Molecular characterization ofFasciola giganticain Punjab, Pakistan to infer the dispersal route among the neighbouring countries of the Indian subcontinent

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in millions of dollars production losses to the livestock industry. To understand the dispersal origin and the spread patterns of F. gigantica is important for preventing the disease. A total of 53 Fasciola flukes collected from buffalo and goat in the Punjab province of Pakistan, were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase ( pepck ) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta ( pold ). A significant genetic difference between F. gigantica from buffalo and goats in Pakistan was indicated by the genetic analysis of two distinct mitochondrial markers [NADH dehydrogenase subunit 1 ( nad1 ) and cytochrome C oxidase subunit 1 ( cox1 )]. Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes were clustered in haplogroup A. Fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, or possibly come from neighbouring Middle East countries. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica regarding the development of sustainable parasite control strategies.