Genetic Characterization of Gigantocotyle explanatum Based on First Internal Transcribed Spacer (ITS-I) of Ribosomal DNA from Buffaloes of Peshawar, Pakistan

Abstract

Background: Paramphistomiasis is a gastrointestinal platyhelminth infection that presents high risk for livestock sectors in terms of morbidity, reduced milk production and meat. Therefore, characterization of these parasites on molecular level is preliminary to understand their successful propagation and relationship with host, in the form of resistance. This approach has proven a powerful tool to find out the taxonomic level of specie responsible for disease that may provide a foundation for the development of vaccines and drugs. ITS-I rDNA spacer region are used for characterization of digenean parasite at different systematic levels and find out the genetic differences within species and with other trematodes from different geographical regions. Objective: The specific objective of the present study was to genetically characterize the Gigantocotyle explanatum liver fluke based on rDNA (ITS1) from Peshawar, Pakistan. Methodology: Adult flukes of G. explanatum were collected from the bile ducts of infected buffaloes and were preserved in 70 percent ethanol. Genomic DNA from all specimens was successfully extracted through chloroform-phenol method and Polymerase Chain Reaction was done to amplify the required region. After confirmation of amplified product by Gel electrophoresis, it was sequenced. Similarity index was checked with other reference sequences by using BLAST and CLUSTALW. The phylogenetic analysis and genetic distance were computed in MEGA 7 by Maximum Likelihood Method. Results: The total amplified sequenced length was of 1286 bp, and for intraspecific and interspecific genetic variation of 400 bp were analyzed. Current species of G. explanatum from Pakistan varied of 12 bp and formed a sub-clade with Paramphistomum cervi (KJ459934), a rumen parasite of China with 92% similarity, and Watsonius watsoni (KC763806) also shared 72 % similarity. Two species of Gastrodiscoides hominis (EU887294) from India also make a sub-clade with 78% similarity index. Comparison with other paraphyletic clades as follow Diplodiscus mehrai (KX506857) and Diplodiscus japonicus (KX506855) from Russia with 78% bootstrap values. Chiorchis (MF370224) of Columbia make distinct position with 78% identity along with G. explanatum species. Genetic pair distance of current species was calculated 0.0000 to 0.03093 with the species of Paramphistomidae and 1.21492 with Cladorchiidae. Conclusion: In summary our current results showed no intraspecific variation in the ITS1 region of G. explanatum from Peshawar, Pakistan and varied 12 bp from Paramphistomum cervi (KJ459934) rumen parasite of China. Phylogenetic analysis reveals high rate of variation with other species found in western countries like Gastrodiscoides hominis (MF370224) of Columbia. This characterization will be helpful to formulate a clear difference among digenean trematodes at their molecular level. The current molecular data will set bases and contribute in further studies on different paramphistomes, particularly for G. explanatum .