First identification of the F200Y SNP in the β-tubulin gene linked to benzimidazole resistance in Ancylostoma caninum

Abstract Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstract

Download Full-text

Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

Parasites & Vectors ◽

10.1186/s13071-019-3828-6 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 17

Author(s):

Pablo D. Jimenez Castro ◽

Sue B. Howell ◽

John J. Schaefer ◽

Russell W. Avramenko ◽

John S. Gilleard ◽

...

Keyword(s):

Anthelmintic Resistance ◽

Multiple Drug Resistance ◽

Amplicon Sequencing ◽

Multiple Drug ◽

Tubulin Gene ◽

Ancylostoma Caninum ◽

The Usa ◽

Hookworm Infections ◽

Β Tubulin

Abstract Background The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum. Methods Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene. Results Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases. Conclusions These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.

Download Full-text

Teladorsagia Circumcincta Beta Tubulin: The Presence of the E198L Polymorphism on Its Own is Associated with Benzimidazole Resistance

10.21203/rs.3.rs-28759/v1 ◽

2020 ◽

Author(s):

María Martínez Valladares ◽

Elora Valderas-García ◽

Javier Gandasegui ◽

Philip Skuce ◽

Alison Morrison ◽

...

Keyword(s):

Phenotypic Variability ◽

Linear Regression Analysis ◽

Significant Variable ◽

Multivariate Linear Regression Analysis ◽

Tubulin Gene ◽

Phenotypic Resistance ◽

Pyrosequencing Assay ◽

Teladorsagia Circumcincta ◽

Benzimidazole Resistance ◽

Β Tubulin

Abstract Background: Benzimidazole 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.Methods: The benzimidazole phenotypic resistance was measured by the Faecal Egg Count Reduction Test (FECRT) and the Egg Hatch Test (EHT) using a discriminating dose in 39 sheep flocks. Around 1,000 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. Results: The percentage of resistant flocks was 32% by FECRT or 26% by EHT, however F200Y and E198A SNPs were absent in T. circumcincta . The amplification 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 = 0.000) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4 and 80.7% before treatment, and from 82.3 to 92.8% after treatment. Very 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 = 0.000), 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 = 0.000). 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. Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta .

Download Full-text

Two benzimidazole resistance-associated SNPs in the isotype-1 β-tubulin gene predominate in Haemonchus contortus populations from eight regions in China

International Journal for Parasitology Drugs and Drug Resistance ◽

10.1016/j.ijpddr.2016.10.001 ◽

2016 ◽

Vol 6 (3) ◽

pp. 199-206 ◽

Cited By ~ 16

Author(s):

Zongze Zhang ◽

Robin B. Gasser ◽

Xin Yang ◽

Fangyuan Yin ◽

Guanghui Zhao ◽

...

Keyword(s):

Haemonchus Contortus ◽

Tubulin Gene ◽

Benzimidazole Resistance ◽

Β Tubulin

Download Full-text

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

Parasites & Vectors ◽

10.1186/s13071-020-04320-x ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

María Martínez-Valladares ◽

Elora Valderas-García ◽

Javier Gandasegui ◽

Philip Skuce ◽

Alison Morrison ◽

...

Keyword(s):

Phenotypic Variability ◽

Linear Regression Analysis ◽

Significant Variable ◽

Multivariate Linear Regression Analysis ◽

Tubulin Gene ◽

Phenotypic Resistance ◽

Pyrosequencing Assay ◽

Teladorsagia Circumcincta ◽

Benzimidazole Resistance ◽

Β Tubulin

Abstract Background Benzimidazole 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. Methods The 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. Results The 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. Conclusions The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Download Full-text