigation into the possible development of BZ resistance in hookworms, we have cloned and sequenced the beta-tubulin isotype-1 genes of the canine hookworm Ancylostoma caninum and the two human hookworm species A. duodenale and N. americanus. The genomic sequences are highly conserved as evidenced by a similar structure of exons and introns; the 10 exons are of the same length in all three species and code for the same amino acids. The genomic sequences were then used to develop a real-time PCR assay for detecting polymorphisms in codons 167 and 200 in all three species. Hookworm specimens previously obtained from Pemba Island school children who had demonstrated a reduced response to treatment with mebendazole were then examined using the real-time PCR assay. None of the samples revealed significant levels of polymorphisms at these loci. If BZ resistance is present in the hookworm populations examined, the results do not support the hypothesis that changes in codons 167 and 200 of beta-tubulin isotype-1 are responsible for any resistance. (J.M. Schwenkenbecher).
Beta-tubulin and benzimidazole resistance in the sheep nematode Haemonchus contortus
Molecular and Biochemical Parasitology, 1991
We have compared benzimidazole (BZ) susceptible (s) and resistant (R) strains ofHaemonchus contortus from sheep by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), Western blotting and ELISA techniques. The s strain bound more drug per mg protein than the R strain. BZ binding could be resolved into high-affinity and low-affinity binding. Low-affinity binding in parasite preparations devoid of tubulin was observed, but high-affinity binding occurred only in preparations containing tubulin. Resistance was associated with a decrease in the high affinity component. The s and R strains were shown by ELISA to contain similar total amounts of tubulin. By 2-D PAGE, the fl-tubulin isoform pattern of the s strain was different from that of the R strain, but the ottubulin isoform patterns of the 2 strains were similar. BZ resistance was associated with a decrease in high-affinity BZ binding to tubulin and an alteration in fl-tubulin isoform pattern.