Sequence analysis of the second internal transcribed spacer [ITS2] region of rDNA for species identification of trichostrongylus nematodes isolated from domestic livestock in Iran

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification. Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism [RFLP] profile was designed to differentiate Trichostrongylus species. A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings. Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species

Comparison of five simple methods for DNA extraction from Echinococcus granulosus protoscoleces for PCR-amplification of ribosomal DNA

Cystic hydatid disease is an important zoonosis, affecting humans and animals and is a significant public health and economic problem throughout the world and Iran. Since extraction of DNA from the parasite is a primary and crucial step which has a principal effect on PCR results, in the current study five simple methods for DNA extraction from protoscoleces of Echinococcus granulosus were applied and compared with each other. After collecting hydatid cysts from an abattoir, DNA samples were extracted from two cyst isolates from sheep, two from goats and two from camels using five different methods involving the use of glass beads, mechanical grinder, freeze-thaw, boiling and crushing. For all DNA samples extracted, one PCR assay based on amplifying rDNA-ITS1 region was performed and amplicons resolved on 1.5% agarose gels. The methods were compared regarding to DNA and PCR bands, time and cost effectiveness and laborious amount. The target DNA was successfully amplified from all samples using all methods produced an expected band size. All methods showed some advantages and disadvantages in PCR gels. The boiling method, which was the most time and cost effectiveness method, achieved the thickest bands in the PCR following grinder, crushing, freeze-thaw and glass beads. Boiling and crushing methods were the most suitable methods regarding their amplicon quality, easiness, quickness and cost effectiveness