A PCR-RFLP FOR THE DISTINCTION BETWEEN FASCIOLA HEPATICA AND FASCIOLA GIGANTICA IN SOHAG
Refaat Mohammad khalifa*, Hanaa Ahmad El-Hady, Eman Khalaf Omran and Noha Samir Ahmad
Parasitology departments, Faculties of Medicine, Assiut* and Sohag Universities
One of the neglected food-borne-diseases is fascioliasis; it is a serious parasitic disease infecting humans and animals worldwide and tops all the zoonotic helminths. In Egypt, human and animal fascioliasis is an endemic clinical and epidemiological health problem. The causative agents, Fasciola hepatica and Fasciola gigantica, which their geographical distributions is overlapped. Moreover, in humans, liver flukes classification cannot be achieved by clinical, pathological or immunological method. The differential diagnosis between Fasciola hepatica and Fasciola gigantica infection is very important because of their different transmission and epidemiological characteristics. A simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the common restriction enzyme Avall is described to distinguish between both fascioloid species. In our study, adults of F. hepatica and F. gigantica were isolated from liver of naturally infected sheep and cattle brought from various regions of Sohag Governorate. The 2 species were identified based on morphological criteria and all doubtful samples were discarded and (QIA amp DNA Mini Kit) used, a new method to isolate DNA from the worm and Restriction fragment length polymorphism patterns of worms were obtained after digestion of the PCR products with AvaII restriction enzymes. The result of a regular PCR experiment for the amplification of the selected 28S rDNA fragment with the designed primer set yielded identical 648- bp-long PCR products for F. hepatica and F. gigantica. Where as the RFLP profile obtained from F. hepatica revealed three fragments of 628, 358 and 300 bp, F. gigantica generated clearly distinguishable four fragments corresponding to 628, 541, 358 and 300 bp fragments. This result refers that two species are present in our locality, F. gigantica and F. hepatica, as there are different band patterns generated after digestion or presence of intermediate forms as there are three similar fragments. This assay is a simple, rapid and reliable method for differentiating Fasciola spp, and can be used for diagnostic and epidemiological purposes.
December 2011