Investigation of genetic diversity in fasciola spp isolates obtained from cattle and sheep by multiplex PCR and sequence analysis

Fasciolosis is a highly prevalent helminthic infection caused by Fasciola hepatica and F. gigantica. With the aim of identifying hybrid Fasciola flukes, multiplex PCR was performed to amplify phosphoenolpyruvate carboxykinase (pepck) gene. Furthermore, to determine Fasciola haplotypes, mitochondrial nad1 was amplified and aldo sequence and phylogenetic analyses were performed. Adult Fasciola isolates were collected from 51 cattle and 51 sheep after slaughtering from the abbatoires in Elazig province of Turkey, genomic DNA was isolated, and one-step multiplex PCR was subsequently performed for amplifying pepck. Isolates that generated a 510 bp band were identified as F. gigantica, those that generated a 241bp band were identified as F. hepatica, and those that generated both bands were identified as hybrid (aspermic) flukes. Further, multiplex PCR data identified four isolates as F. gigantica and 84 as F. hepatica. Fourteen hybrid isolates (five cattle and nine sheep) were identified. On unidirectional DNA sequence analysis of nad1 PCR products, three sequences were identified as F. gigantica and 99 as F. hepatica. Besides, four of 87 haplotypes de-tected for F. hepatica nad1 sequences were found to be previously reported, while the remaining 83 are unique to this study. To summarize, this study reports for the first time the existence of hybrid Fasciola flukes and 83 unique haplotypes of F. hepatica in Turkey.