Identifying the genotype of parasitic nematodes Teladorsagia circumcincta resistant to benzimidazole anthelmintics

The purpose of the research is to identify strains of parasitic nematodes Teladorsagia circumcincta resistant to benzimidazole anthelmintics using molecular genetic methods.

Materials and methods. Animals: The Southern sheep aged 8 to 12 months. Parasitic nematodes of the family Trichostrongylidae were isolated from sheep abomasums. Male and female nematodes were preserved in separate labeled Eppendorf tubes at -20 ^оС. Molecular genetic testing. Native genomic DNA was isolated using a commercial kit DNA-Extran-2 (Synthol, Moscow) for DNA extraction from microgram quantities of tissues. Nested PCR. For DNA amplification, a T-100 BioRad thermal cycler and a commercial Master Mix reagent kit, Evrogen, were used.  The resulting DNA fragments were used as the main component for a restriction reaction with RsaI endonuclease (SibEnzyme, Novosibirsk). PCR-based detecting the resistant genotype of T. circumcincta. Multiplex-PCR was performed as recommended by Silvestre et al., 2000, using a commercial Master Mix reagent kit, Evrogen. The electrophoresis results were analyzed in the GelDoc gel-documenting system, Bio-Rad. The obtained fragments were sequenced at the Genome Collective Use Center of the Federal State Budgetary Scientific Institution “Research Centre for Medical Genetics” (Moscow).

Results and discussion. To identify β-tubulin gene mutations that lead to benzimidazole anthelmintic resistance in parasitic nematodes of the species T. circumcincta, molecular genetic testing was conducted using nested PCR, restriction fragment length polymorphism analysis, and multiplex-PCR methods. The results of the presence of nucleotide substitutions in the β-tubulin gene were confirmed by Sanger sequencing.

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