Genotyping of Fasciola hepatica Isolated from Domestic Animals in the West of Iran

Abstract

The most common parasites responsible for fascioliasis are F. hepatica and F. Gigantica. The fascioliasis is important in terms of health and economic. The west of the Iran is one of the major poles of animal husbandry and the prevalence of fascioliasis is high. The aim of current study is to determine Fasciola spp. in the west of Iran. In the present study, 45 samples were collected from slaughterhouses in the three provinces in the west of Iran including Ilam, Lorestan and Kermanshah. The flukes were detected using morphological methods. DNA of all samples was extracted. ITS1 Was identified by PCR and PCR-RFLP technique using Rsa1 restriction enzyme. Then, 15% of the sample was sequenced. Based on ITS1 sequence (ribosomal internal transcribed spacer), all samples showed 700bp bands. The results of sequencing showed a similarity between 99% to 100%. Using Rsa1 restriction enzyme, all samples produced three distinct bands including 60bp, 100bp and 360bp. These results demonstrated that the most common fasciola in the west of Iran in sheep and cattle is F. Hepatica. The results of present study showed that Just F. Hepatica is polluter in Livestock in the west of Iran. Further studies by new molecular markers for accurate identification of fasciola species will be useful in control and prevention of fascioliasis.

Molecular Identification of Fasciola Isolated from the Liver of Meat Animals in Fars Province, Iran

“…Polymerase chain reaction (PCR) using the lyophilized PCR PreMix manufactured by BIONEER Company, South Korea, was carried out in 20 μ l of the total volume, containing 10 μ l of PreMix, 4 μ l of distilled water, 4 μ l of DNA, 1 μ l of forward primer, and 1 μ l of reverse primer. Then, the Its1 gene amplification was made using the specific primers ITS1-F primer and primer sequence of 5-TTGCGCTGATTACGTCCCTG-3 and ITS1-R primer with a primer sequence of 5-TTGGCTGCGCTCTTCATCGAC-3 [ 16 ] with a temperature plan of (94°C for 3 min), annealing [(94°C for 90 sec, 55°C for 90 sec, and 72°C for 2 min and 30 cycles], and extension step: (72°C for 10 min), and the Its1 gene was replicated in 700 bp in length [ 17 ]. To confirm the PCR implementation in each stage, the obtained products were electrophoresed on a 1.5% agarose gel and compared to the marker.…”

“…According to the manufacturer, instruction, the tubes were incubated at 37°C for 7 h, to ensure full cutting of fragments. For analyzing the digestion products, 15 μ l of each product in addition to 2 μ l of loading buffer was electrophoresed on 3% agarose gel [ 17 ].…”