PCR- RFLP بررسي تخم تريكوستر نژيلوس هاي جدا شده از گوسفندان منطقه اصفهان به روش

Study on four trichostrongylus species in isfahan by PCR-RFLP

Background: Trichostrongyliases as zoonotic diseases are of major public health and socio-economic importance. Accurate identification of these parasites is the first step in strategic planning for the prevention, control and treatment of the parasitic disease. The aim of this study is using PCR-RFLP technique for genotyping. Materials and methods: samples were collected from elementary tract content of 70 sheep in Isfahan and Khorasgan slaughterhouses. Identification of Trichostrongylus was determined based on morphological character and key identification after direct and. flotation methods . For genotyping studies, DNA was extracted and the ITS-2 of ribosomal DNA of each species with specific primers was amplified by PCR and restricted with the endonucleases Dral, RsaI, HinfI. The profile was visualized in agarose gel under ultraviolet transillumination. Results: The eggs of most species are morphologically indistinguishable at the generic level .rDNA-ITS2 fragment size of all species were the same size about 330bp, however, there were differences among species in their PCR-RFLP patterns. The two fragments produced with RsaI in all species were the same size about 138bp,190bp. The PCR product T.probulurus with Dral remained unrestricted. Restriction with DraI produced two fragments in the PCR product of T. axei and T.colubriformis about 110bp, 215bp, however, the larger of the two fragments in T.vitrinus is smaller in size than in T.axei and T.colubriformis had a size about/45bp,185bp.The two fragments produced with Hinfl in T.colubriformis had a size about 90bp,238bp, By contrast other Trichostrongylus remained unrestricted. Conclusion: Based of our results, from genotyping studies using PCR-RFLP technique, it can be concluded that four species had been identified .This study provides a pattern to distinguish Trichostrongylus species in Isfahan region using the Dral, Rsal, Hinfl restriction endonucleases.