F200Y polymorphism of the β-tubulin isotype 1 gene in Haemonchus contortus and sheep flock management practices related to anthelmintic resistance in eastern Amazon

AbstractDevelopment of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

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Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus

Veterinary Parasitology ◽

10.1016/j.vetpar.2011.11.068 ◽

2012 ◽

Vol 186 (3-4) ◽

pp. 344-349 ◽

Cited By ~ 52

Author(s):

Virginie Barrère ◽

Luis Alvarez ◽

Gonzalo Suarez ◽

Laura Ceballos ◽

Laura Moreno ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Haemonchus Contortus ◽

Systemic Exposure ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Tubulin Isotype ◽

Encoding Gene ◽

Β Tubulin

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In-silico comparison of inhibition of wild and drug-resistant Haemonchus contortus β-tubulin isotype-1 by glycyrrhetinic acid, thymol and albendazole interactions

Journal of Parasitic Diseases ◽

10.1007/s12639-020-01274-w ◽

2020 ◽

Author(s):

Athithan Velan ◽

Muddasarul Hoda

Keyword(s):

Haemonchus Contortus ◽

In Silico ◽

Glycyrrhetinic Acid ◽

Drug Resistant ◽

Tubulin Isotype ◽

Β Tubulin

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Variation in the β-tubulin isotype 1 gene at codon 167 and 200 responsible for benzimidazole resistance in Haemonchus contortus in sheep of Krishna District, AP, India

Veterinarski arhiv ◽

10.24099/vet.arhiv.170318a ◽

2018 ◽

Vol 88 (3) ◽

pp. 323-333

Author(s):

Kaplaywar Sathish ◽

◽

Chennuru Sreedevi ◽

Krovvidi Sudhakar ◽

Pattipati Malakondaiah ◽

...

Keyword(s):

Haemonchus Contortus ◽

Benzimidazole Resistance ◽

Tubulin Isotype ◽

Β Tubulin

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The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand

Veterinary World ◽

10.14202/vetworld.2021.764-768 ◽

2021 ◽

Vol 14 (3) ◽

pp. 764-768

Author(s):

Opal Pitaksakulrat ◽

Monticha Chaiyasaeng ◽

Atchara Artchayasawat ◽

Chatanun Eamudomkarn ◽

Sorawat Thongsahuan ◽

...

Keyword(s):

Haemonchus Contortus ◽

Control Strategies ◽

Integrated Approach ◽

Southern Region ◽

Nucleotide Polymorphisms ◽

Benzimidazole Resistance ◽

Genotype Frequencies ◽

Tubulin Isotype ◽

Central Regions ◽

Β Tubulin

Background and Aim: Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene. Materials and Methods: A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol. Results: The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001). Conclusion: This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the β-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.

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High levels of benzimidazole resistance and β-tubulin isotype 1 SNP F167Y in Haemonchus contortus populations from Ceará State, Brazil

Small Ruminant Research ◽

10.1016/j.smallrumres.2016.11.023 ◽

2017 ◽

Vol 146 ◽

pp. 48-52 ◽

Cited By ~ 6

Author(s):

Jessica Maria Leite dos Santos ◽

Jomar Patrício Monteiro ◽

Wesley Lyeverton Correia Ribeiro ◽

Iara Tersia Freitas Macedo ◽

José Vilemar de Araújo Filho ◽

...

Keyword(s):

Haemonchus Contortus ◽

Benzimidazole Resistance ◽

Tubulin Isotype ◽

Β Tubulin

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Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing

Parasitology ◽

10.1017/s003118200800543x ◽

2009 ◽

Vol 136 (3) ◽

pp. 349-358 ◽

Cited By ~ 84

Author(s):

G. von SAMSON-HIMMELSTJERNA ◽

T. K. WALSH ◽

A. A. DONNAN ◽

S. CARRIÈRE ◽

F. JACKSON ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Haemonchus Contortus ◽

Anthelmintic Resistance ◽

Allele Frequencies ◽

Detection Methods ◽

Resistance Testing ◽

Β Tubulin

SUMMARYBenzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in β-tubulin genes have been associated with BZ-resistance. In the present study, we investigated β-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000–10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the ‘egg hatch test’, and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.

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