Dataset Information

Molecular characterization of ?-tubulin gene associated with benzimidazole resistance in larvae of field isolates of Parascaris (Nematoda: Ascarididae).

ABSTRACT: Since the past 2 decades, an increasing number of resistance to the Benzimidazoles (BZs) have been reported in nematode parasites of livestock. More recently, detection of single-nucleotide polymorphisms (SNPs) at codons of 167, 198 or 200 of the ?-tubulin gene has been attributed to the occurrence of resistance. In the present study, we investigated the presence of those SNPs in the ?-tubulin isotype-1 gene in different isolates of Parascaris in horse. Also, the mitochondrial (mt) and ribosomal genes were sequenced for species confirmation of the isolates. The analysis of sequences inferred from COII gene confirmed that those isolates were P. equorum. The distance between mt genes obtained here and several ascarid species in equids and other hosts suggests the need for the combination of more genetic data with morphologic and other diagnostic measures. The analysis on ?-tubulin isotype-1 gene revealed no resistance-related SNPs or substitutions at the expected codon positions and selection pressure with BZs has not occurred for Parascaris worms. Although the molecular data showed the susceptibility of Parascaris isolates against BZs, other mechanisms of resistance should be also investigated to confirm the validity of molecular results.

SUBMITTER: Malekpour SH

PROVIDER: S-EPMC6841862 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Molecular characterization of β-tubulin gene associated with benzimidazole resistance in larvae of field isolates of <i>Parascaris</i> (Nematoda: Ascarididae).

Malekpour Seyed Hossein SH Rakhshandehroo Ehsan E Yektaseresht Azadeh A

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20190724 4

Since the past 2 decades, an increasing number of resistance to the Benzimidazoles (BZs) have been reported in nematode parasites of livestock. More recently, detection of single-nucleotide polymorphisms (SNPs) at codons of 167, 198 or 200 of the β-tubulin gene has been attributed to the occurrence of resistance. In the present study, we investigated the presence of those SNPs in the β-tubulin isotype-1 gene in different isolates of <i>Parascaris</i> in horse. Also, the mitochondrial (mt) and ri ...[more]

PMID: 31749539

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Project description:BackgroundThe development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles now being the most widely used substance to control Parascaris infections. However, over-reliance on one drug class is a risk factor for the development of AR. Consequently, benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the β-tubulin genes. The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. This study investigated whether these SNPs exist in the P. equorum population and tested the hypothesis that BZ resistance can develop from pre-existing SNPs in codons 167, 198 and 200 of the β-tubulin isotype 1 and 2 genes, reported to be associated with AR in strongylids. The efficacy of the oral paste formula fenbendazole on 11 farms in Sweden was also assessed.MethodsTwo isotype-specific primer pairs were designed, one on either side of the codon 167 and one on either side of codons 198 and 200. A pool of 100,000 larvae was sequenced using deep amplicon sequencing by Illumina HiSeq. Faecal egg count reduction test was used to assess the efficacy of fenbendazole.ResultsNo SNPs were observed in codons 167, 198 or 200 of the β-tubulin isotype 1 or 2 genes of P. equorum, even though 100,000 larvae were sequenced. Faecal egg count reduction testing of fenbendazole showed that this anthelmintic was still 100% effective, meaning that the likelihood of finding high allele frequency of SNPs associated with benzimidazoles resistance in P. equorum was low. Unexpectedly, the allele frequencies observed in single worms were comparable to those in pooled samples.ConclusionsWe concluded that fenbendazole does not exert selection pressure on the β-tubulin genes of isotypes 1 and 2 in P. equorum. The fact that no pre-existing SNPs were found in codons 167, 198 and 200 in P. equorum also illustrates the difficulties in generalising about AR mechanisms between different taxonomic groups of nematodes.

Project description:Infections by parasitic nematodes inflict a huge burden on the health of humans and livestock throughout the world. Anthelmintic drugs are the first line of defense against these infections. Unfortunately, resistance to these drugs is rampant and continues to spread. To improve treatment strategies, we must understand the genetics and molecular mechanisms that underlie resistance. Studies of the fungus Aspergillus nidulans and the free-living nematode Caenorhabditis elegans discovered that a beta-tubulin gene is mutated in benzimidazole (BZ) resistant strains. In parasitic nematode populations, three beta-tubulin alleles, F167Y, E198A, and F200Y, have long been correlated with resistance. Additionally, improvements in sequencing technologies have identified new alleles - E198V, E198L, E198K, E198I, and E198Stop - also correlated with BZ resistance. However, none of these alleles have been proven to cause resistance. To empirically demonstrate this point, we independently introduced the F167Y, E198A, and F200Y alleles as well as two of the newly identified alleles, E198V and E198L, into the BZ susceptible C. elegans N2 genetic background using the CRISPR-Cas9 system. These genome-edited strains were exposed to both albendazole and fenbendazole to quantitatively measure animal responses to BZs. We used a range of concentrations for each BZ compound to define response curves and found that all five of the alleles conferred resistance to BZ compounds equal to a loss of the entire beta-tubulin gene. These results prove that the parasite beta-tubulin alleles cause resistance. The E198V allele is found at low frequencies along with the E198L allele in natural parasite populations, suggesting that it could affect fitness. We performed competitive fitness assays and demonstrated that the E198V allele reduces animal health, supporting the hypothesis that this allele might be less fit in field populations. Overall, we present a powerful platform to quantitatively assess anthelmintic resistance and effects of specific resistance alleles on organismal fitness in the presence or absence of the drug.

Project description:BackgroundBenzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta.MethodsThe benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L.ResultsThe percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT.ConclusionsThe E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Dataset Information

Molecular characterization of ?-tubulin gene associated with benzimidazole resistance in larvae of field isolates of Parascaris (Nematoda: Ascarididae).

Publications

Molecular characterization of β-tubulin gene associated with benzimidazole resistance in larvae of field isolates of <i>Parascaris</i> (Nematoda: Ascarididae).

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