ArticleTropical animal health and production2025
Molecular characterization and phylogenetic analysis of Fasciola gigantica from West Bengal, India using mitochondrial DNA (cox1 and nad1) as markers.
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Abstract
Molecular identification and characterization of Fasciola has been a recent trend which is the most reliable technique over morphological identification of this parasite. In the light of this consideration, the current study was aimed carry out molecular characterization of Fasciola gigantica from cattle and buffalo from West Bengal, India using mitochondrial cytochrome c oxidase subunit 1 (cox1) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) as markers. The flukes (n = 24) were primarily identified based on their morphology. Genomic DNA was extracted from the flukes and Polymerease Chain Reaction (PCR) was performed with specific primers to detect and amplify 180 bp and 520 bp of mitochondrial cox1 and nad1 regions respectively of the adult flukes collected from liver and bile ducts of slaughtered cattle and buffalo. The amplicons were consequently sequenced and the sequence data were analysed. PCR, multiple sequence analysis and phylogenetic tree reconstruction confirmed the presence of F. gigantica flukes in West Bengal region. All the present Fasciola sequences phylogenetically clustered in F. gigantica clade for both cox1 and nad1 regions with respect to the basal group Paragonimus westermanii (Gen bank acc.no-AY190066 and AF051840). Intra-specific multiple sequence analysis of experimental samples exhibited no nucleotide site variation for both mitochondrial cox1 and nad1regions. On the other hand, inter-specific multiple sequence analysis revealed five base substitutions with other F. gigantica of GenBank derived for both cox1 and nad1 regions. Similarly intra-species amino acid alignment revealed zero amino acid site variation among the present experimental Fasciola flukes for both cox1 and nad1 regions. Furthermore, inter-specific amino acid sequence analysis revealed single amino acid sequence polymorphism in mitochondrial cox1 region and three amino acid sequence polymorphisms in mitochondrial nad1 regions among F. gigantica of West Bengal isolates with other F. gigantica of different isolates. The present study revealed a higher level of species specific homogeneity coexisting in mitochondrial cox1 and nad1 region. Multiple sequence alignment also revealed significant similarity sites which can also be used to identify F. gigantica. All sequenced Fasciola of West Bengal isolates were identified as F. gigantica and the sequence analysis also revealed the relatedness between isolates from West Bengal, India and other different countries. Comparison of p-distance revealed that the sequences of F. gigantica isolated from cattle and buffalo of West Bengal diverged from each other by values ranging between 0.000 and 0.742 for mitochondrial cox1 gene while the values for nad1 gene was 0.000–0.000. This is the first report of molecular identification of F. gigantica from cattle and buffalo of West Bengal (India) isolates using mitochondrial cox1 and nad1 as molecular marker. Finding of our study based on multiple sequence analysis, phylogenetic tree analysis and low level of genetic p-distance study suggests that host variations are not likely to be useful characters for genetic differences of F. gigantica in a relatively confirmed geographical area.
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